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"Identification of key differentially expressed mRNAs and microRNAs in non‘small cell lung cancer using bioinformatics analysisWEIWEI WANG SHANSHAN WANG and LEI PANDepartment of Pulmonary and Critical Care Medicine Beijing Shijitan Hospital Capital Medical University Beijing PR ChinaReceived September Accepted January 103892etm20209105Abstract Nonsmall cell lung cancer NSCLC is a leading cause of mortality worldwide However the pathogenesis of NSCLC remains to be fully elucidated Therefore the present study aimed to explore the differential expression of mRNAs and microRNAs miRNAsmiRs in NSCLC and to determine how these RNA molecules interact with one another to affect disease progression Differentially expressed genes DEGs and differentially expressed miRNAs DEMs were identified from the GSE18842 GSE32863 and GSE29250 datasets downloaded from the Gene Expression Omnibus GEO database Functional and pathway enrichment analysis were performed based on Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG databases STRING Cytoscape and MCODE were applied to construct a proteinprotein interaction PPI network and to screen hub genes The interactions between miRNAs and mRNAs were predicted using miRWalk and a miRNAmRNA regulatory network was constructed The prognostic value of the identified hub genes was then evaluated via KaplanMeier survival analyses using datasets from The Cancer Genome Atlas A total of DEGs and DEMs were identified from the GEO datasets The enriched pathways and functions of the DEGs and target genes of the DEMs included osteoclast differentiation cell adhesion response to a drug plasma membrane extracellular exosome and protein binding A subnetwork composed of genes was extracted from the PPI network and the genes in this subnetwork were mainly involved in the cell cycle cell division and DNA replication A miRNAgene regulatory network was constructed with miRNAgene pairs based on DEMs and DEGs KaplanMeier survival analysis Correspondence to Professor Lei Pan Department of Pulmonary and Critical Care Medicine Beijing Shijitan Hospital Capital Medical University Tieyi Road Haidian Beijing PR ChinaEmail panleibjsjthcnKey words nonsmall cell lung cancer bioinformatics microRNA miRNAmRNA network KaplanMeier plotindicated that the expression of ubiquitin E2 ligase C cell division cycle protein DNA topoisomerase IIα aurora kinase A and B cyclin B2 maternal embryonic leucine zipper kinase slit guidance ligand phosphoglucomutase endomucin cysteine dioxygenase type dihydropyrimidinaselike miR‘130b miR‘ and miR‘ was significantly associated with overall survival of patients with lung adenocarcinoma In the present study a miRNAmRNA regulatory network in NSCLC was established which may provide future avenues for scientific exploration and therapeutic targeting of NSCLCIntroductionLung cancer is one of the most common types of cancer and is the leading cause of cancerassociated mortality worldwide accounting for of all deaths due to cancer Approximately of lung cancers are nonsmall cell lung cancer NSCLC In turn NSCLCs are comprised of lung adenocarcinoma LUAD of lung cancers lung squamous cell carcinoma LUSC of lung cancers and large cell carcinoma of lung cancers Despite advances in diagnostics surgery and medication in recent decades the average 5year survival rate of patients with NSCLC remains as low as This poor prognosis is a consequence of high rates of tumor metastasis and recurrence and numerous signaling pathways having been identified to be involved in these processes Thus an enhanced understanding of the molecular mechanisms controlling NSCLC progression is required to improve the low survival rate The development of highthroughput sequencing has allowed for comprehensive comparisons of gene expression profiles thereby identifying differentially expressed genes DEGs between tumor and normal tissues Changes in expression levels usually indicate pathological states as proteins encoded by DEGs may be involved in tumorigenesis and tumor progression microRNAs miRNAsmiRs are short noncoding RNA molecules that mediate the posttranscriptional regulation of mRNAs via binding to complementary sequences in the 'untranslated region UTR of mRNAs and suppressing their translation or mediating their degradation An individual miRNA may regulate hundreds of different mRNA molecules highlighting the existence of miRNAmRNA regulatory 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCnetworks Depending on how it is expressed a specific miRNA may therefore act to suppress or promote oncogenesis via specific effects on relevant target mRNAs Indeed miRNA profiling efforts have been used to identify specific miRNA signatures associated with particular tumor subtypes thereby allowing for cancer diagnosis treatment planning and prediction of patient prognosis Bioinformatics analysis of gene expression microarray data provides a useful tool for revealing numerous previously unrecognized mRNAs and miRNAs that may be implicated in the pathogenesis of cancer or other diseases In the present study gene expression datasets were analyzed using an integrated bioinformatics approach in order to identify DEGs and differentially expressed miRNAs DEMs between NSCLC tumors and healthy control tissues Functional enrichment and proteinprotein interaction PPI network analyses were performed to better establish the functions of these mRNAs and miRNA and these approaches were combined with an analysis of mRNAmiRNA interactions to screen hub genes and miRNAs in this regulatory network Through this approach the present study aimed to further elucidate the molecular mechanisms of NSCLC to identify potentially novel therapeutic strategies for its treatmentMaterials and methods Microarray data information The datasets used in the present study were obtained from the Gene Expression Omnibus GEO database www ncbinlmnihgovgeo The original gene expression profiles from the datasets GSE18842 GSE32863 and GSE29250 were used and the clinical information of the patients was obtained from the original s The GSE18842 dataset included samples tumors and controls and all samples were paired except tumors and control The platform used for the GSE18842 dataset was GPL570 HGU133_Plus_2 Affymetrix Human Genome U133 Plus Array The GSE32863 dataset included samples tumors and controls and all samples were paired The platform used for the GSE32863 dataset was the GPL6884 Illumina Human WG6 v30 expression beadchip The GSE29250 dataset included samples tumors and controls and all samples were paired The platform used for the GSE29250 dataset was the GPL8179 Illumina Human v2 MicroRNA expression beadchipIdentification of DEGs To compare gene expression profiles the GEO2R tool httpwwwncbinlmnihgovgeogeo2r accessed March which is based on the limma package in R was used to individually identify DEMs and DEGs in each dataset To control for type I error as a result of multiple comparisons within each dataset the false discovery rate FDR determination feature automatically included in the GEO2R tool was employed Significant DEGs were those that remained significant after FDR correction when tested via multiplecomparisons ttests and fold change FC2 and P005 were set as the cutoff criteria Any probes that did not correspond to a specific gene symbol were then filtered from the resultant dataGene function analysis For gene ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathway analyses of DEMs and DEGs the database for annotation visualization and integrated discovery DAVID database v68 httpdavidabccncifcrfgov was used focusing specifically on humans and using all genes as an enrichment background Significant enrichment in these analyses was determined based on an adjusted Pvalue of as established via the BenjaminiHochberg method These Pvalues were determined on the basis of a cumulative hypergeometric distribution calculating qvalues based on the BenjaminiHochberg procedure as a means of controlling for multiple testing For comparisons of hierarchical clustering of enriched terms clusters were designated as groupings that had a similarity score with the most significant term within a given cluster being selected to represent the cluster as a wholePPI network construction and analysis The online Search Tool for the Retrieval of Interacting Genes and proteins STRING database v110 stringdborg was used for PPI network construction PPI pairs with a combined score ‰¥ were used to generate the network Cytoscape v340 cytoscapeorg was used to visualize the regulatory interactions between these genes and CentiScaPe v22 Center for Biomedical Computing University of Verona Italy was used to analyze network distributions based on topological properties The Molecular Complex Detection application MCODE v16 was used to identify and extract subnetworks from the global PPI network based on the kcore algorithm The genes with a degree ‰¥ in this regulatory network were identified as hub genes as described previously Prediction of the mRNA‘miRNA interactions An online tool called miRWalk httpmirwalkummuniheidelbergde which integrates predictive outputs of TargetScan and miRDB was used to predict DEG and DEM interactions A score ‰¥ was considered as the critical criterion for the miRWalk predictive analysis Only the target mRNAs predicted by all tools miRWalk TargetScan and miRDB were used for further analysis By overlaying identified DEGs and these predicted mRNA targets a miRNAmRNA regulatory network was constructed and then visualized using CytoscapeAnalysis of datasets from the cancer genome atlas TCGA TCGA is an online database that may be used to research and explore publicly available datasets cancergenomenihgov accessed March including RNA sequencing RNAseq data from TCGA samples of different types of cancer In the present study the tumor types were limited to LUAD and LUSC RNAseq data and clinical data from LUAD and LUSC samples from TCGA datasets were used Based on the approach previously outlined by Li and Dewey a PERL program was used to multiply the ˜scaled estimate™ by yielding transcripts per million TPM values for all gene expression as TPM values were thought to be a more reliable means of comparing gene expression than fragments per kilobase of TPMmapped reads or reads per kilobase of TPMmapped reads values In the present study to improve the reliability of the analysis the expression of hub genes was validated in TCGA datasets using Gene Expression Profiling Interactive Analysis GEPIA v10 httpgepiacancerpkucn For each of the hub genes patients were 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Figure Volcano plots and clustering heat maps of the differentially expressed genes between the non small cell lung cancer and normal tissues A1 Volcano plots of differentially expressed mRNA from the GSE18842 dataset A2 Volcano plots of differentially expressed mRNA from the GSE32863 dataset A3 Volcano plots of differentially expressed miRNA from the GSE32863 dataset Each graph represents the expression of the gene plotted by log2 fold change on the horizontal axis and log10q value on the vertical axis Red represents upregulation green represents downregulation and blue represents no significant difference B1 Clustering heat maps based on GSE18842 dataset B2 Clustering heat maps based on the expression of mRNAs in GSE32863 dataset B3 Clustering heat maps based on the expression of miRNAs in GSE32863 dataset Orange represents upregulation and blue represents downregulation miR microRNA hsa Homo sapiens UP upregulation DW downregulation NoDiff no difference NA not available Some miRNAs in the databases have probes but no datastratified into groups based on expression levels of each gene and differences in patient survival were analyzed generating hazard ratio HR and CI values as well as logrank Pvalues for each comparison ResultsIdentification of DEGs and DEMs In the GSE18842 and GSE32863 datasets and DEGs were identified respectively of which and were upregulated and and were downregulated Fig 1A and B A total of DEGs were shared between these datasets upregulated and downregulated The GSE32863 dataset yielded a list of DEMs of which were upregulated and were downregulated Fig 1C Functional enrichment analysis of overlapped DEGs and target genes of DEMs To assess the biological roles of these DEGs and target genes of DEMs KEGG and GO enrichment analyses were performed The top enriched terms for each analysis were compiled in Fig KEGG pathway enrichment analysis indicated that the DEGs and target genes of DEMs were mainly enriched in osteoclast differentiation complement and coagulation cascades Staphylococcus aureus infection and pertussis Fig 2A GO analysis in the category biological process suggested these DEGs and target genes of DEMs were primarily enriched in ˜cell adhesion™ ˜response to drugs™ and ˜extracellular matrix organization™ Fig 2B GO analysis in the category cellular component suggested that the DEGs and target genes of DEMs were mainly enriched in ˜plasma membrane™ ˜extracellular exosome™ and ˜extracellular localization™ Fig 2C In the category molecular function the DEGs and target genes of DEMs were mostly enriched in ˜protein binding™ ˜identical protein binding™ and ˜calcium ion binding™ Fig 2D PPI network construction and analysis of modules The overlapping DEGs which were shared between the datasets GSE18842 and GSE32863 indicated a distinct set of interactions and networks PPI pairs with a combined score ‰¥ were used to generate the network A PPI network was constructed using of the DEGs and the resultant network had nodes and edges Fig 3A There were upregulated and downregulated genes among the DEGs A total of nodes had a degree of and were designated as hub genes including interleukin Jun protooncogene JUN 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure Functional enrichment analysis of overlapped DEGs and target genes of DEMs A Biological process analysis in the GO database B Cellular component analysis in the GO database C Molecular function analysis in the GO database D Pathway enrichment analysis in the KEGG database The horizontal axis represents the ratio of genes and the vertical axis represents different terms in functional enrichment analysis hsa Homo sapiens DEGs differentially expressed genes DEMs differentially expressed microRNAs GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes ECM extracellular matrix neg negativeubiquitin E2 ligase UBE2C cell division cycle protein CDC20 DNA topoisomerase IIα TOP2A aurora kinase A AURKA AURKB cyclin B2 CCNB2 kinesin family member 20A FBJ osteosarcoma oncogene and maternal embryonic leucine zipper kinase MELK The topology parameters of these hub genes in the PPI network are presented in Table I Furthermore a subnetwork containing nodes and edges was extracted from the global PPI network Fig 3B The results of the KEGG and GO analyses for the genes in the subnetwork are presented in Table II The most significantly enriched terms in this network were ˜cell cycle division™ and ˜DNA replication™ associated with cancer confirming the relevance of the present analysis to NSCLC progression and prognosisSurvival analysis of hub genes Survival analysis was performed for the hub genes based on TCGA data Increased expression of of the hub genes UBE2C CDC20 TOP2A AURKA AURKB CCNB2 and MELK was significantly associated with poorer overall survival OS in patients with LUAD Fig miRNA‘gene network Using the miRWalk application putative DEM targets as established by the TargetScan and miRDB databases were identified A total of putative target mRNAs overlapped with the DEG dataset yielding miRNAgene pairs based on DEMs and the DEGs that were putative targets These were used to construct an overlapping regulatory network Fig A total of upregulated DEMs were predicted to downregulate DEGs whereas decreased expression of DEMs was predicted to be associated with increased expression of DEGs TCGAbased survival analysis suggested that none of the hub genes were associated with OS in patients with NSCLC However specifically among patients with LUAD those with elevated expression of slit guidance ligand SLIT3 phosphoglucomutase PGM5 endomucin EMCN cysteine dioxygenase type CDO1 dihydropyrimidinaselike DPYSL2 miR‘130b and miR‘ had a significantly higher OS compared with patients who had low expression of these genes By contrast elevated miR130b and miR127 expression was associated with poorer OS in patients with LUAD Fig Furthermore the differences of the DEGs and DEMs in miRNAgene networks of NSCLCs with or without KRAS mutation were examined The results suggested that the expression levels of TOP2A P00357 Fig 7A AURKA P00409 Fig 7B and MELK P00190 Fig 7C were significantly lower in KRAS mutation groups compared to KRAS wildtype groups Fig 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Table I Topology parameters of the genes with a degree ‰¥ in the protein‘protein interaction networkGene IL6 JUN UBE2C CDC20 TOP2A AURKA AURKB CCNB2 KIF20A FOS MELK IL6 interleukin JUN Jun protooncogene UBE2C ubiquitin E2 ligase CDC20 cell division cycle TOP2A DNA topoisomerase Iiα AURKA aurora kinase A CCNB2 cyclin B2 KIF20A kinesin family member 20A FOS FBJ osteosarcoma oncogene MELK maternal embryonic leucine zipper kinaseBetweenness Closeness 974x104 919x104 801x104 715x104 767x104 694x104 717x104 704x104 720x104 872x104 697x104 Stress Degree DownDownUpUpUpUpUpUpUpDownUpMCODE_Score Regulation Figure PPI network and subnetwork A Global PPI network and B the core subnetwork extracted from the global network Red represents upregulation and green represents downregulation The bigger the node the higher the degree PPI proteinprotein interactionDiscussionCancer is a genetic disease wherein cumulative mutations drive the multistep progression towards oncogenesis eventually culminating in unrestrained cancer growth NSCLC remains one of the most common and deadliest forms of cancer making the elucidation of the molecular mechanisms governing this disease paramount In the present study DEMs and DEGs associated with NSCLC were identified via a bioinformatics analysis yielding DEGs and DEMs 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCMCM GTTP MCM CDC GTTP CDC AKRUANEF BNCCMCM CDCMCM x x x noitacilper AND sisoiem etycoO elcyc lleC ash ash ashseneG eulavP oitar eneG syawhtapGGEK A mreTkrowtenbus eht ni seneg eht rof sisylana tnemhcirne OGdna yawhtapGGEK II elbaTseneG eulavP oitar eneG PByrogetac ni sisylana OG B mreTMPSA CEBU BKRUA GTTP CDCGTTP CDC AKRUA AKRUA CRBI FPNEC CRBKLEMI FPNEC XPT AKRUA CRBIKEN BNCC XPT KEN XPT KEN BNCCACDCBNCC x x x noisivid raelcun citotim fo noitisnart MG noisivid llec OGOGOGelcyc llec citotimNEF MCM MCM AAKI CDCCEBU BKRUA GTTP CDC SNGIAKRUABKRUA CDC CRBI FPNECCEBU BKRUA GTTP CDC ACDCAKRUAAPOT MCM MCMMCM MCM CDCMPSA BKRUA AKRUA SMYT x x x x x x x noisehoc ditamorhc retsis noitanitiuqibu nietorp OGOGssecorp cilobatac tnednepedxelpmoc gnitomorpesahpana noitacilper AND OGOGnietorp tnednepednitiuqibu ni ssecorpdevlovni cilobatac noitazinagro eldnips fo noitisnart SG OGOG gnidniwnuAND OGnoitacilper ANDni devlovnielcyc llec citotimAFIKAFIK BKRUA CDC AKRUA AFIK MPSA BKRUA AKRUA CRBI FPNEC PASUN XPT CRPCRBI FPNEC KEN CRP ACDC BKRUA CDC AKRUA CRBI NEF APOT BNCC ACDC FPNECCDC AAKI XPT CRP SMYT MCM CEBU SNGIMCM x x x m ydobdimsalpoelcun eldnips OGOGOG seneG eulavP oitar eneG CCyrogetac ni sisylana OG C mreT\x0cEXPERIMENTAL AND THERAPEUTIC MEDICINE CDCMPSA AKRUA CRBI APOT BNCC FPNECACDC PASUN AAKI XPT KEN CRPCDC SMYT MCM MCM BKRUA BKRUA AKRUA CRBI PASUNCDC FPNEC XPT KENBKRUA AKRUA CRBI PIRT NEF AFIKCDC AKRUA CRBI PASUN XPT KENMCM AKRUA XPT CRP BNCCAKRUA FPNEC KEN BNCC CDC SNGIGTTPKLEM ACDC CRPCRP x x x x x x x xelpmoc regnessap emosomorhc noteleksotyc elubutorcim elubutorcim eldnips elop eldnips elubutorcim emosortnec OGOGOGOGOGOG suelcun OGseneG eulavP oitar eneG CCyrogetac ni sisylana OG C deunitnoC II elbaT mreTPIRT AFIK NEF KLEMAPOT BNCC ACDC CDC MCMCEBU MCMBKRUA AFIKGTTP PIRT KLEM CDC AKRUA APOTCRBI MCMFPNEC CEBU BKRUA MCM AKRUA XPT PASUN AAKI XPT KEN CRP KENSNGIAFIK CRBI PASUN CRPAPOT MCM KLEM BKRUA AKRUA CDCCDC SNGCRBIIKENBKRUA AKRUAKLEM AFIKBKRUA AKRUA AKRUA XPT KENCRPMCM CDC x x x x x x x x x x ytivitca esanik eninoerhtenires gnidnib esanik nietorpnietorp ytivitca esanik gnidnib enires enotsih elubutorcim ytivitca esacilehAND ™™ gnidnib ytivitca esanik nietorpnigiro noitacilper AND gnidnib emyzne OGOGOGOGOGOGOGOG gnidnib nietorp gnidnibPTA OGOG emosomorhcinimortnec FPNEC elcyc noisivid sisenikotyc fo Enitiuqibu CEBU rotaluger nietorp CRP esatehtnys etalydimyht SMYT αiI esaremosiopot AND rotcaretni rotpecer enomroh dioryht APOTPIRT tinubus xelpmoc SNGI A rebmemyli maf nisenik AFIK esanik reppiz enicuel cinoyrbme lanretam nietorp detaicossa eldnips dna raloelcun rotcaf noitaelcun elubutorcimXPT detaicossa elcyc noisivid MCM llec CDC noitarapes ditamorhc retsis fo tnenopmoc ralullec CC llec ACDC selubutorcim eldnips rof rotcaf ylbmessa MPSA B esanik XPT esanikdetaler rotaluger GTTPGTTPAMNI KEN tnenopmoc A esanik arorua xelpmoc ecnanetniam emosomorhcinimAKRUA esaelcunodne cfi arorua ssecorp lacigoloib PB noitcnuf ralucelom FM semoneGdna seneG fo aidepolcycnEotoyK MCM gniniatnoc taeper PAI larivolucab CRBI iceps‘erutcurts pafl NEF tnenopmoc xelpmoc ecnanetniamGGEK elcyc ygolotnO eneG OGnoisivid llec CDC sneipas Bnilcyc omoHBNCC ash KLEMFnietorp esagil erem PASUNBKRUA SNGIseneG eulavP oitar eneG FMyrogetac ni sisylana OG D mreT 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure KaplanMeier survival analysis of the hub genes in the proteinprotein interaction network The KaplanMeier survival analysis based on the expression of A UBE2C B CDC20 C TOP2A D AURKA E CCNB2 F MELK and G AURKB The horizontal axis represents overall survival months and the vertical axis represents the percentage of survival The dotted line indicates the upper and lower boundaries of the confidence interval UBE2C ubiquitin E2 ligase CDC20 cell division cycle TOP2A DNA topoisomerase IIα AURKA aurora kinase A CCNB2 cyclin B2 MELK maternal embryonic leucine zipper kinase HR hazard ratio TPM expressionbased on overlapping hits in the GSE18842 GSE32863 and GSE29250 datasets These hits included upregulated and downregulated genes as well as upregulated and downregulated miRNAs Through functional enrichment analyses it was determined that these DEGs were primarily associated with processes including ˜osteoclast differentiation™ ˜complement and coagulation cascades™ ˜cell adhesion drug responses™ ˜plasma membrane™ ˜extracellular exosome™ and ˜protein binding™ In addition a DEG PPI network was generated and a significant subnetwork module was identified that contained genes associated with the cell cycle DNA replication and oocyte meiosis with the GO terms enrichment for ˜mitotic nuclear division™ ˜cell division™ ˜G2M transition of mitotic cell cycle™ ˜spindle™ ˜midbody™ ˜nucleoplasm™ ˜ATP binding™ ˜protein binding™ and ˜microtubule binding™ Cell cycle dysregulation is known to be a key factor linked to tumor development and progression Recent studies indicated that microtubule binding is linked to tumor metastasis and drug resistance Complement activation and coagulation cascade activation are similarly able to promote tumor development as a consequence of their ability to mediate the recruitment of myeloid cells that support tumor growth To summarize the identified DEGs may regulate the proliferation invasion migration and drugresistance of cancer cells through these pathways thus affecting the occurrence and development of NSCLC The investigation of these DEGs may pave a way towards novel targeted therapies for NSCLC\x0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Figure miRNAmRNA regulatory network The triangles represent miRNAs and the circles represent mRNAs Red indicates upregulation and green indicates downregulation miRNA microRNA miR microRNA hsa Homo sapiensBased on the PPI network hub genes with high degrees of interaction degree ‰¥ were extracted A prognostic analysis of these hub genes was performed using the online tool GEPIA The results revealed that patients with LUAD who had upregulation of UBE2C CDC20 TOP2A AURKA AURKB CCNB2 and MELK had a worse prognosis The expression of UBE2C has been previously indicated to be upregulated in lung cancer and the results of the present study suggested that it was associated with poor survival Similar observations have previously been made in ovarian cancer breast cancer and gastric cancer UBE2C is involved in the progression of the cell cycle and transcription and upregulation of UBE2C may induce an enhanced growth and colony formation of tumors as well as decreased autophagy in cancer cells Furthermore UBE2C reduces the sensitivity of cells to common chemotherapy drugs for lung cancer including cisplatin and docetaxel UBE2C may be used as a therapeutic target for NSCLC Another oncogene in several types of tumor and a hub gene identified in the present study was CDC20 CDC20 is an important regulator of the cell cycle and altered expression or functional impairment may induce mitotic arrest to prevent activation of adenomatous polyposis coli and hence increase premature anaphase manifesting as aneuploidy in daughter cells CDC20 was observed to be upregulated at mRNA and protein levels in NSCLC and was significantly correlated with tumor size pleural invasion and histological classification Of note knockdown of CDC20 caused inhibition of growth migration ability and formation of colonies in lung cancer cells as well as cell cycle arrest in G2M phase and induction of apoptosis making this oncogene a potential target molecule to address NSCLC therapy Upregulation of CDC20 has been associated with shorter OS in patients with LUAD but not in patients with LUSC which is consistent with the results of the present study TOP2A encodes for a DNA topoisomerase involved in torsional dynamics during replication and transcription which is also associated with cell proliferation TOP2A has been indicated to be upregulated in numerous types of tumor including breast nasopharyngeal and renal cell carcinomas and is associated with poor prognosis therefore TOP2A has important roles in cancer The ability of NSCLC cells to proliferate and invade tissues is associated with elevated TOP2A expression Several anticancer agents have been developed to target this gene and the development of drug resistance has been associated with mutation of TOP2A 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure KaplanMeier survival analysis of the hub genes in the miRNAmRNA regulatory network The KaplanMeier survival analysis based on the expression of A SLIT3 B PGM5 C EMCN D CDO1 E DPYSL2 F MIR130B G MIR1181 and H MIR127 The horizontal axis represents overall survival months and the vertical axis represents the percentage of survival The dotted line indicates the upper and lower boundaries of the confidence interval miRNAmiR microRNA SLIT3 slit guidance ligand PGM5 phosphoglucomutase EMCN endomucin CDO1 cysteine dioxygenase type DPYSL2 dihydropyrimidinaselike TPM transcripts per million HR hazard ratioFigure Violin plots indicating the differences in microRNAgene networks with or without KRAS mutation The expression levels of A TOP2A B AURKA and C MELK were significantly lower in KRAS mutation groups compared to KRAS WT groups P005 TOP2A DNA topoisomerase IIα AURKA aurora kinase A MELK maternal embryonic leucine zipper kinase WT wildtypeAURKA and AURKB are highly conserved serinethreonine kinases the former of which is associated with regulating centrosome duplication and spindle formation and the latter of which is important for regulating chromatin modifications and suppressing cytokinesis NSCLC prognosis is known to be associated with elevated expression of these genes In the present study TCGA dataset analysis suggested that the prognosis of patients with LUAD was associated with AURKA and AURKB which require further clinical trial validation CCNB2 is a cyclin gene that activates cyclindependent kinase to drive the G2M cell cycle transition and inhibition of CCNB2 leads to cell cycle arrest It has been previously confirmed that CCNB2 is upregulated in tissue and serum samples from patients with NSCLC Elevated CCNB2 mRNA levels are known to be closely associated with tumor differentiation grade and histological type and upregulation of CCNB2 at the protein level has been significantly associated with the degree of differentiation tumor size lymph node metastasis distant metastasis and clinical stage Previous studies of NSCLC have suggested that there was no statistically significant correlation between the levels of CCNB2 protein and mRNA in NSCLC The results of the present study indicated that upregulation of CCNB2 mRNA was a poor prognostic biomarker in patients with LUAD while a previous study suggested that the protein levels of CCNB2 may serve as an independent prognostic marker in NSCLC Therefore the role of CCNB2 in NSCLC should be further elucidatedMELK is a serinethreonine kinase that has been indicated to be highly expressed in several human cancer types prostate breast brain colorectal and gastric cancer and glioblastoma multiforme stem cells Elevated expression of MELK is associated with the degree of tumor malignancy and with poor survival in cervical cancer breast cancer and gastric cancer Furthermore the present study suggested that upregulation of MELK is associated with the progression of NSCLC miRNAs regulate a wide array of target mRNAs via 'UTR binding and subsequent translational repression As a 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE result complex miRNAmRNA networks may govern a wide range of biological pathways making miRNAs critical for the progression of numerous types of cancer In the present study a total of DEMs were identified and miRWalk‘mediated predictive analyses were performed to identify those DEMs that were predicted to interact with DEGs yielding hub miRNAs and associated mRNAs including miR1275p miR1345p miR130b3p miR1181 miR1453p miR1533p CDO1 SLIT3 and PGM5 Survival analyses revealed that dysregulation of miR1275p miR130b3p miR1181 CDO1 SLIT3 PGM5 EMCN and DPYSL2 were significantly associated with the prognosis of patients with LUADmiR127 has previously been indicated to function as either a promoter or suppressor of cancer development depending on the specific context Based on the NSCLC network established in the present study miR127 was among the most prominent regulatory miRNAs suggesting it serves complex regulatory functions in the context of NSCLC In a previous study miR127 expression was indicated to be elevated in LUAD and associated with poor prognosis consistent with the results of the present study High levels of miR127 induce epithelialtomesenchymal transition rendering tumor cells with stem celllike properties and propagate tumor resistance to epidermal growth factor receptor inhibitor The aggressiveness of the cancer was associated with a circuit involving miR127 NFκB and tumor necrosis factor αinduced protein which are markers of inflammation miR130b has also been documented in several other types of tumor with upregulation observed in prostate cancer while downregulation was identified in thyroid carcinomas In the present study miR130b3p expression was determined to be significantly increased in NSCLC and associated with poor survival although this was specifically restricted to patients with LUAD in the TCGA dataset warranting further investigation miR134 has been indicated to be differentially regulated in lung cancer and other types of cancer including gastric cancer breast cancer and oral cancer with certain studies reporting increased expression in lung cancer while other studies observed that it was downregulated miR134 may function to either promote or suppress tumor progression highlighting complex mechanisms warranting further investigationmiR1181 has been observed to be downregulated in nasopharyngeal carcinoma ovaria

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

"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

Wntcateninmediated signaling is a key pathway regulating tissue growth and development and tumorigenesis and has received increasing attention in recent years In addition to participating in healthy tissue and an development ectopic activation of the pathway can cause a variety of tumors and other pathologies The pathway plays a critical role in many processes such as proliferation differentiation apoptosis migration invasion epithelial“mesenchymal transition and cancer cell stemness The importance of the Wnt signal is selfevident This review describes the underlying mechanism of Wnt signaling pathway and highlights the latest findings on the relationship between Wnt signaling pathway and tumorigenesis In addition the potential relationship between miRNAs and Wnt signaling is presented Furthermore we discuss the intrinsic link between Wnt signaling and cancer cell stemness which shed light on the malignant progression of tumor cells Finally cancer treatment strategies based on the canonical Wnt signaling pathway are summarized hoping to help clinical development Keywords Wntcatenin tumor miRNAs cancer stem cell target therapeutic strategyIntroductionWnt signaling pathway plays a crucial role in embryonic development adult tissue homeostasis and cancer1 Due to the role in cell fate and tissue development increasing attention has been paid to Wnt signaling pathway in regenerative medicine2 Moreover the pathway is also involved in many pathological processes such as proliferation differentiation apoptosis migration invasion epithelial mesenchymal transition EMT and cancer cell stemness Notably the aberrant activation of Wntcatenin signaling results in the expression of several modulators that antagonize the antitumor activity of T cells which might lead to the failure of cancer immunotherapy3 Since Wnt signaling plays a pivotal role in tumorigenesis it is a promising target for the development of cancer therapeuticsSince the discovery about years ago many reports on the relationship between Wnt signaling and malignant tumor progression have been published The Wnt family consists of cysteinerich protein species which deliver canonical Wnt signaling through the interaction with Frizzled FZD and coreceptor low density lipoproteinrelated receptors and LRP56 thereby regulating cell fate growth and tissue repair4 After a series of posttranslational modifications such as porcupine palmitoylation lipid modification and glycosylation in the endoplasmic reticulum the Wnt precursor gradually maturates into the functional Wnt ligand Then the transmembrane protein Wntless Wls binds Wnt ligand and reaches to Cancer Management and Research “ Wen 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 the work 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 For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWen Dovepressthe plasma membrane via the Golgi apparatus Finally Wnt is detached from the cell membrane as components of exosomes or lipid protein ps15 As shown in Figure Wnt ligandbinding membrane receptor complex induces constitutive activation of Wntcatenin signaling which delivers downstream signaling LRP receptor phosphorylation recruits Axin to degrade Dishevelled DVL protein activation leads to inactivation of destruction complex which leads to the stability and accumulation of catenin6 Subsequently catenin is translocated into nucleus and interacts with the transcription coactivators like Tcell factor TCF and lymphoid enhancer factor LEF proteins to regulate the expression of Wnt target genes such as AXIN2 cycling and cMYC7 When Wnt signal is turned off the scaffold protein Axin adenomatous polyposis coli APC glycogen synthase kinase 3 GSK3 and casein kinase 1α CK1α form a multimeric destruction complex which induces catenin phosphorylation and ubiquitination degradation8Other mechanisms have been identified downstream of the canonical Wnt signaling pathway including the Hippo signaling cascade downstream nuclear effectors YAPTAZ Under Wnt OFF conditions YAPTAZ is an essential component of the destruction complex It recruits E3 ubiquitin ligase transducin repeatcontaining protein TrCP to promote catenin degradation in which YAP TAZ is a negative regulator of Wnt signaling However when Wnt is ON LRP56 induces the dissociation of YAP TAZ from Axin resulting in nuclear accumulation of YAP TAZ and enhancement of the expression of proproliferative genes in which YAPTAZ is a positive regulator of Wnt signaling9 Furthermore members of the Rspondin Figure An overview of Wnt signaling pathway As illustrated on the upper left side of Figure in the absence of Wnt ligand destruction complex induces the phosphorylation of catenin and the phosphorylated catenin is recognized and ubiquitinated by YAPTAZrecruited TrCP and subsequently subjected to proteasomal degradation In addition the E3 ubiquitin ligases Cullin 3KLHL12 recognize DVL and induce its degradation On the membrane ZNRF3RNF43 targets Frizzled receptor for ubiquitination and lysosomal degradation In the nucleus the inhibitory complex Groucho and histone deacetylases HDAC bind to TCFLEF transcription factors resulting in the inhibition of the Wnt signaling gene expression as shown in the lower left side of Figure On the upper right side of Figure the phosphorylated LRP receptor recruits Axin and DVL protein to the plasma membrane Activated DVL inactivates the destruction complex In this process the combination of DVL and ASPM inhibits the recognition of Cullin 3KLHL12 catenin with the assistance of histone modifying coactivators such as Pygopus Pygo and CREB binding protein CBPp300 interacts with the transcription factors TCFLEF to regulate target gene expression Frizzled ubiquitination is inhibited through the interaction of RSPO with LGR46 and ZNRF3 RNF43 œUb stands for ubiquitination and œps stands for phosphorylation Abbreviations LRP56 lowdensity lipoproteinrelated receptors and DVL dishevelled TCF Tcell factor LEF lymphoid enhancer factor APC the adenomatous polyposis coli GSK3 the glycogen synthase kinase 3 CK1α casein kinase 1α TrCP transducin repeatcontaining protein RSPO Rspondin LGR5 leucinerich repeat containing Gproteincoupled receptors ASPM abnormal spindlelike microcephaly associated HDAC histone deacetylases Pygo Pygopus CBP CREB binding proteinsubmit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alleucinerich RSPO ligand family were identified as positive effectors of Wnt signaling10 In the absence of RSPO the two homologues E3 ubiquitin ligases ZNRF3RNF43 ubiquitinated Frizzled inducing Fzd endocytosis When RSPO forms complex with repeatcontaining Gproteincoupled receptors LGR “ and ZNRF3 RNF43 however the Frizzled ubiquitination process is inhibited thereby inducing the activation of the Wnt signaling cascade Recently the abnormal spindlelike microcephaly associated ASPMDishevelled signal axis was proposed to participate in the generation of canonical Wnt signaling ASPM interacts with Wnt regulator DVL blocking the recognition for DVL by the ubiquitin ligase complex Cullin 3KLHL12 inhibiting the ubiquitination and subsequent degradation of DVL and maintaining a high level of Wnt activity11Besides the canonical pathway the Wnt signal also has an additional noncanonical activation pathway which is independent of catenin The noncanonical Wnt signaling pathway targets different receptors and activates multiple intracellular targets among which the most widely studied are Wntplanar cell polarity PCP and WntCalcium Ca2 pathways As shown in Figure in the WntPCP pathway Wnt ligand binds to receptor proteins to recruit and activate DVL which subsequently activates the small GTPases Rho or Rac triggering downstream Rhoassociated kinase ROCK and cJun Nterminal kinase JNK recruitment allowing cytoskeleton reanization12 In the WntCa2 pathway Fzd receptor is activated which induces the activation of DVL Then DVL recruits phospholipase C PLC which converts phosphatidylinositol 45bisphosphate PIP2 to diacylglycerol DAG and inositol 145triphosphate Figure An overview of noncanonical Wnt signaling pathway Noncanonical Wnt signaling is divided into WntPCP and WntCa2 pathways In WntPCP Wnt ligand binding receptor proteins trigger the activation of the ROCK and JNK downstream kinases allowing cytoskeletal reanization In WntCa2 noncanonical Wnt signaling increases the intracellular levels of DAG and IP3 by recruiting DVL stimulates the intracellular Ca2 release and activates downstream PKC CaN and CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal andor transcriptional responses Abbreviations ROCK Rhoassociated kinase JNK cJun Nterminal kinase PLC phospholipase C PIP2 phosphatidylinositol 45bisphosphate DAG diacylglycerol IP3 inositol 145triphosphate PKC protein kinase C CAN calcineurin CaMKII Ca2calmodulindependent protein kinase II DAAM1 the formin protein dishevelled associated activator of morphogenesis RHOA Ras homolog family member A RAC1 Rasrelated C3 botulinum toxin substrate AP1 activator protein1 CDC42 Cell division cycle protein NFAT nuclear factor of activated T cellsCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressIP3 IP3 stimulates intracellular Ca2 release and DAG and Ca2 together activate downstream protein kinase C PKC calcineurin CNA and Ca2calmodulindependent protein kinase II CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal and or transcriptional responses1314 Although noncanonical Wnt signaling is also an indispensable factor in the development of cancer this review mainly focuses on canonical Wnt signalingThere is growing evidence that Wntcatenin signaling is one of the main driving factors of some malignant tumors such as colorectal cancer liver cancer and breast cancer This review therefore focuses on the latest research progress on the correlation between the Wnt signal and tumors and the potential relationship between the Wnt signal and miRNAs In addition the possible relationship between the Wnt signal and CSC is discussed Finally cancer treatment strategies based on the canonical Wnt signal are summarizedWnt Signaling in Colorectal CancerColorectal cancer CRC is the third most common cancer in the world and the fourth most deadly cancer after lung liver and stomach cancer with the highest incidence in developed countries Constitutive activation of Wnt catenin signaling is a functional marker of colorectal cancer in which APC mutations occur in approximately of CRCs APCbased therapy may be therefore a promising strategy for the treatment of colorectal cancer15 It was reported that APC silencing drives hyperproliferation in the intestine and eventually forms colon adenomas Restoring APC gene led to continuous regression without recurrence whereas reestablished crypt homeostasis was found in tumor tissues carrying Kras and p53 mutations8 Wnt signaling is thus a promising target for the development of cancer therapy It was recently found that metastasisassociated in colon cancer MACC1 served as a transcriptional target of Wntcatenin signaling The DBC1 Deleted in breast cancer1 coactivator promotes selfrenewal capacity and drug resistance in colon cancer by regulating LEF1catenindependent enhancer in the MACC1 intron16 Furthermore Deptor The DEP domain containing mTOR interacting an mTOR inhibitor promotes cancer cell proliferation and survival in CRC and is a potential target for novel cancer therapies Deptor is a direct target gene for WntcatenincMyc signaling17 Silencing Deptor induces differentiation and inhibition of CRC cells by increasing ketone production and decreasing the expression of B lymphoma MoMLV insertion region Bmi1 while increasing mTOR activation A combination of AktmTOR and Wntcatenin inhibitors thus can exert a potent antitumor effect In addition to inhibition of positive regulators for Wnt signaling a negative regulator was also found as a possible target for Wnt signaling18 Vset and transmembrane domain containing 2A VSTM2A is a secreted protein that inhibits Wntcatenin signaling in colon cancer by directly inhibiting LRP6 activity and inducing LRP6 endocytosis and degradationCancer metastasis is the main cause of death in CRC patients Tocopherol alpha transfer proteinlike TTPAL and chromatin anizer special ATrich binding protein SATB1 are possibly involved in the invasion and metastasis of CRC Gou found19 that the upregulation of TTPAL is associated with a poor prognosis of colon cancer in which TTPAL directly interacts with thyroid receptorinteracting protein TRIP6 and inhibits the ubiquitination and degradation of TRIP6 TRIP6 competitively binds to the membraneassociated guanylate kinase with inverted domain structure1 MAGI1 tumor suppressor and dissociates catenin from MAGI1 Free catenin enters the nucleus to activate the oncogenic Wntcatenin signal increasing the ability of cancer cells to migrate and invade In addition overactivation of Wnt signaling promotes the interaction between TCF7L2catenin complex and SATB1 leading to SATB1 expression20 SATB1 regulates the expression of tumor growth and metastasisassociated genes regulating the interconversion of colon cancer in the invasion phenotypeWntcatenin signaling affects the malignant progression of colon cancer by regulating tumor suppressoractivator It is therefore pivotal to further explore Wnt signal related targets with a view to revealing a network of Wnt signaling in cancer and providing more options for cancer treatments The roles of Wnt signaling in the development of colon cancer were summarized in this section hoping to understand Wnt signaling from a new perspectiveWnt Signaling in Liver CancerAccording to the survey by World Health anization™s International Cancer Research Center in liver cancer is one of the world™s highmortal cancers and hepatocellular carcinoma HCC is the second leading cause of cancer death worldwide catenin is expressed in the adult liver which mediates Wnt signaling and regulates the transcription and translation of genes around liver cells affecting various aspects of liver biology Here we mainly describe the roles submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alto regulate of Wnt signaling in hepatic metabolic partitioning and liver regeneration RSPOLGR45ZNRF3RNF43 module is the main signaling complex in the Wntcatenin signaling pathway liver metabolism development and regeneration21 Knocking down LGR45 in the mouse liver resulted in a loss of catenin signaling and a defect in hepatic metabolic partitioning Supplementing RSPO1 or knocking down Znrf3Rnf43 extends Wntcatenin signal gradient in the liver in a reversible and LGR45dependent manner resuming the liver defect phenotype In addition liver regeneration is possibly regulated by type I transmembrane protein TMEM9 In the model of liver injury induced by CCl4 TMEM9 enhances the rapid assembly of the vATPase lysosomal proton pump activates cathepsin activity in lysosomes induces APC degradation and activates Wntcatenin pathway leading to the promotion of liver regeneration22 Wntcatenin signaling is therefore essential for liver development whereas the abnormal activation of the catenin signal is also a sign of promoting the development of hepatocellular carcinoma and other liver diseases As shown in Figure approximately of HCC exhibited Wntcatenin signaling activity among them about of hepatocellular carcinomas had Catenin 1 CTNNB1 mutations23 which highlighted the importance of CTNNB1 in the progression of liver cancer Together with the activation levels of other signals in HCC we might hypothesize that the crosstalk between Wnt signaling and multiple signaling pathways promotes the development of liver cancer24 In addition in a mouse hepatocyte model catenin activation per se is not sufficient to induce liver cancer and other mutation events must be involved in the development of liver cancer such as mutations in a telomerase reverse transcriptase gene TERT25 or those in the gene mesenchymal epithelial transition factor MET26 This phenomenon may be related to the weak catenin activity in the mouse hepatocyte model We found that there are weak mutations in the CTNNB1 in HCC and HCA hepatocellular adenoma for example the mutation takes place at K335N387 S45 or T41 of the CTNNB127Wnt signaling plays a crucial role in the growth and development of liver However its aberrant activation may promote the occurrence of hepatocellular carcinoma and other liver lesions It is noteworthy that a liverspecific Wntcatenin signaling pathway exists in liver cells Glypican3 GPC3 is a heparan sulfate proteoglycan specifically expressed on the surface of liver cancer cells Li reported28 that GPC3 regulates the activation of Wnt signaling by dual mechanisms and promotes proliferation of hepatocellular carcinoma When FZD is not abundant GPC3 acts as an alternative Wnt receptor and recruits Wnt at the cell surface through the Nterminal CRD domain leading to the promotion of Wnt signaling When FZD is abundant GPC3 and FZD interact through their heparin sulfate HS chains and the two molecules form a triple complex with Wnt to synergistically stimulate Wnt signaling GPC3 might function as a bridge to stabilize the interaction between Wnt and FZD Further studies promote the elucidation of the roles of Wnt signaling in the development of liver cancer and provide a theoretical basis for GPC3 targeted therapyWnt Signaling in Breast CancerWnt signaling is involved in the malignant progression of breast cancer such as proliferation invasion metastasis and drug resistance In healthy breast tissue inhibition of Figure Status of Wntcatenin signal activation in HCC A Activation ratio of Wntcatenin signaling in HCC B Mutation frequency of common highmutation genes in HCCCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressWntcatenin activity leads to developmental disorders reduced cell proliferation during pregnancy29 and However there is increasing evidence that aberrant activation of the Wnt signal promotes the development of breast cancer It was demonstrated30 that bone marrow tyrosine kinase on chromosome X BMX is upregulated in breast cancer It inhibits the degradation of catenin by promoting GSK3 phosphorylation thereby disrupting normal Wnt signaling and promoting the malignant progression of breast cancer Rational regulation of Wntcatenin signaling is therefore a key to the treatment of breast cancer although it is difficult to specifically control the signaling In theory inhibition of Wntcatenin signaling can prevent tumor progression Surprisingly it is likely that silencing canonical Wnt signaling drives cancer cells into dormancy resulting in the hindrance of tumor cells from cancer immunoediting by the host immune system31 It was reported that Dickkopf1 DKK1 inhibits lung metastasis by antagonizing noncanonical Wnt signal of breast cancer DKK1 however could promote breastto bone metastasis by regulating canonical Wnt signal in osteoblasts Taken together it is not straightforward to manipulate canonical Wnt signaling for the treatment of metastatic cancerWnt Signaling and MicroRNAsIn recent years several studies have revealed the potential connection between noncoding RNA and Wnt signaling Table summarizes the effect of miRNAs on Wntcatenin signaling in cancerIt has been reported that miRNA expression is also controlled by Wnt signaling In HCC CTNNB1 binds to the miR18396182 promoter region and increases the transcription of the miRNAs thereby enhancing tumor cell invasion It is noteworthy that there is a mutual feedback loop between some miRNAs and Wntcatenin signaling In colon cancer miR452 induces Wnt signaling by targeting GSK3 Since T cell factorlymphatic enhancement factor is an effective transcription factor in the miR452 promoter in turn miR452 transcription is promoted by Wnt signaling forming a positive feedback loop46 In addition a negative feedback loop was found between the miRNA and Wnt signaling pathway miR145 a tumor suppressor miRNA can interact with TCF4catenin complex recruit polycomb repressive complex PRC2 histone trimethylase and suppress Wnt signal transduction Recruitment of PRC2 however results in a high level of methylation at the miR145 promoter region which downregulates the expression of miR14547 It was further demonstrated that abnormally downregulated miR145 was inversely correlated with its inhibitory regulators TCF4 and SUZ12 Suppressor of Zeste Protein Homolog indicating that miR145 forms a double negative regulatory loop with the negative regulator of colorectal cancer It is therefore imperative to further analyze the network and regulation of miRNA and Wnt catenin signaling pathway which would provide insights into the development of miRNAbased drugsWnt Signaling in Cancer StemnessIt is hypothesized that malignant tumors grow in a layered manner and cancer stem cells CSCs having selfrenewal characteristics and multidirectional differentiation ability play important roles in the tumor development and progression CSCs continuously maintain the replacement of new CSC tumorigenic subpopulations and differentiate into nonCSC progenies with high proliferative ability48 It is assumed that cancer originates in part from CSCs de Sousa e Melo demonstrated that in colorectal cancer Lgr5GFP tumor cells had higher tumorigenicity than Lgr5GFPˆ’ tumor cells showing that Lgr5 cells had tumorinitiating ability49 In addition depletion of Lgr5 cells triggers differentiated cancer cells to continuously reverse to cells in a tumorpropagating state and replenish the Lgr5 CSC pool In another study a similar conclusion was provided Mature leukemia cells could reacquire clonogenic and leukemogenic properties through dedifferentiation It is worthy of note that CSC plasticity contributes to the interconversion of leukemia status through interfering with endogenous PU1 an ETS family pioneer transcription factor required for myelopoiesis50 However CSCs that transformed from differentiated cells require additional factors mutations inflammation or changes in the microenvironment for their stemness features51 For example after hepatotoxin induction hepatocytes produce tumor nodules and express progenitor cell markers52 Targeting CSCs and preventing the generation of CSCs from nonCSCs are thus a key for the development of cancer therapyThere is a wealth of evidence that CSCs are closely related to tumor invasion metastasis and drug resistance The differentiation gradient between CSCs and their non CSC progenies results in tumor heterogeneity which indicates that cells of different genotypes can exist in the same tumor including subgroups with transferability Owing to the unique nature of tumors CSCs have become the leading cause of drug resistance in cancer treatment Wnt submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alTable Effect of miRNAs on WntCatenin Signaling in CancermiRNAmiR100miR125bmiR181a5pmiR3773pmiR504miR1301miR31943pmiR186miR4543pmiR27amiR221222miR1945pmiR31miR5905pTargetsUp“““““““““TMEM170B“““EcadherinDownDKK1 ZNRF3ZNRF3 RNF43 DKK3 APC2catenin TCF4XIAP ZEB2FZD7BCL9BCL9catenin MCRS1RPRD1A AXIN2 DKK3 SFRP1cateninWIF1 SFRP2 DKK2 AXIN2SOX17Dkk1 AXIN1 GSK3Wnt1 catenin NcadherinEffectCell TypeActivationActivationInhibitionInhibitionInhibitionInhibitionInhibitionInhibitionActivationInhibitionActivationactivationActivationInhibitionColorectal cancerColorectal cancerColorectal cancerColorectal cancerHCCHCCHCCHCCBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerRef[][][][][][][][][][][][][][]Abbreviations XIAP Xlinked inhibitor of Apoptosis ZEB2 zinc finger Ebox binding homeobox BCL9 Bcell CLLlymphoma MCRS1 microspherule protein RPRD1A regulation of nuclear premRNA domaincontaining 1A SFRP1 secreted frizzledrelated protein TMEM170B transmembrane protein 170B WIF1 Wnt inhibitory factor Ecadherin epithelial cadherin Ncadherin neuralcadherincatenin signaling plays an essential role in the reprogramming and cancer cell stemness and is an indispensable cytokine network for promoting the progression of CSCs Reilein found that the cell types of CSCderivatives depend in part on the magnitude of spatially graded Wnt pathway activity53 The latest report54 proves the remarkable heterogeneity in Wnt activity in HCC cells among which WntactivityhighALDH1EpCAM triplepositive cells are the most stemlike and highly tumorigenic cells in all CSC populations This type of cells is called œsuperpotent CSCs spCSC The ASPMDishevelled signal axis is highly activated in spCSCs and is heterogeneous in HCC tissues Future studies would reveal the underlying mechanism of Wnt and stem cell heterogeneity in liver cancer and provide new insights into CSCbased tumor treatment strategyWnt signaling contributes to reprogramming and maintenance of the CSC status such as the epithelial“mesenchymal transition Chang found that in epithelial cells the SRYrelated highmobilitygroup box gene Sox15 tumor suppressor interacted with the cateninEcadherin complex and then interacted with the proximal promoter region of caspase3 CASP3 Caspase3 inactivates the Twist family bHLH transcription factor Twist which inhibits Wnt signaling for CSC phenotype acquisition In interstitial cells Twist1 forms a complex with cateninTCF4 and binds to the proximal promoter region of ATPbinding cassette G2 ABCG2 which induces the expression of stem cellrelated genes and promotes Wnt signaling to induce the CSC phenotype In the presence of Wnt signaling EMT regulates the CSC phenotype by inducing the conversion of two types of complexes55 In addition to activated Wnt signaling inactivated Wnt signaling can also promote malignant progression of CSCs especially tumor recurrence Autocrine inhibition of WNT signaling induces the resting state of CSCs and tumor dormancy After the primary tumor undergoes periodic proliferation and cancer immunoediting a small fraction of the progenies are transformed into latency competent cancer LCC cells and these cells exhibit Soxdependent stem phenotype LCC cells actively silence WNT signaling to enter into a quiescent state by expressing Sox2 target gene DKK1 The cancer cells then gain longterm viability through immune escape When the function of cancer immunoediting is somehow hampered LCC cells randomly enter cell cycle triggering the next metastatic exp

Thyroid_Cancer

thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples œedgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients™ prognosis was verified using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identification of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore it™s very important to find eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression profiles of specific cancers are easy to obtainwhich helps us better find more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to define and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenverified However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identification of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionprofiles of THCA patients were accessed from TCGA databaseand modules associated with THCA were identified by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression profiles of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentification and Confirmation ofTHCAAssociated GenesœedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionprofiles In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients™ prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset œglmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentification of THCAAssociatedModulesAs shown in A a total of DEGs were identifiedlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of ‰ ‰ log k and log Pk were calculated for linearmodels™ construction respectively is the squared value of thecoefficient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the first time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and significance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were verified to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identification of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology fitting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene significance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore — GHR —GPR125 — Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was verified Figure 2E From the heatmaps of the expression profiles of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3A“C THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3D“FFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patients™prognosis F Heatmap of the genes expression profilesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model A“C Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset D“F ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identificationof the important genes during tumorigenesis and the definitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq profiles andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been verifiedto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is firstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction It™sreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereverified as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further verified by ROC and KaplanMeier curvesAs the expression profiles of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and it™s necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the finalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci “ Carling T Udelsman R Thyroid cancer Annu Rev Med “ 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma “pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope “ Zhao QJ Zhang J Xu L Liu FF Identification of a fivelong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol “ 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentification ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival“ 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA ™ endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res “ Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit “ 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is specifically expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model “ 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identification of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics “ 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep“ 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117“ 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConflict 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 Zhao Zhang Shao and Fang 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'

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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

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

ORIGINAL Clinical Translational Immunology e1165 101002cti21165wwwwileyonlinelibrarycomjournalcti Elly Marcq1 Bianca von Scheidt2 Ashleigh S Davey2Novel combination immunotherapy for pancreatic cancerpotent antitumor effects with CD40 agonist andinterleukin15 treatmentJonas RM Van Audenaerde12Amanda J Oliver2 Jorrit De Waele1 Delphine Quatannens1 Jinthe Van Loenhout1Patrick Pauwels13 Geert Roeyen4 Filip Lardon1 Clare Y Slaney25Michael H Kershaw25  Phillip K Darcy25  Evelien LJM Smits17 1Center for Oncological Research CORE Integrated Personalized Precision Oncology Network IPPON University of AntwerpWilrijk Belgium2Cancer Immunotherapy and Immune Innovation Laboratory Peter MacCallum Cancer Centre Melbourne VIC Australia3Department of Pathology Antwerp University Hospital Edegem Belgium4Department of Hepatobiliary Endocrine and Transplantation Surgery Antwerp University Hospital Edegem Belgium5Sir Peter MacCallum Department of Oncology The University of Melbourne Parkville VIC Australia6Department of Oncology and Multidisciplinary Oncological Centre Antwerp Antwerp University Hospital Edegem Belgium7Center for Cell Therapy and Regenerative Medicine Antwerp University Hospital Edegem Belgium Marc Peeters16CorrespondenceELJM Smits Center for Oncological ResearchCORE Integrated Personalized PrecisionOncology Network IPPON University ofAntwerp CDE T431 Universiteitsplein Wilrijk BelgiumEmail eveliensmitsuantwerpenbe Contributed equally as senior authorsReceived May Revised July Accepted July 101002cti21165Clinical Translational Immunology e1165AbstractObjectives With the poorest 5yearsurvival of all cancersimproving treatment for pancreatic cancer is one of the biggestchallenges in cancer research We sought to explore the potentialof combining both priming and activation of the immune systemTo achieve this we combined a CD40 agonist with interleukin15and tested its potential in pancreatic cancer Methods Responseto this combination regimen was assessed in pancreatic ductaladenocarcinoma mouse models and a thorough analysis of thetumor microenvironment was performed Results We demonstratedprofound reduction in tumor growth and increased survival ofmice with the majority of mice being cured when both agentswere combined including an unprecedented 8fold dose reductionof CD40 agonist without losing any efficacy RNAseq analysisshowed involvement of natural killer NK cell and Tcellmediatedantitumor responses and the importance of antigenpresentingcell pathways This combination resulted in enhanced ltrationof tumors by both T cells and NK cells as well as a striking increaseT cells over Tregs We also observed ain the ratio of CD8significant increase in numbers of dendritic cells DCs in tumorDCs with crossdraining lymph nodes particularly CD103T cells andpresentation potential A criticalinvolvement of NK cells in the antitumor effect was highlightedImportantly strong immune memory was established with anT cells only when both interleukin15 andincrease in memory CD8the CD40 agonist were combined Conclusion These novelpreclinical data support initiation of a firstinhuman clinical trialrole for CD8ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alwith this combination immunotherapy strategy in pancreaticcancerKeywords CD40 agonist combination immunotherapy interleukin natural killer cells pancreatic cancer T cellsINTRODUCTIONPancreatic ductal adenocarcinoma PDAC is thethird most lethal cancer worldwide with a 5yearsurvival of barely It is even projected tobecome the second leading cause of cancerrelated death by To date it remains one ofthe most aggressive and challenging malignanciesbecause of a complex tumor microenvironmentincluding a strong desmoplastic reaction4 lowimmunogenicity56 and a molecular signature infavor of the tumor driven by loss of multipletumor suppressor genes7 Despite all efforts madein the past almost no improvement in survival hasbeen achieved rendering PDAC a disease whichrepresents the very definition of an urgent unmetneed for novel therapeutic approaches to finallyimprove the outcome of patientsAbout of patients are not eligible for curativesurgical resection because of locally advanced ormetastatic disease at the time of diagnosis Hencepatients are treated with either FOLFIRINOX emcitabinenabpaclitaxel depending on theirphysical fitness Howeverthese treatments areassociated with majortoxicity issues and havelimited impact89 New promising approaches thatare successful in other cancer types such as antiPD1and antiCTLA4 have shown little improvementover treatment with gemcitabine1011 This highlightsthe need for other novel compounds to enter thebattle arena of PDACIn this context the use of CD40 agonists inPDAC has been explored during the past decadeboth as a single modality1213 and in combinationwith current firstline treatments1415 or othercompounds such as checkpoint inhibitors PD1and CTLA416 Antitumor responses have beenreported in both mice and humans and there is ageneral consensustherapyprovides the necessary immunepriming signals toconverttumormicroenvironmenthot‚ammatory microenvironment17 In addition allstudies demonstrated that combination therapyinvolving CD40 agonists provided more potentresults in terms of tumor growth suppression andthat CD40 agonistimmunogenicdesirablecoldthetoaextended survival1516 These data support furtherinvestigation of combination approaches withother promising candidates to unlock the fullpotentialSinceinterleukin IL15 is an essential cytokine foractivation and maintenance of immune effectorcells there is a strong rationale for combiningimmunepriming agents with this moleculetherapyagonistCD40ofbothTcellproliferationto malignantin maintainingWe have previously shown in vitro that IL15stimulated natural killer NK cells can kill bothPDAC tumor cells and stromal pancreatic stellatecells which are responsible for the poor responseto treatment18 IL15 is a versatile cytokine whichstimulatesandgeneration of cytotoxic T lymphocytes as well asactivation and expansion of natural killer NKcells Furthermore it has the capability to induceTcell memory cells thereby playing a crucialCD8immuneroleresponsespossibletheseprevention offeatures render IL15 a highly attractive cancerimmunotherapeutic as confirmed by its high rankin the NCI™s top immunotherapeutic drugs withthe greatest potential for broad usage in cancertherapy22 MoreoverIL15 needs to be transpresented by the IL15Rα on dendritic cells DCsto its target to be effective2023 Since it has beendemonstrated that CD40 agonists also increase theexpression of IL15Rα on DCs we hypothesisedthatinenhanced immune activation and increased antitumor effects24combining both agents mightlonglastingandrelapse19“ AlltumorresultcellsIn this we show for the first time in micewith pancreatic tumors that when CD40 agonistantibody and IL15 are combined they exhibitsynergistic effectsin terms of enhanced antitumor efficacy resulting in profound increases inlongterm survival with complete cure in themajority of cases Moreover an unprecedentedstriking dose reduction of CD40 agonist waspossible by the addition of IL15 The antitumoreffect was found to be mediated predominantlysupported byby CD8dendritic cells DCincreased amounts of CD103ThewithT cells and NK cellscrosspresentingcapacityunique Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancertumors by both celltypes wasltration ofcommensurate with a reduction in the amount ofregulatory T cells These noveltranslationalpreclinical data provide a solid rationale toinitiate a clinicalinvestigating this novelimmunotherapy combination strategy for patientswith one oftumorsnowadaysthe hardestto treattrialRESULTSCombined IL15 and CD40 agonist therapyresults in increased antitumor efficacyin vivosignificantreduction ofWe sought to investigate whether the combinedtreatment of IL15 and a CD40 agonist antibodymay lead to augmented antitumor responses inPDAC To investigate this C57BL6J mice bearingeither Panc02 or KPC tumors were treated with IL andor a CD40 agonist antibody deliveredintraperitoneally when tumors reached a size of“ mm2 Figure 1a We demonstrated that thecombination of these agents resulted in decreasedtumor volumes and increased survival of mice inboth the Panc02 and KPC tumor models In thefirsttumor model Panc02 we observed asignificant decrease in tumor volume when micewere treated with either single agent Howevertreatment with the combination regimen resultedin a furthertumorvolume and a significant increase in survival with out of mice being completely tumor freeFigure 1b d and f and Supplementary figure 1AC E and G In the second tumor model KPC weobserved very similar results with reduced tumorvolumes and increased survival of mice with thecombination being significantly betterthanuntreated control or either singleagent therapyIn this experiment seven out of mice werecompletely tumorfree following combinationtreatment Figure 1c e and g and Supplementaryfigure 1B D F and H Remarkably while weobserved similar responses between the Panc02and KPC tumor modelsthe dose of CD40agonist used in the Panc02 model five doses of µg was eight times lower than in the KPCfirst dose µg with four consecutivemodeldoses of µg In summary we show here thatthe combination ofIL15 and CD40 agonisttherapy has profound antitumor activities andcombining both agentsto significantsynergistic effectsleadsDistinct gene expression profiles pointtowards combined immune priming andimmune activation following therapyoftheclusteringTo unravel the effects caused by the single agentsRNA sequencing wasand their combination performed on the more resistant KPC tumorsharvested on day in the treatment scheme Firstprincipal component analysis PCA demonstrateda clear distinction between the isotype IL15 andcombination group while the CD40 agonisttreatment revealed a bigger overlap with theisotype control Figure 2a To investigate in moredetail the effect of therapy on immunerelatedgenes we performed a PCA using a verifiedNanostring immunerelated gene panel whichdemonstrateddifferenttreatment groups Figure 2b The top up anddownregulated immunerelated genesshowedthat genes involved in antigen presentation Thelper immune type responses and NK cellcytotoxicity were modulated Figure 2cToconfirm this gene set enrichment analysis GSEAwas performed showing that gene sets involved inNK cellmediated cytotoxicity Figure 2d and eand Supplementary figure 2A the IL122 pathwayFigure 2f and g and Supplementary figure 2Band the CD8 TCR downstream pathway Figure 2hand i and Supplementary figure 2C were clearlyamong the top upregulated pathwaysin thegroups when IL15 was administered while theCD40promotedantigen processing and presentation pathwaysFigure 2j and k and Supplementary figure 2DImportantlythese features of both IL15 andCD40 agonisttherapy were retained in thecombination grouptreatmentstronglyagonistT cells are responsible for antitumorCD8efficacy following therapyTo gain more insightinto the immune cellsresponsible for the observed antitumor responseswe depleted several immune cell populations intumorbearing mice in both tumor models usingspecific depletion antibodies We monitored theeffect the depletions had on both tumor growthkinetics and survival of mice Figure 3a“d andSupplementary figure Upon depletion of CD4T cells we observed in both PDAC models nosignificant difference in survival of mice betweenthe depleted and nondepleted groups indicatingT cells do not play a significant role inthat CD4ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Combining IL15 with a CD40 agonist results in decreased tumor volume and increased survival C57BL6j mice were injected witheither × Panc02 or KPC cells subcutaneously When tumors reached a size of “ mm2 mice were randomised and treated withisotype control IL15 CD40 agonist or IL15 CD40 a Treatment scheme showing timing of dosing is indicated for IL15 µg with blackarrows and for CD40 agonist or the corresponding isotype with red arrows five doses of µg for Panc02 or first dose µg andconsecutive four doses µg for KPC b c Tumor growth kinetics are depicted [n or mice per group representative data of Panc02or KPC independent experiments] Oneway ANOVA with Bonferroni post hoc d e Survival of Panc02 n and KPC n micetreated as indicated Pooled data of Panc02 or KPC independent experiments Survival was determined by tumor size reaching mm2Logrank test f g Waterfall plots showing the change in tumor area relative to baseline after days Panc02 n or days KPCn Pooled data of Panc02 or KPC independent experiments All data represent mean 06 SEM P ˂ P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerFigure RNA profile of KPC tumors KPC tumors were harvested on day of the treatment schedule for subsequent RNA isolation andsequencing a PCA plot showing separation of samples based on all genes b PCA plot showing separation of samples based on immunerelated genes Nanostring mouse immunerelated genes c Volcano plot showing significantly differentially expressed immunerelated genesbetween isotype control group and the combination group d“k GSEA plots for differentially expressed genes are showing enrichment for genesinvolved in a n tumorsgroupª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Immune cell depletion C57BL6j mice bearing Panc02 or KPC tumors were treated with isotype control or the IL15 CD40 agonistcombination regimen alone or with depleting antibodies against CD4 CD8 asialoGM1 NK cell depletion a b Tumor growth kinetics ofPanc02 or KPC tumors either nontreated isotype treated with the combination regimen only no depletion or combination and depletionantibodies Oneway ANOVA with Bonferroni post hoc Data points represent mean 06 SEM n “ micegroup representative data of twoindependent experiments c d Waterfall plots showing the change in tumor area relative to baseline after days e“k Survival of Panc02or KPCbearing mice either nontreated isotype treated with the combination regiment no depletion or the combination and depletionantibodies against CD4 e f CD8 g h asialoGM1 i j or CD8 asialoGM1 k l Data pooled from two independent experiments withn or Panc02 or n “ KPC LogRank test ns P ‰¥ P P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic canceroftheeffectantitumorthe antitumor response elicited by the IL15 andCD40 agonist combination treatment Figure 3eT and NK cells wereand f However when CD8depletedthecombination treatment was significantly reducedin both tumor models Figure 3k and l WhenT cells alone were depleted there was aCD8significant increase in tumor growth and reducedsurvival of mice Figure 3g and h Howeverdepletion of NK cells only led to a mixed responseIn the Panc02 tumor model we clearly observed asignificant decrease in survival of mice followingdepletion of NK cells although interestingly therewas no effect on tumor growth In the KPC tumormodelthere was a trend towards decreasedsurvival of mice following NK cell depletionalthough this was notsignificantP In conclusion theseT cells wereexperiments demonstrated that CD8the predominant effector cell type responsible forthe observed antitumor effects with a clearcontribution of NK cells in the Panc02 modelFigure 3i and jstatisticallyCombination therapy increases intratumoral ltration of immune cellsintheKPCutilisedto monitorTo further explore the anticancer effects of thecombination regimen multicolour flow cytometrytumorltratingwaslymphocytestumor modelSupplementary figure We observed strikingdifferences between the different treatment armsthat were tested Figure 4a The first observationwas that the antitumor effect was not due to ahigher total number of ltrating lymphocytesbut rather caused by significant differences inwhich immune cellsubsets were present Thefrequency of ltrating NK and NKT cells wassignificantly higher in the combination therapygroup compared to the isotype and CD40 agonisttreatment groupsIL15 alone demonstrated anincreased frequency of both cell types althoughthis was not statistically significant compared tocontroltreated miceIn terms of neutrophilnumbers although they were abundantly presentwethetreatment groups For T cells we observed anincreased number offollowingcombination therapy compared to the othertreatment groups This increased number of T cellsT cellscould be attributed to predominantly CD8which werethecombination therapytreated mice compared to alltotal T cellssignificantlydifferenceobservedbetweenhighernoincellsseveralon NK and NKTtreatment arms while there was nootherT cellsdifference in numbers of ltrating CD4Interestingly we also observed a significantdecrease in numbers of regulatory T cells Tregspresent in the tumor when CD40 agonist was partof the treatment We also measured expression ofCD69 on immune cells as an indicator ofactivation We observed clear upregulation of thismarkerfollowingTcombination therapy however not on the CD8cells The results were confirmed by geneexpression analysis ofrelevant genesFigure 4b Here FoxP3 transcription was indeeddownregulated when Tregs were less abundantand the combination regimen demonstratedthe strongest upregulation of NKT cellrelatedgenes such as granzyme A and B The analysisrevealed a strong immune activation signature asIFNγ and CD69 were upregulatedIL12Puttingthecombination treatment of CD40 agonist andIL15 had a more profound antitumor effect asit caused a significant increase in the amountof antitumor immune cells NK NK T and CD8T cells compared to control orsingleagenttreatmentcommensurate with adecrease in immunosuppressive Tregs resulting inan enhanced CD8Treg ratio within the tumorsobservationsthat wastogetherIL18theseCombination therapy results in increasedamounts of CD103crosspresenting DCsDendritic cells are known to play critical roles inantigen processing and presentation and are keyplayers in the activation of both NK and T cellsWe further explored their presence in the KPCtumor model Here we observed a significantincrease in number of DCs in the tumor only inthecombination therapy group Figure 5aDCs theFurthermore the amount of CD103subtype responsible for crosspresentation wasIL15 caused a significantdetermined HereDCs in theincrease in the number of CD103tumor while this was significantly lower in thegroups following treatment with CD40 agonistFigure 5b To further investigate how the DCsbehaved we analysed the presence of these cellsin the tumordraining lymph nodes TDLN andobserved a 3fold increase in number of DCs whenCD40 agonist was administered Figure 5c Thecrosspresenting DCsfrequencyincreasedconditionsFigure 5d suggesting that these DCs capturedoftwofoldCD103undertheseª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alFigure Characterisation of tumorltrating lymphocytes C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter a Tumors were harvested at day posttreatment initiation Singlecell suspensions were acquired afterenzymatic digestion for flow cytometry analysis Immune cell populations indicated as fold change of absolute number of cells and CD69T cells Data pooled from three independent experiments n “16group Oneway ANOVA withexpression MFI on NK NKT and CD8Bonferroni P P ‰¤ P ‰¤ P ‰¤ b Heatmap of gene expression of relevant genes for the quantified immunesubsets n tumorsgroup Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerantigens at the tumor site and migrated to theTDLN to activate NK and T cells Furthermoregene signatures showed a higher expression ofCD80 CD83 and CD86 when the combinationtherapy was administeredindicating that likelyantigenpresenting cells like DCs are activated andmaturein mRNA encodingexpression of CXCR3 CXCL9 and CXCL10 notCXCL11 as chemotactic chemokines suggests theirinvolvementin the trafficking of antitumorimmune cells to the tumor site Figure 5eincreaseTheInduction of immune memorythe goals ofimmunotherapy isOne oftheinduction of strong immunological memory toprevent future relapse We observed in our studyan increased number of effector and centralT cells in KPC tumors when treatedmemory CD8with the combination regimencompared toisotypearm treatmentssingleFigure 6aTo investigate whetherfunctional immune memory was induced tumororand bcontrolFigure Characterisation of DCs in tumor and TDLN C57BL6j mice bearing KPC tumors were treated with isotype control IL15 CD40agonist or the combination of the latter Tumors or TDLN were harvested at day posttreatment initiation Singlecell suspensions were acquiredafter enzymatic digestion for flow cytometry analysis a b DCs or CD103DCs in TDLN Data pooledfrom three independent experiments n “16group Oneway ANOVA with Bonferroni P P ‰¤ P ‰¤ e Heatmapof gene expression of relevant genes for the quantified immune subsets n tumorsgroupDCs in tumors c d DCs or CD103ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et alfree mice from the combination treatment groupwere rechallenged with the same tumor cell typeas they were originally inoculated with Here weobserved clear induction of immune memory inboth PDAC models with out of micebecoming tumor free for the Panc02 tumor modeland all mice becoming tumor free for the KPCtumor model compared to naı¨ve control miceFigure 6c“fDISCUSSIONWith high resistance to current firstline treatmentand failure of numerous clinicaltrials PDACpatients are in desperate need for new treatmentoptions25 There is a general consensus that CD40agonist therapy triggers the necessary immunepriming signalling to convert immunogenic coldcancerenhancementto itstargetthe effects are ofofto hot However when applied intumorsshortpancreaticduration warrantingthistherapy1217 IL15 is a powerful stimulator of NKT cells and induces CD44hi memorycells and CD8T cells This cytokine needs to be transpresentedby the IL15Rα on DCsto beeffective2023 Since it had been demonstrated thatthe CD40 agonist also increases the expression ofIL15Rα on DCs we hypothesised that combiningboth agents may resultin enhanced immuneactivation and increased antitumor effects In thisstudy we demonstrated thatIL15 and CD40agonist administered alone can elicit a powerfulimmune response However when combinedenhanced andthesestronglyimportantly exceeded survivalrates of CD40agonist with gemcitabine and nabpaclitaxel oreffects wereFigure Rechallenge experiments C57BL6j mice cured from Panc02 or KPC tumors after treatment with IL15 CD40 agonist were reinjected with the same tumor type at the contralateral side of the abdomen a b Tumor kinetics and survivallogrank test of micerechallenged with Panc02 tumor cells n c d Tumor kinetics and survival of mice rechallenged with KPC tumor cells n e f FlowEffector or Memory T cells of KPC tumorbearing mice after days following treatment n cytometry quantification of intratumoral CD8Oneway ANOVA with Bonferroni P ‰¤ P ‰¤ P ‰¤ Vol e1165Page ª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc 0cJRM Van Audenaerde et alNovel immunotherapy for pancreatic cancerstudy26 A limitation ofCD40 agonist and gemcitabine and nabpaclitaxeltogether with PD1 and CTLA4 blockade in similarpancreatic mouse models1516 In addition whenCD40 agonist was combined with antiangiogenicdrugs blocking VEGFA and angiopoietin survival rates did not equal the ones observed inthese studiesthisincluding ours may be that the mouse models donot completely mimic the human situation sincetumorsubcutaneouslyNevertheless CD40 agonists given as a singlemodality have shown beneficial effect also inhuman clinical trials with PDAC patients131427 andour previous studies have demonstrated that uponIL15 stimulation human NK cells can kill bothpancreatic cancer and stromal stellate cells in anautologous human ex vivo setting18cells wereinjectedThe potential of this combination regimen isnot just limited to PDAC since IL15 and CD40agonist therapy has been tested by others in micebearing established CT26 and MC38 colorectaltumors The authors showed promising resultsalbeit with less surviving mice compared to ourstudy28 This might be due to the fact that wegave in total five doses of CD40 agonist instead offour as in the other studies Furthermore resultsof otherinvestigators using this combinationtherapy in a prostate cancer model TRAMPC2demonstrated similar numbers of surviving mice aswe found underscoring the enormous potentialof the combination approach24 Of note bothcolorectal cancer and prostate cancer have asignificant better 5year overall survival of andthesignificance of our findings in pancreatic cancerwith a 5year survival of barely Strikinglyin this study we also demonstrated that IL15potentiates CD40 agonist treatment causing an fold dose reduction in one of the PDAC mousemodels which has not been reported so far Thisimportant dose reduction could be of greattranslationalimportance as lower doses mightsignificantly decrease adverse events in patientsunderscoringrespectivelyWe observed thatthe combination therapy‚uenced several immune cell types in favor ofincreased antitumor efficacy As also observed byothers in prostate cancer there was an increase innumber of intratumoral effector immune cellsT cells that boththat included NK cells and CD8contributed to enhancing tumor control but thesestudies did not look beyond these immune cells24In our more extensive analysis we also observed areduction in number of Tregs known for theirCD8anTcellsfavorableestablishesreceptor activationimmunosuppressive potential and an increasedfrequency of DCsfor priming T cells andactivating NK cells Especially our finding ofDCs withstrongly increased amounts of CD103crosspresenting potentialin the tumordraininglymph nodes elucidates on the mechanism behindour observed antitumor effectscompriseimportantcompartment ofthe adaptive immune systemwith wellestablished antitumor effects Upon Tcellthey produce effectorcytokines like IFNγ and kill cancer cells in anantigenspecific manner via the granzymeperforinsystem3132 Increased numbers of CD8T cell incancer are therefore linked to better outcome andprognosis With our combination strategy weT cellsobserved an increased number of CD8whichtumormicroenvironment for an anticancer response Wefound these cells were the most important playerin our therapy models since depletion of thesecells virtually abrogated the antitumor responseBoth CD40 and IL15 already as single modalitiesTcell numbers butinduced an increase in CD8the combination therapy induced significantlyhigher intratumoral ltration of these effectorcells This might be due to certain chemokinessince our unique gene expression analysis showeda higher expression of CXCR3 and its ligandsCXCL9combinationtreatment These chemokines are known to beresponsible for chemoattraction of both T cellsand NK cells33 Moreover these two chemokinesareandchemotherapeutic efficacy in PDAC in patients34Interestinglyweredownregulated following combination therapyHowever the CCL5CCR5 axis in PDAC has beenshown to correlate with promotion of migrationand invasiveness of the pancreatic cancer cellsand thus downregulation could be actuallythebeneficialCX3CR1 axis is associated with early recurrenceafter surgery with poor patient prognosis36 Theactivation marker CD69 was not shown to beT cells This may be dueupregulated on the CD8to the fact that tumors were harvested at day posttreatment and CD69 is considered to be anearly activation marker which disappears after days37 Among the CD8subsets wememory T cellsfound higher presence of CD8which is linked to immune memoryIL15 hasbeen described to be important for the inductionin our models35 Furthermorecorrelated with increased survivalfollowingandCXCL10TcellCCL5andCX3R1aª The Authors Clinical Translational Immunology published by John Wiley Sons Australia Ltd on behalf ofAustralian and New Zealand Society for Immunology Inc Vol e1165Page 0cNovel immunotherapy for pancreatic cancerJRM Van Audenaerde et aland maintenance of these cells and combiningthis modality with for instance PDL1 blockademightincrease the number of CD8memory T cells38furtherNatural killer cells have become an increasinglyimportant target for cancer immunotherapy sincethey have demonstrated to mediate successful andefficient antitumor responses3940 They play animportant role in pancreatic cancer as we haveshown here and although depletion of NK cellsdid not have as drastic an effect on response toT cells it isCD40 and IL15 combination as CD8likely that they still play an important role in thistherapy given the increase in both their activationand numbers in the tumor This is an importantobservation since a higher frequency of NK cells isclearly linked to better survival41 IL15 is a strongstimulator of NK cells23 and its effect is stronglyincreased by the addition of CD40 agonists thatupregulate IL15Rα on DCs28 which is necessaryT cells andfor transpresentation of IL15 to CD8NK cells20 Moreover since expression of IL12 IL and IL18 is upregulated this may lead toin the tumorformation of NK memory cellsmicroenvironment which needsto be furtherexplored42ahighhaveTregsimmunesuppressivepotential43 When CD40 agonist was administeredwe observed a significantreduction in thenumber of Tregs within the tumor as confirmedby others in other solid tumor models2644 Themechanism by which CD40 agonists cause Tregreduction still needs to be elucidated althoughone study indirectly points towards the blockageofmyeloidderivedsuppressor cells and the Tregs45 In addition thehighlyfollowingcombination therapy was very encouraging sincemetaanalysis showed that this is associated withimproved overall survival in cancer patients46 andresponse to therapy4748CD8TreginteractionincreasedbetweenratioFinally reduction of Tregs in PDAC allows DCsimmuneto induce a more potent antitumorT cells49 Ourresponse largely mediated by CD8data demonstrated that DCs in general increasedby 3fold in the tumordraining lymph nodes as aofresultTheirtheimportancein cancer has been extensivelydemonstrated as they function as the generals ofour immune system by capturing tumor antigensand presenting them to T cells thereby elicitingspecific immune responses5051 A very importantDCs which aresubset of these DCs are the CD103combinationtherapyTbycellstheyactivateconsidered to reside in peripheral tissues Uponthey migrate to the lymph nodesactivationwhereantigenpresentation52 Moreover they have found to bethe only APCs to transport intact antigens totumordraining lymph nodes and prime tumorT cells53 Hence our novel dataspecific CD8DC in the tumorshowing a doubling in CD103draining lymph nodes support the increased antitumor responses we observ

Thyroid_Cancer

"Many cancerassociated single nucleotide polymorphisms SNPs are located in the genomic regionsof long noncoding RNAs lncRNAs Mechanisms of these SNPs in connection to cancer risk are not fullyunderstoodMethods Association of SNP rs140618127 in lncRNA LOC146880 with nonsmall cell lung cancer NSCLC wasevaluated in a casecontrol study of individuals The mechanism of the SNP™s biologic influence was exploredwith in vitro and in vivo experiments including plasmid transfection siRNA knockdown flow cytometry assessmentand assays of cell proliferation migration invasion and colony formationResults Association analysis showed that A allele of SNP rs140618127 was associated with low risk of NSCLC in theChinese population Lab experiments indicated that SNP rs140618127 contained a binding site for miR5395p andthe binding between miR5395p and LOC146880 resulted in declined phosphorylation of an oncogene ENO1 Thereduced phosphorylation of ENO1 led to decreased phosphorylation of PI3K and Akt which is further linked to thedecline in cell proliferation and tumor progressionConclusion The study demonstrates that SNP rs140618127 in lncRNA loc146880 provides an alternate binding sitefor microRNA miR5395p which affects the phosphorylation of ENO1 and activation of the PI3K and Akt pathwayKeywords NSCLC lncRNA SNP miRNA ENO1BackgroundLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in the world []The majority oflungcancer NSCLC which accounts for around of alllung cancer is nonsmall cell Correspondence qianbiyunsjtueducn Tienan Feng and Nannan Feng contributed equally to this work1Hongqiao International Institute of Medicine Shanghai Tongren HospitalClinical Research Institute Shanghai Jiao Tong University School of MedicineShanghai China7Second Affiliated hospital of Chengdu Medical College China NationalNuclear Corporation Hospital Chengdu Sichuan ChinaFull list of author information is available at the end of the lung cancer cases Genetic factors may play an importantrole in an individual™s susceptibility to NSCLC Longnoncoding RNAs lncRNAs are a class of noncodingtranscripts with nucleotides or more Increasingevidence suggests thatlncRNAs are involved in theoccurrence oflung cancer due to their functions asoncogenes or tumor suppressors [] Our previous studies indicated that a lncRNA on chromosome 17q243named LOC146880 was expressed higher in tumortissues than in adjacent normal tissues and high expression was associated with poor prognosis of NSCLC []Singlenucleotide polymorphisms SNPs in the noncoding regions of the genome have been shown to affect 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 0cFeng Journal of Experimental Clinical Cancer Research Page of rs140618127 the ˜A™ allelecancer risk via regulating the transcription andor changing the structure of lncRNA [“] A previous studyidentified SNPs in more than humanlncRNAs and a large number of SNPs were predicted tohave a potentialimpact on the microRNA miRNAlncRNA interaction [] Here we report the identificationof SNP rs140618127 in LOC146880 as a new susceptiblelocus to NSCLC Bioinformatics analysis predicts thatin LOC146880variantprovides an altered secondary structure which maycreate a binding site for microRNA miR5395p []sequestering its action on other molecules Shiraishi conducted a GWAS on lung adenocarcinoma andidentified SNP rs7216064 in BPTF 17q243 in association with the cancer risk OR p 7e11 []Seow confirmed that SNP rs7216064 was associated with the risk of lung cancer based on a GWASstudy of Asian female nonsmokers [] We found thatSNP rs140618127 was in strong linkage disequilibriumwith SNP rs7216064 LD r2 and this lncRNASNP was associated with lung cancer risk OR p in our casecontrol study of individualsToSNPrs140618127 in NSCLC development and progressionweconsequence ofLOC146880 and miR5395p interaction and found thatthe microRNA behaved like a tumor suppressor []which prevented LOC146880 from interacting withprotein ENO1 an oncogene product [] reducing itsphosphorylation As a resultthe phosphorylation ofPI3KAKT was also reduced after the suppression ofENO1 phosphorylation [] which further inhibitedtumor growth and metastasisleading to a betterprognosis of NSCLCthe molecular mechanism ofexploreevaluated thatthe biologicalMaterials and methodsStudy populationsSuspected NSCLC individuals had histopathological orcytologically confirmed diagnosis according to theWorld Health anization classification These studysubjects including suspected individuals diagnosed withlung cancer or normal were recruited from the ChinaMedical University CMU Distributions of the basiccharacteristics ofthe study subjects are provided inTable At recruitment an informed consent wasIlluminated Only ifthe subject agreed heshe wasincluded This study was approved by the InstitutionalReview Board at CMUSNP selection and genotypingSNPs with r2 were considered to be in the same LDblock With this criterion one SNP was selected in eachLD block and genotyped using the TaqMan genotypingmethod in the ABI RealTime PCR systemApplied Biosystems For quality control we implemented several measures in our genotyping assays including each plate contained both case and controlsamples technicians were blinded to the casecontrolstatus of the samples both positive and negativecontrol no DNA template samples were included ineach 384well plate and nearly of the sampleswere assayed in duplicate and the concordances werebetween and Cell linesHuman NSCLC cell lines A549 and PC9 and humanlung epithelial BEAS2B cells were purchased from theCell Bank of Type Culture Collection at the ChineseAcademy of Sciences Shanghai Institute of Biochemistryand Cell Biology These celllines were passaged forfewer than months All the cells were tested for mycoplasma and were found to be free from infection Thecells were maintained in DMEM supplemented with FBS and grown without antibiotics in an atmosphere of CO2 and relative humidity at °C² and ² RACE and coding prediction of LOC146880We used ² and ² RACE to determine the transcripinitiation and termination sites of LOC146880tionalwith a SMARTe RACE cDNA Amplification kit Clontech The Alignment File of a fulllength sequence ofLOC146880 obtained from ² and ² RACE is availableupon requestConstruction of reporter plasmids transient transfectionsand luciferase assaysA reporter plasmid in the psiCHECK2 vector Promegawas created which contains a 1000bp LOC146880 exonregion flanking rs140618127 [G] or rs140618127 [A]with the restriction enzymes XhoI and NotI FermentasA549 and PC9 cells were seeded at — cells per wellin 24well plates and ng of the reporter plasmid and pmol of miR5395p mimic Ambion were cotransfected into the cells h later using Lipofectamine Invitrogen These cells were collected h aftertransfection Renilla luciferase activity was measured andused to normalize the efficiency of transfectionRNA extraction and qRTPCR analysisTotal RNA from the NSCLC tissue specimens and celllines used in this study was extracted using the TRIzolreagent Firststrand cDNA was synthesized using theSuperScriptInvitrogenRelative RNA levels determined by qPCR were measuredon an ABI sequence detection system AppliedBiosystems using the SYBR Green method Βetaactinwas employed as an internal control for the quantification of LOC146880 and the mRNA levels of other genesreverse transcriptase kitII 0cFeng Journal of Experimental Clinical Cancer Research Page of For miRNA quantification small nuclear RNA U6 wasused as an endogenous control The relative expressionof RNA was calculated using the comparative CtmethodSubcellular fractionationCytosolic and nuclear fractions of A549 and BEAS2Bcells were prepared and collected according to theinstructions ofthe NuclearCytoplasmic Isolation kitBiovision LOC146880 was mainly detected in thenuclear fraction although it was also present in the cytoplasm Fig S1belowBrieflydescribedRNA pulldown and mass spectrometry analysisRNA pulldown assays were performed following theprotocolbiotinylatedLOC146880 or antisense LOC146880 was incubated withcellular protein extracts from A549 cells and streptavidin beads were then added Recovered proteins associated with LOC146880 or antisense LOC146880 wereexcised and proteomics screening was accomplished bymass spectrometry analysis on a MALDITOF instrumentIn vitro transcription ofLOC146880 and its deletion fragments were analyzedwith primers containing the T7 promoter sequenceBruker DaltonicsPlasmid construction and transfectionTo construct a lentiviral vector expressing humanLOC146880 NR_026899 a fulllength of LOC146880cDNA containing rs140618127 [G] or rs140618127 [A]was commercially synthesized GeneChem and subcloned into the AgeI and NheI sites of the GV367IRESPuromycin lentiviral expression vector GeneChem Toproduce lentivirus containing LOC146880 T cellswere cotransfected with the vector described above andlentiviral vector packaging system GeneChem using Lipofectamine Infectious lentiviruses were collectedat h after transfection and filtered through 045μmPVDF filters for analysis of genotype After conformation these lentiviruses were designated to LOC146880[G] or LOC146880 [A] We used the GV367IRESPuromycin empty vector as a negative control Theviruscontaining pellet was dissolved in DMEM and aliquots of the solution were stored at ˆ’ °C A549 andPC9 cells were infected with concentrated virus in thepresence of polybrene SigmaAldrich The supernatantwas replaced with complete culture medium after hfollowed by selection with puromycin and the expression of LOC146880 in infected cells was verified byqPCRRNA immunoprecipitation assaysRIP experiments were performed using the Magna RIPProteinRNABindingkitMillipore Antibodiesagainst ENO1 Abcam orcontrol proteins were diluted at Total RNA inputcontrol and precipitation with the isotype control IgGfor each antibody were assayed simultaneously The coprecipitated RNAs were detected by RTqPCRImmunoprecipitationCell lysis and immunoprecipitationCells were homogenized in — RIPA buffer supplemented with ProteasePhosphatase Inhibitor CocktailPierce Cell lysates were centrifuged and the supernatants were prepared for immunoblotting or immunoprecipitation withbelowImmunoblot signal was detected using Clarity WesternECL Substrate Thermo FisherantibodiesdescribedtheTable Characteristics of lung cancer patients and healthy controlsVariablesP valueControlN NSCLCN Age at dx year ‰¥ GendermalefemaleSmoking statusnoyesSNP rs140618127G alleleA allele adjusted by smokinggenderage OR valueP valueOR value “ “ “ “ “ “ “ “ 0cFeng Journal of Experimental Clinical Cancer Research Page of from cells orImmunoblot assaysProtein extractsimmunoprecipitationsamples were prepared using detergentcontaining lysisbuffer Total protein μg was subjected to SDSPAGE and transferred to PVDF membrane MilliporeAntibodies against ENO1 Abcam ab155102 ENO1phosphorylated at Cterminalinhibitory site Tyr44StressMarq Bioscience spc965D PI3K CST 13666SPI3K phosphorylated at Tyr458 CST 4228S AKTCST 2938S AKT phosphorylated at Ser473 CST9018S PCNA CST 2586S NFkB CST 8242SVimentin Abcam ab92547 βCatenin CST 8480S Ecadherin CST 14472S NCadherin Abcam ab18203and βActin SigmaAldrich A1978200UL were usedMembranes were incubated overnight at °C withprimary antibody diluted and proteins were detected with the Odyssey near infrared dualcolor laserimaging system LICORAnalysis of cell proliferation migration invasion cellcycle and colony formationCells were seeded in 96well flatbottom plates with cells in μl cell suspension in each well Afterculture cell viability was measured with the CCK8assay Each experiment with six replicates was repeatedthree times For cell cycle analysis cells were collectedand fixed in ethanol overnight at °C Singlecellsuspensions were labeled with μgml Propidium Iodide Sigma and analyzed by flow cytometry BeckmanCoulter For colony formation cells were seededin 65mm culture dishes and allowed to grow until visible colonies formed in complete growth medium weeks Cell colonies were fixed with methanol stainedwith crystal violet and counted Invasion assays wereperformed in Millicell chambers in triplicate The 8μmpore inserts were coated with μg of Matrigel BD Biosciences Cells — were added to the coated filtersin serumfree medium PMI1640 medium containing FBS was filled in the lower chambers as a chemo attractant After h at °C in an incubator suppliedwith CO2 cells that migrated through the filters werefixed with methanol and stained with crystal violet Cellnumbers in three random fields were counted The migration assay was conducted in a similar fashion withoutcoating the filters with MatrigelExperiments on xenograft animalsTen male BALBc mice weeks old were kept in aspecific pathogenfree grade environment All animalexperiments were approved by the Animal Care and UseCommittee of Shanghai Jiao Tong University School ofMedicine Shanghai China All applicable guidelines ofthe Animal Care and Use Committee of Shanghai JiaoTong University School of Medicine for the care and usethe hind flank regions ofof animals were followed PC9 cells of rs140618127 [A]and rs140618127 [G] type were collected and resuspended in PBS at a concentration of — cellsmLand mixed with Matrigel® at a ratio of respectivelyThe mixture mL was subcutaneously injected intotwo sides ofthe micers140618127 [A] and rs140618127 [G] cells in the samemouse Tumor size was measured once every daysusing a Vernier caliper across its two perpendicular diameters and tumor volume was calculated using the following formula V 12ab2 where V is the tumorvolume a is the largest diameter and b is the smallestdiameter After weeks oftreatment all mice weresacrificed and their tumors were collected and weighedHistological evaluation ofsamples wasperformedthe tumorHistopathological analysesTumor tissues from the animals were fixed in paraformaldehyde BOSTER Wuhan Chinafor h atroom temperature The fixed tissues were then dehydrated in a graded series of alcohol cleaned in xyleneand embedded in paraffin A rotary microtome was usedto section paraffin the blocks into 4μm thick sectionsThe sections were deparaffinized and stained withhematoxylin and eosin HE A light microscopeOlympus was used to examine the stained tissuesectionsStatistical analysisThe association between SNP rs140618127 and NSCLCrisk was analyzed under an additive model using the unconditionallogistic regression model adjusted for agesex and smoking status Results of laboratory experiments were presented as Means ± SD Student™s t testwas used to compare means between two groups andANOVA was employed for comparison of more thantwo groups Repeated ANOVA was employed for comparison of more than two groups which contained repeated measure data All the statistical analyses wereperformed using Statistical Product and Service Solutions SPSS software version and GraphPad PrismVersion GraphPad Software San Diego CA USAResultsSNP rs140618127 G A in LOC146880 and NSCLC riskSNP in LOC146880 rs140618127 is in strong linkagedisequilibrium with SNP rs7216064 r2 which is aGWASdiscovered risk allele for NSCLC We found thatSNP rs140618127 G A in the exon of LOC146880chr17 was associated with the risk of NSCLC the ˜A™ allele compared to ˜G™ had an adjusted oddsratio OR “ in a casecontrol study of subjects Table Stratified analyses suggested 0cFeng Journal of Experimental Clinical Cancer Research Page of that this effect was more evidence in those who were ‰¥female and nonsmokers Supplemental years oldTable S1 The minorfrequency of SNPrs140618127 is low globally but can be high as in some American populations see Suppl alleleEffects of LOC146880 with rs140618127 [a] on cellproliferation and behaviorsWe examined the effects of LOC146880 on cell proliferation by its allele at rs140618127 and found that overexpression of rs140618127 [A] in the NSCLC celllinesA549 and PC9 both with the G allele at rs140618127substantially reduced the rate of cell proliferation whencompared with rs140618127 [G]Fig 1A Colonyformation ability in both A549 and PC9 cells wasmarkedly suppressed by rs140618127 [A] when compared with rs140618127 [G] Fig 1B Overexpression ofrs140618127 [A] significantly suppressed the invasionand migration of NSCLC cells Fig 1C 1D Tumorsize in a xenograft animal model of PC9 was decreasedin both genotype groups but the decline in tumor sizewas greater for rs140618127 [A] than for rs140618127[G] P There was no significant difference intumor size between the vector control group and wildtype rs140618127 [G] Fig S2 HE staining showedthat tumors of rs140618127 [A] possessed less malignantmorphology Fig 1E Together these results indicatethat rs140618127 [A] can inhibit the growth of lung cancer more in vitro and in vivo compared to rs140618127[G]such a possibilityInteraction between LOC146880 and miR5395pEvidence suggests that SNPs in lncRNAs may generatenew interacting sites between lncRNAs and other transcripts such as miRNAs [] Using an online softwarelncRNASNP httpbioinfolifehusteducnlncRNASNP[] we found that several SNPs in LOC146880 werepredicted to haveand SNPrs140618127 was indicated to lie within a putative binding site for miR5395p The G A mutation atrs140618127 was predicted to change the local foldingstructures and free energy of LOC146880 which mightcreate a binding site for miR5395p Following this prediction we investigated whether miR5395p interactswith LOC146880 based on its genotype at rs140618127Luciferase reporter assays showed that in comparison tothe construct containing rs140618127 [G] the constructwith the ˜A™ allele had significantly reduced luciferase activity in the presence of miR5395p suggesting moreinteraction of miR5395p with LOC146880 [A] thanwith LOC146880 [G] Fig 2A The interaction betweenmiR5395p and LOC146880 [A] could be blocked bythe miR5395p inhibitor miR5395p is constitutivelyexpressed in both A549 and PC9 cells In cells stablyoverexpressing LOC146880 miR5395p only decreasedthe levels of LOC146880 with rs140618127 [A] not alleleG indicating that allele A is a target of miR5395p Fig2Bthe RNA pulldown assay weInteraction between LOC146880 and ENO1isolated aUsingLOC146880 withrs140618127[G]protein complexMass spectrometry analysis showed that there were threeproteins in this complex and the most abundant onecompared to antisense one was ENO1 Fig 3A Wethen selected ENO1 for validation detecting ENO1 inthree independent RNA pulldown assays RNA immunoprecipitation RIP assays also showed enrichment ofLOC146880 in the complexes precipitated with ENO1antibody as compared with IgG or another irrelevantantibody indicating that ENO1 may be a key target protein of LOC146880 Fig 3b Next we evaluated the consequences of the interaction between LOC146880 andENO1 We found that ENO1 mRNA expression andprotein level were not significantly different P see Fig S3 in the cells overexpressing LOC146880 withrs140618127[A] or rs140618127[G] in the presence ofmiR5395p Fig 3C 3D However HE staining ofxenograft tumors in mice showed that phosphorylatedENO1 was higher in rs140618127 [G] than in [A] Fig3E The expression of CMYC a downstream target ofENO1 was decreased remarkably when the cells weretransfected with a siRNA against ENO1 Fig 3FRegulation of PI3KAKT signal by LOC146880 via ENO1phosphorylationWe found that ENO1 phosphorylation was markedly decreased in cells overexpressing rs140618127 [A] as compared with those overexpressing rs140618127 [G] in thepresence of miR5395p mimics Fig 4A Fig S4 andFig S5 Using insilico prediction tools [ ] weidentified a phosphorylation site at Tyr44 in the proteinbased on the PDB database Fig S5 [] We next investigated the impact of altered LOC146880 levels on thedownstream signal of ENO1 Since our results describedaboveoverexpressionincreased cell proliferation migration and invasion wefocused our investigation on the PI3KAKTNFkB signaling The total amount of PI3K and AKT proteins wasnot significantly different between cells overexpressingrs140618127 [A] and [G] However we observed thatprotein phosphorylation levels affected the expression ofdownstream molecules in the PI3KAKT signaling inA549 Fig S4 Cells overexpressing LOC146880 withrs140618127 [A] showed substantial decreases in NFkB PCNA Vemintin and Ncadherin levels while theirβcatenin and Ecadherin levels weresignificantlyincreased when compared withthesamecellsLOC146880indicatedthat 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs140618127[A] inhibits NSCLC cell proliferation and EMT process A proliferation assay of different samples which were compared byrepeated ANOVA B clone formation ability of different samples which were compared by ANOVA C wound healing assay of different sampleswhich were compared by ANOVA D cell invasion assay of different samples which were compared by ANOVA e cancer protective effect ofrs140618127[A] in xenograft animals which were illuminated by repeated ANOVA and ttest 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs14061812[G][A] expression of different condition in A549 and PC cell lines a luciferase activity of different conditions which wereilluminated by ANOVA b LOC146880 expression level of different conditions which were compared by ttestoverexpressing LOC146880 with rs140618127 [G] Fig4B Fig 4C Fig S7 and Fig S8 Immunohistochemical staining of xenograft tumors showed that pPI3KpAKT TWIST NCadh and SNAIL were all significantly higher in rs140618127 [G] than in [A] Fig 4Dand Fig S9DiscussionIn this study we found that SNP rs140618127 inLOC144680 contained a binding site for miR5395pand the binding between miR5395p and LOC146880resulted in declined phosphorylation of an oncogeneENO1 which was found to be a downstream target ofLOC146880 Furthermore the reduced phosphorylationof ENO1 led to decreased phosphorylation of PI3K andAkt which was linked to the decline in tumor cell proliferation and progress The entire process of how SNPrs140618127 influences NSCLC is depicted in Fig Our casecontrol study supports the notion that SNPrs140618127 genotype [A] may have a protective effecton NSCLC compared to genotype [G] In a previousstudy we found that LOC146880 expression was significantly higher in NSCLC tumors than adjacent normaltissuesforLOC146880 []a possible oncogenicsuggestingroleThere has been an increasing interest in understandingthe mechanisms of rare genetic variants in lncRNAs inrelation to the complex traits and diseases [ ] Ingle suggested that genetic polymorphisms in lncRNAMIR2052HG offer a pharmacogenomic basis for the response of breast cancer patients to aromatase inhibitortherapy [] Tang indicated that SNP rs9839776in SOX2OT was significantly associated with breast cancer possibly via influencing the expression of SOX2OT[] Redis demonstrated that the GWASidentifiedSNP rs6983267 on 8q24 is in a lncRNA gene calledCCAT2 which regulates cancer cell metabolism in anallelespecific manner through binding to the cleavagefactor I complex This complex is implicated in anallelespecific regulatory mechanism of cancer metabolism orchestrated by alleles ofthe lncRNA [ ]Russell et alidentified a neuroblastoma susceptibilitylocus rs9295534 located in the upstream enhancer of atumor suppressor CASC15S The SNP could decreasethe transcriptional activity of CASC15S and be associated with the disease outcome [] Wang foundthat SNP rs965513 a locus on 9q22 in the FOXE1 geneand lncPTCSC2 was associated with the risk of papillary thyroid carcinoma []In this study we found that a lncRNA could regulatethe function of a protein via its phosphorylation with 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 promotes ENO1 activation in a variantspecific manner A pulldown assaymass spectrum of identificationsilver staining BRIP assay with A549 and PC9 cell lines which were compared by ANOVA C D ENO1 mRNA expression and protein level of rs140618127[G][A]by overexpression plasmid transfection which were compared by ttest E HE staining of ENO1 phosphorylation in vivo which were comparedby ttest F ENO1 LOC146880 and cMYC expression level after ENO1siRNA transfection which were compared by ttestlittle influence on gene expression or protein concentration Similar findings have been reported before in whichthe phosphorylation site of a protein can be blocked by alncRNA leading to decreased phosphorylation For example NFkB can be inhibited by a long noncodingRNA which directly blocks IKB phosphorylation inbreast cancer [] LncRNA can also bind to proteinsincreasing or decreasing their phosphorylation viaanother protein LncRNA DANCR and PANDAR influence the phosphorylation of serine in RXRA and SFRS2via GSK3β and P53 in breast and ovarian respectively[ ] GSK3β™s phosphorylation in breast cancer wasreported to be reduced by lncRNA NLIPMT [] Thephosphorylation of ULK1 can be suppressed by lncRNAHOTAIR in NSCLC [] LINC00675 enhances the phosphorylation of vimentin on Ser83 to suppress gastriccancer progression [] Our finding of a lncRNA™s impact on the phosphorylation of a protein was quiteunique and interesting because it is achieved by a microRNA through a polymorphic site in LOC146880In our study we found that a G to A transition atrs140618127 in LOC146880 could turn into a bindingsite for a microRNA and miR5395p was indeed the target Interestingly the wildtype of LOC146880 had no 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 regulates PI3KAKT signaling via ENO1 A549 PC9 A ENO1pENO1 PI3KpPI3K AKTpAKT protein level usingrs140618127[G][A] overexpression plasmid transfection B PCNA NFkB protein level using rs140618127[G][A] overexpression plasmidtransfection C βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection D HEstaining of pPI3K pAkt TWIST NCadhersin and SNAIL 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig Diagrammatic sketch of rs14061812mediated NSCLC tumorigenesis LOC146880 rs14061812[G] increases ENO1™s phosphorylationresulting in activating PI3KAKT signaling pathway and NSCLC tumorigenesis while LOC146880rs14061812[A] binds to miR5395p decreasesENO1™s phosphorylation resulting in deactivating PI3KAKT signaling pathway and NSCLC tumorigenesisinteraction with the microRNA at all This SNP has notbeen reported before in any studies [] However miR is known to be a tumor suppressor [ ] Ourfinding of miR539™s binding to loc146880 provided newinsights into a possible mechanism that explains the biologic function of miR539 as a tumor suppressor Thiseffect takes place when a microRNA and lncRNA interact through a polymorphic site which results in changesin phosphorylation in a protein ENO1 that the lncRNAmay target on Low levels of LOC146880 did not influence the mRNA expression or protein levels of ENO1but suppressed the phosphorylation of ENO1 ENO1 is ametabolic enzyme involved in the synthesis of pyruvateIt also acts as a plasminogen receptor and mediates theactivation of plasmin and extracellular matrix degradation In tumor cells ENO1 is upregulated and supportsthe Warburg effect The protein is located on the cellsurface where it promotes cancerinvasion and issubjected to substantial posttranslational modificationsnamely acetylation methylation and phosphorylation[] Reduced phosphorylation of ENO1 lowers thePI3KAkt signal which results in slower cell migrationor invasion of NSCLCThe SNPbased interaction between miRNA andlncRNA in regulation of protein function has beenhypothesized and predicted by YaRu but fewstudied have provided evidence [] Our study was thefirst to show the interaction between LOC146880 andmir5395p in the NSCLC and to elucidate the downstream mechanism involving tumor growth and metastasis The modulation model of the lncRNA and miRNA isnot the classical competing endogenous RNAs ceRNAHow LOC146880 interacts with ENO1 to regulate itsphosphorylation and downstream signals remains to beelucidated Although the ˜A™ allele of rs140618127 is lowin general some racial groups still have a relatively highfrequency In some Caucasian populations the ˜A™ allelefrequency is close to ConclusionsWe found in a casecontrol study of Chinese thatSNP rs140618127 in LOC146880 was associated with therisk of NSCLC People with the G allele of rs140618127had higher risk than those with the A allele Our in vitroand in vivo experiments demonstrated that LOC146880was an oncogene and the G allele of rs140618127 hadstronger oncogenic effects on lung cancer cells than theA allele in LOC146880 This differential effect appearedto come from the binding of a microRNA miR539 tothe A allele but not the G allele at rs140618127 ThemicroRNA binding prevented the lncRNA™s interactionwith its downstream target ENO1 which led to the reduction of ENO1 phosphorylation and suppression ofthe PI3KAKT signaling resulting in lower tumor cellproliferation and less aggressive cell behaviors 0cFeng Journal of Experimental Clinical Cancer Research Page of Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s13046020016525Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestAdditional file Fig S1 Locations of LOC146880 A549 BEAS2BLOC146880 locals mainly in cytoplasm The comparison between twogroups using ttestAdditional file Fig S2 Comparison of tumor size between vectorcontrol group and the wide type There was no significant difference oftumor size between vector control group and the wide typers14061812[G]Additional file Fig S3 Comparison of ENO1 protein level ofrs140618127[G][A] by overexpression plasmid transfection which werecompared by ttestAdditional file Fig S4 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of A549 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S5 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of PC9 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S6 Predicting site of phosphorylation of ENO1A predicting phosphorylation site of ENO1ENO1 chain A usingNetPhos B predicting phosphorylation site of ENO1ENO1 chain Ausing PhosphoELM BLAST C The predicting phosphorylation site ofENO1 chain A using data of PDB databaseAdditional file Fig S7 Comparison of PCNA and NHkB protein levelof using rs140618127[G][A] overexpression plasmid transfection whichwere compared by ttest A results of A549 cell lines B result of PC9cell linesAdditional file Fig S8 Comparison of βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection which were compared by ttest A results ofA549 cell lines B result of PC9 cell linesAdditional file Fig S9 Comparison of the HE staining of pPI3K pAkt TWIST NCadhersin and SNAIL which were compared by ttestAdditional file Distribution of

Thyroid_Cancer

"characterize the frequency and clinical features of lungnodules in IgG4 related disease IgG4RD patients as an insight for help with the diagnosis of lung nodulesMethods A retrospective study was carried out in West China Hospital Sichuan University from January toDecember patients with definite IgG4RD were enrolledResults Fifty of patients with definite IgG4RD had radiologically confirmed lung nodules of whom werediagnosed with definite IgG4 related lung disease Lung nodules detected in more than patients were small andsolid always with regular margins Multiple and bilateral distributions was also a major characteristicof these lung nodules Lobulation and speculation were simultaneously detected in patients including patientscombined with pleural indentation Calcification of nodules was detected in only one patient Thirtyseven patientsalso had additional radiological abnormalities of lungs including groundglass opacity thickening of pleura thickening of interlobular septa thickening of bronchial wall pleural effusion mass interstitial changes and mediastinal or hilar lymphadenopathy Most patients were treatedwith glucocorticoids alone or combined with immunosuppressive agents Sixteen patients received a reexamination by chest computed tomography CT scan after treatment of whom showed a decrease in the sizeandor the number of nodulesConclusions The incidence of lung nodules in IgG4RD patients can be high For an IgG4RD patient with lungnodules the possibility that the lung nodules related to IgG4RLD is high It is hard to differentiate IgG4 relatedlung nodules from other lung diseases in particular lung cancer Radiological characteristics and positive responsesto glucocorticoids and immunosuppressive agents can help with the differential diagnosis For these patientsregular followup is also importantKeywords Clinical characteristics IgG4 related disease IgG4 related lung disease Lung nodules RadiologicalcharacteristicsBackgroundClinical presentation of nodules in the lungs of patientscan be a challenge for diagnosis especially when thenodules are small and asymptomatic The nodules maybe difficult to biopsy for histopathology because of size Correspondence liuyihuaxiyiyuan126com Yan Xie and Anji Xiong contributed equally to this work1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu ChinaFull list of author information is available at the end of the and location for example very small nodules deeply imbedded in the lung The first diagnosis willlikely besome form of neoplasm When that diagnosis is determined to be incorrect many physicians especially respiratory physicians and thoracic surgeons may beunsure of the next step toward diagnosis and treatmentAffected patients of course will be anxious for a confirmed diagnosis since they will likely assume the smallnodules are an early stage of lung cancer A recently recognized diseaseimmunoglobulin G4related disease 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 0cXie BMC Pulmonary Medicine Page of IgG4RD must be considered for such presentation ofasymptomatic small nodules particularly in the lungsliver or lymph nodes []IgG4RD is a chronic fibrotic inflammation characterized by the involvement of multiple ans The mostcommon manifestations of the disease include swellingof salivary and lacrimal glands lymphadenopathy andtype autoimmune pancreatitis AIP Other anssuch as lung bronchi kidney retroperitoneum thyroidheart mesenterium meninges and skin can also be involved Frequently but not always serum IgG4 levelswill be elevated [] IgG4 is the rarest of the IgG subclasses generally has relatively low antigen affinity andis unable to bind complement component 1q C1q andactivate the complement cascade [] IgG4 can exchangeFab arms by swapping a heavy chain and attached lightchain with a heavylight chain pair from another IgG4molecule which providesthe antiinflammatory activity attributed to IgG4 antibodies [] Akey pathological feature of IgG4RD nodules is a denselymphoplasmacytic infiltrate anized into a storiformpattern which frequently forms a tumefactive mass thatmay destroy an involved an [] When the tumefactive mass occurs in the lungs it may present as a noduleor groundglass opacity on radiology [] IgG4relatedlung disease IgG4RLD is the lung involvement ofIgG4RD which was first described in in a patientwith interstitial pneumonia autoimmune pancreatitisand IgG4positive plasma cells in the interstitium []The IgG4RLD presentation can be heterogeneous andits radiologic manifestations are often extensive evenwhen clinically asymptomatic []the basisforAlthough lung nodules have been described in to of IgG4RLD in a few case reports and reviews todate [ ] no study has employed a systematic analysis of IgG4RD patients with lung nodules Here wepresent retrospective data of the clinical and radiologicalfeatures of IgG4RD patients from a single medical center™s experience Our goal is to provide clinical information and insight for the diagnosis of IgG4RD in patientswho present with asymptomatic nonneoplastic smalllung nodulesMethodsPatients informationPatients with definitive IgG4RD were selected from alldepartments of West China Hospital Sichuan Universityfrom January to December The clinical serological and radiographic imaging characteristics andtreatment responses of the patients were analyzedOur study was performed according to the rules of thehospital™s medical ethics committee Informed consentwas obtained in accordance with the institutionalguidelinesDiagnosis of IgG4RDThe diagnosis of IgG4RD was made according to thecomprehensive diagnostic criteria for IgG4RD publishedin by Umdehara [] The criteria for definiteprobable and possible IgG4RD are as followsDefinite1 Probable1 Possible1 Diffuse or localized swelling or masses in single ormultiple ans Elevated serum IgG4 mgdl Histopathology of biopsied nodules showsa Marked lymphocyte and plasmacyte infiltrationand fibrosisb Infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cells and IgG4 plasma cells per 40X fieldChest CT protocolsCT images were available for all patients involved in thisstudy Because of the retrospective nature of this studychest CT scanning protocols were varied The scanningwas performed on one of the six machines ranging from16detector to 128detector CT scanners Philips Medical Systems Best the Netherlands or Siemens MedicalSystems Erlangen Germany with patients in the supineposition For patients who underwent contrastenhancedexamination an intravenous nonionic contrast mediumIopamidol mgml ml was given and imaging started s later after injection To minimize motion artifacts CT images were acquired during a singlebreathhold The main parameters were shown as follows tube voltage 100120kv tube current mA slice thickness mm section interval mmDefinition of lung nodules and IgG4RLDCT images of patients™ chests were analyzed by radiologists The lung nodules were defined as rounded or irregular opacity well or poorly defined measuring up to cm in diameter [] Nodules were categorized aslarge or small as follows large cm ‰ diameter ‰ cmor small diameter cm If the diameter of the lung lesion is larger than cm in the CT images the lesionwas defined as a massIgG4RLD is defined as IgG4RD diagnosed as abovethat includes diffuse or localized swelling or masses inone or both lobes of the lungs with histopathology thatincludes marked lymphocyte and plasmacyte infiltrationand fibrosis andor infiltration of IgG4 plasma cells defined as IgG4 plasma among all IgG plasma cellsand IgG4 plasma cells per × field 0cXie BMC Pulmonary Medicine Page of StatisticsStatistical analyses were performed with GraphPadPrism version GraphPad Software Inc La Jolla CAUSA Descriptive data are reported as median interquartile range and frequencies are percentages Continuous variables without normal distribution wereexpressed as median and interquartile IQR For comparison between groups the unpaired Student™s ttestwas used for continuous variables and chisquare testwas used for categorical variables All statistical testswere 2sided and results with P values were considered statistically significantResultsFrom January to December a total of patients were diagnosed with definitive IgG4RD in WestChina Hospital Sichuan University of whomhad CTconfirmed lung nodules were included in thisretrospective studyPatient description and clinical presentationDemographic and clinical data for all patients are summarized in Table The median age of the patients withlung nodules was “ years at diagnosis with amale female ratio of Almost half of the patientshad their initial diagnosis of IgG4RD in the Departmentof Rheumatology The duration of symptoms beforediagnosis averaged months range “ monthsFortytwo of the patients had no pulmonary infectionor a history of cancer and other chronic pulmonary diseases And more than half of the patients had ahistory of smokingFirst symptomsFor patients with lung nodules the most common firstsymptoms were salivary gland swelling and lacrimal gland swelling Seven patients presented withcough as their first symptom There were several raresymptoms including fever gum swelling low back painand edemaExtrapulmonary ans involvedMost patients had multiple sites or ans involved median number IQR1“ Twentyone patients had atleast three extrapulmonary ans or sites affected Themost common involved extrapulmonary ans includedsalivary gland lacrimalgland pancreas and kidney Other sites included liver nasal sinus peritoneum pituitaryskinthyroid glands bile ducts gingiva andpericardiumlymph node Serological characteristicsSerum levels of IgG4 erythrocyte sedimentation rateESR Creaction protein CRP and complements werequantified Serum IgG4 concentrations were measuredin all patients in this study with a median value of mgdl IQR426“ for patients with lung nodulescompared to mgdl IQR371“ for IgG4RD patients without lung nodules Table Elevated ESR andserum CRP levels were found in and patients respectively among patients with lung nodules versus patients with elevated ESR and patients with elevatedCRP among IgG4RD patients without lung nodulesTwentyfour patients with lung nodules versus without had reduced serum complement Twenty patientswith lung nodules versus without had antinuclearantibody ANA was defined as positive Therewere no statistically significant differences in serologicalmeasurements between IgG4RD patients with and without lung nodules Table Comparison of clinical characteristicsThe general clinical characteristics of patients with lungnodules were compared with those of patients withoutlung nodules As the results indicated in Table significant differences were observed in smoking history between the two groups while no significant differences inage gender ratio duration of symptoms before diagnosis and the number of extrapulmonary involved answere observedRadiological characteristics of the lungsFifty patients included in this study had lung nodules revealed in chest CT images The radiological characteristics of lungs among the patients with lung nodulesare summarized in Table and Table Five patients had large lung nodules Fig 1a with diameters ranging from to cm while almost all patients showed smalllung nodules Fig 1beMost patients had multiple nodules Only patients had single small nodules For the distribution oflung nodules patients had bilateral nodules accounting for more than half of the cases When consideringthe pulmonary lobe involvement the number of patientswith nodules located only in the upper lobe and middleor lower lobe was and respectively The rest patients had nodules randomly distributed in differentlobes at the same timeFor the lesion densities on CT solid nodules can bedetected in almost all patients Only patientsshowed groundglass nodules all of which were smallnodules Nodules in out of the patients showed bothgroundglass and solid patterns Fig 1d Fig 1e Mostpatients showed regular and welldefined nodulemargins Only patients had nodules with irregular or 0cXie BMC Pulmonary Medicine Page of Patients with lung nodulesn “Patients without lung nodulesn “““““Table Clinical characteristics of IgG4RD patientsParametersAge medianIQR yearsMenwomen n Smoking history n Duration of disease medianIQR monthsSerum IgG4 medianIQR mgdlElevated ESR n Elevated CRP n Reduced C3 n Reduced C4 n Positive ANA n First symptoms n Salivary gland swellingLymphadenopathyLacrimal gland swellingCoughJaundiceDysuriaFeverAbdominal painGum swellingLow back painEdemaFatigueDiarrheaNumber of extrapulmonary ans involved medianIQRExtrapulmonary an involvement n “Salivary glandssubmandibular gland and parotid glandLymph nodeLacrimal glandPancreasKidneyLiverNasal sinusSkinBile ductsPeritoneumPituitaryThyroid glandsGingivaPericardiumBloodP value“Data are expressed as medianinterqurtile range for contious variables and frequenciespercentages for categorical variablesIgG4RD IgG4 related disease ESR Erythrocyte sedimentation rate CRP Creactive protein C3 Complement C4 Complement ANA Antinuclear antibody IQRInterquartile range P 0cXie BMC Pulmonary Medicine Page of Table Radiological characteristics of lungs before drugtherapyPatients with indicated lung nodulesSizesinglemultipleSmall nodule onlyLarge nodule onlyBoth small and large noduleDistributionLateralityUnilateralBilateralLobeUpper lobeMiddle or lower lobeRandomTypeGroundglass nodule onlySolid nodule onlyBoth groundglass and solid noduleMarginIrregular or untidyRegularSecond associated featuresLobulationSpiculationPleural retractionCalcificationChanges of nodules after treatmentsn Smaller or disappearNo differenceOther radiological featuresGroundglass opacityThickening of pleuraThickening of interlobular septaThickening of bronchial wallPleura effusionMassConsolidationInterstitial changesn Mediastinal and hilar lymphadenopathypoorly defined margins Some other features of nodulesincluding lobulation spiculation pleuralindentationand calcification were also explored All these signswere rare in our study Lobulation and speculation Fig1c were simultaneously detected in patients including patients combined with pleural indentation Fig 1cCalcification of nodules was detected in only one patientThirtyseven patients including the patients with definite IgG4RLD showed some other radiological abnormality of lungs Fig which included groundglassopacity2150 thickening of pleura950 thickening ofinterlobular septa thickening of bronchial wall3 pleural effusion450 mass350 consolidation1 interstitial changes550 and mediastinal or hilarlymphadenopathy3250Six patients were diagnosed as definite IgG4RLD Thechest CT findings of these patients were shown in Table All of the patients showed mixed patterns in CTchanges Two patients had single large lung nodulesonly while the rest patients showed multiple smalllung nodules only Mass thickening of pleura thickening ofinterlobular septa groundglass opacity andpleura effusion were detected in and patientsrespectively Enlarged mediastinal or hilar lymph nodewas detected in all IgG4RLD patientsTherapy and responsesThe results of therapy and responses are shown in Table and Table For the treatment of the patients withlung nodules patients received prednisone with orwithout additional antiinflammatory or immunosuppressive drugs with prednisone only with prednisone and cyclophosphamide with prednisone andmycophenolate mofetil or methotrexate and withprednisone and azathioprine Three patients received asurgical treatment only and patients with mild diseasereceived symptomatic therapyMost patients improved to some degree aftertreatments during the duration of hospitalization Sixteen patients received a reexamination of chest CT scanafter treatment The durations between CT scans wereat least month Ten patients showed a decrease in thesize andor the number of nodules while patientsshowed no difference between pre and posttherapy CTimagesDiscussionLung nodules are small rounded lesions with at leasttwothirds ofits margins surrounded by lung parenchyma and not associated with atelectasis or lymphadenopathy In this study we focused on the lung nodules ofIgG4RD patients and IgG4RD patients presented with lung nodules in CT images As far as weknow there hasn™t been any study concerned about theincidence of lung nodules in IgG4RD patients Previousstudies have reported that lung nodules were incidentallyidentified in approximately “ ofthe socialdemographically population [ ] Our result revealed 0cXie BMC Pulmonary Medicine Page of Table Radiologic findings in patients with definite IgG4RLDPatientsMass Thickening ofNoGroundglassopacityYespleuraYesSmallnoduleLargenoduleSolitaryThickening ofinterlobular septaPleuraeffusionEnlarged mediastinal or hilarlymph nodeYesSolitaryYesYesMultipleMultipleMultipleMultipleYesYesYesYesYesYesYesYesYesYesYesYesYesYesYesthat the incidence of lung nodules in IgG4RD patientswas much higherComparing the clinical characteristics of patients withlung nodules with that of patients without lung nodulesno significant difference was found in terms of age gender ratio duration of symptoms before diagnosis number of extrapulmonary involved ans and serologicalcharacteristics indicating that these factors have no association with the progress of nodule formation Forclinical symptoms only patients were observed withcough Most patients especially patients with small nodules only were relatively asymptomatic despite substantial burdens of disease within thelung Clinicalsymptoms of lung disease depend on the location andsize of lesions and are often nonspecific for the diagnosisof some lung disease including IgG4RLDIn our study six patients were diagnosed as definiteIgG4RLD All of the patients had lung nodules Consistent with results of previous studies conducted byInoue [] and Sun [] this result revealedthat nodular lesion can be a common manifestation ofIgG4RLD Together with lung nodules some otherchest CT findings including mass solid nodules roundshaped glass opacity thickening of bronchovascular bundles and interlobular septa alveolar interstitial changeslike honeycombing and bronchiectasis lobar or segmental consolidation and lymph node enlargement et alwere often seen in IgG4RLD patients And these CTchanges always present as various mixed patterns [ “] In this study except lung nodules we also detected mass groundglass opacity thickening of pleurapleura effusion mediastinal and hilar lymphadenopathyand thickening of interlobular septa in IgG4RLD patients It is worth noting that IgG4RLD related nodulelesion reported previously are always single solid andlarge type together with or without multiple small nodules [ “] And these nodules are always solidand with spiculated or irregular margins In our studyonly IgG4RLD patients had large nodules while ofthe IgG4RLD patients presented as multiple smallnodules And only one of the patients had noduleswith irregular margins As pulmonary biopsy results areFig Radiological manifestations of lung nodules in IgGRD patients via chest computed tomography scans a A single large solid nodule withirregular margin was detected in the right lung Thickening of pleura and tracheal traction were also noted b Multiple small and solid lungnodules were scattered in both lungs c A single small and solid nodule was shown in the right lung apex Lobulation spiculation and pleuralindentation of the nodule can be noted Thickening of pleura and the bronchial wall can also be observed de Multiple solid and groundglassnodules were shown in both lungs 0cXie BMC Pulmonary Medicine Page of Fig Other radiological manifestations of lungs in IgGRD patients via chest computed tomography scans a Thickening of the left pleura canbe observed A small nodule near the thickened pleura can also be noted bc Thickening of interlobular septa and pleura effusion in the rightlung can be observed d Mass and groundglass opacity were shown ef Small lung nodules combined with groundglass opacity consolidationthickening of interlobular septa and thickening of bronchial wall can be observedhard to get this inconsistency may be caused by a smallsample size of studies related to IgG4RLD Thus morestudies are needed to clarify the CT imaging features ofIgG4RLDAs lung nodules could also be caused by infection malignancy or some other pulmonary disorder we cannotbe certain that in the absence of definitive nodular biopsies all of the lung nodules in our study were related toIgG4RLD For an IgG4RD patient with lung nodulesthe probability of lung nodules related to IgG4RLD isvery high especially in the absence of other lung diseases In a study reported by Tsushima of IgG4RD patients with lung nodules were confirmed tobe IgG4RLD by biopsy [] In our study most patientshad no pulmonary infection or a history of cancer andother chronic pulmonary diseases For these patientsthe probability of lung nodules related to IgG4RLD isvery high Besides CT findings of our study also revealedthat most of the lung nodules in IgG4RD patients weresmall and solid always with regular margins MultipleTable Treatments of IgG4RD patientsTreatmentsPatients with lung nodulesn Pred onlyPredimmunosuppressiveagentsothersPredCYC PredMMF PredMTX PredAZA IgG4RD IgG4 related disease Pred prednisone CYC cyclophosphamide MMFmycophenolate mofetil MTX methotrexate AZA azathioprine Others surgicaltreatment or symptomatic no coticosteriod or immunosuppressive agentswas givenand bilateral distributions was also a major characteristicof these lung nodules Lung nodules in one patient alsoshowed calcification These radiologic features and distribution of lung nodules were usually regarded as benign [ ]IgG4RLD variedIt is noteworthy that three patients with large lunglobulation spiculation andnodules showed signs ofpleuralindentation all of which may predict an increased risk of malignancy [ ] As radiological findings ofthese signs can often beobserved in lung nodules and masses related to IgG4RLD [ ] Besides we also found that the proportionof lung nodules in smokers was significantly higher thanthat in nonsmokers indicating that smoking may be arisk factor of these lung nodules The proportion of elderly in IgG4RD patients was not small Therefore forIgG4RD patients with lung nodules it™s necessary buthard to differentiate IgG4RLD from lung cancer Asmentioned above the simultaneous presence of otherkinds of lung lesions may provide support for the diagnosis of IgG4RLD and help with the differential diagnosis In our study most patients also had thesimultaneous presence of other CT findings includingthickening of pleura thickening of interlobular septathickening of bronchial wall pleural effusion presenceof an undefined mass interstitial changes consolidationgroundglass opacity and mediastinal or hilar lymphadenopathy which may help to increase the possibilityof IgG4RLDMost IgG4RLD patients have a significant response toglucocorticoid therapy which can help to further distinguish IgG4RD nodules and malignancy [] In ourstudy patients went through a reexamination of CTscan after glucocorticoid therapy with or without 0cXie BMC Pulmonary Medicine Page of immunosuppressive agents Ten patients including patients that had large lung nodules with signs of lobulation spiculation pleural indentation and cavity showedpositive responses A decrease in the size andor thenumber of nodules was observed which helped to further support the diagnosis of IgG4RLDtype oflobectomy forSometimes during a routine medical examinationpatients may present with lung nodules detected without other pulmonary symptoms Since nodules mayresemble bronchoalveolar carcinoma and raise suspicion of malignancy [] case reports indicate that patients with thislung lesions may haveundergone wedge resection orsuspected malignancy only to discover upon tissue examination that the relevant nodules were a consequenceof IgG4RD [ ] In our study a patient with bilateral smalllung nodules was first misdiagnosed astuberculosis and another patient with a single largenodule underwent a middle lobectomy and histological findings were consistent with IgG4RD Thusan understanding of nodules related to IgG4RD isnoteworthy Since IgG4RD may be a multian disease the involvement of other ans and serologicalchange can help with diagnosis in patients with undiagnosed lung nodules In our study only patientshad the pathological proof of IgG4RLD while theother patients were diagnosed with IgG4RD primarilybecause of clinical manifestations in other ansparticularly salivary glandslacrimalgland and pancreas which is consistent with otherstudies [] In Sun™s study of biopsyproven IgG4RLD patientsinvolvement wasproven in only patient with uveitis mastoiditisSince patients with only lung involvement are moreprone to receive a lung biopsy the uncommon extrapulmonary involvement may be caused by selectionbias [] Once the diagnosis of IgGRD is confirmedthe radiological characteristic and treatment responsecan help with the diagnosis ofthus aregular followup is important for these patientsextrapulmonarylymph nodesIgG4RLDOur study had some limitations First most of the lungnodules involved in our study were not histopathologically proven Therefore we were unable to clarify theexact proportion of lung nodules related to IgG4RLDSecond due to the retrospective features of our studythe scan protocols of CT varied and the number of patients with a reexamination of CT was small whichmay further limit the value of our studyConclusionsThe incidence of lung nodules in IgG4RD patients canbe high For an IgG4RD patient with lung nodules thepossibility that the lung nodules related to IgG4RLD ishigh It is hard to differentiate IgG4 related lung nodulesin particularfrom other lung diseaseslung cancerRadiological characteristics and positive responses toglucocorticoids and immunosuppressive agents can helpwith the differential diagnosis For these patients regularfollowup is also importantAbbreviationsIgG4RD IgG4 related disease AIP Autoimmune pancreatitisC1q Component 1q IgG4RLD IgG4related lung disease CT Computertomographic IQR Interquartile range ESR Erythrocyte sedimentation rateCRP Creaction protein ANA Antinuclear antibody C3 Complement C4 Complement Pred Prednisone CYC CyclophosphamideMMF Mycophenolate mofetil MTX Methotrexate AZA AzathioprineAcknowledgementsNot applicableAuthors™ contributionsYX and AX equally contributed to study design data collection andinterpretation and drafting and revisions of the manuscript TM contributedto drafting and revision of the manuscript YL contributed to drafting andrevision of the manuscript All authors have read and approved themanuscriptFundingThis work was not funded by any grantsAvailability of data and materialsAll relevant data in this study are freely available to any scientist wishing touse them for noncommercial purposes without breaching participant confidentiality And relevant data can be obtained by contacting the corresponding authorEthics approval and consent to participateThe study was approved by the Ethical Committee of West China HospitalSichuan University According to the rules of the hospital™s medical ethicscommittee this study fulfilled the criteria of exception to the requirementsof informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Rheumatology and Immunology West China HospitalSichuan University Chengdu China 2Department of MicrobiologyImmunology and Biochemistry University of Tennessee Health ScienceCenter Memphis TN USAReceived August Accepted July ReferencesStone JH Zen Y Deshpande V IgG4related disease N Engl J Med “Vasaitis L IgG4related disease a relatively new concept for clinicians Eur JIntern Med “Doi T Kanatsu K Mayumi M Analisis of IgG immunecomplexes in serofrom patients with membranous nephoropathyrole of IgG4 subclass andlowavidity antibodies Nephron “van der Neut KM Schuurman J Losen M Bleeker WK MartínezMartínez PVermeulen E den Bleker TH Antiinflammatory activity of human IgG4antibodies by dynamic fab arm exchange Science “Inoue D Zen Y Abo H Gabata T Demachi H Kobayashi T Yoshikawa J Immunoglobulin G4related lung disease CT findings with pathologiccorrelations Radiology “Taniguchi T Ko M Seko S Nishida O Inoue F Kobayashi H Interstitialpneumonia associated with autoimmune pancreatitis Gut “ 0cXie BMC Pulmonary Medicine Page of Mahajan VS Mattoo H Deshpande V Pillai SS Stone JH IgG4relateddisease Annu Rev Pathol “ Matsui S Taki H Shinoda K Suzuki K Hayashi R Tobe K Respiratoryinvolvement in IgG4related Mikulicz™s disease Mod Rheumatol “Fujinaga Y Kadoya M Kawa S Hamano H Udea K Momose M et alCharacteristic findings in images of extrapancreatic lesions associated withautoimmune pancreatitis Eur J Radiol “ Umehara H Okazaki K Masaki Y Kawano M Yamamoto M Saeki T et alComprehensive diagnostic criteria for IgG4related disease IgG4RD Mod Rheumatol “ Hansell DM Bankier AA Macmahon H McLoud TC Muller NL Remy JFleischner society glossary of terms for thoracic imaging Radiology “ Gould MK Tang T Liu IL Lee J Zheng C Danforth KN Recent trendsin the identification of incidental pulmonary nodules Am J Respir Crit CareMed “Loverdos K Fotiadis A Kontogianni C Iliopoulou M Gaga M Lung nodulesa comprehensive review on current approach and management AnnThorac Med “Sun X Liu H Feng R Peng M Hou M Wang P Biopsyproven IgG4related lung disease BMC Pulm Med Tsushima K Tanabe T Yamamoto H Koizumi T Kawa S Hamano H et alPulmonary involvement of autoimmune pancreatitis Eur J Clin Investig“Lin W Lu S Chen H Wu Q Fei Y Li M Clinical characteristics ofimmunoglobulin G4related disease a prospective study of Chinesepatients Rheumatology “ Ryu JH Sekiguchi H Yi ES Pulmonary manifestations of immunoglobulinG4related sclerosing disease Eur Respir J “ Zen Y Nakanuma Y IgG4related disease a crosssectional study of cases Am J Surg Pathol “ MacMahon H Naidich DP Goo JM Lee KS Leung ANC Mayo JR et alGuidelines for management of incidental pulmonary nodules detected onct images from the fleischner society Radiology “ Masaki Y Kurose N Yamamote M Takahashi H Saeki T Azumi A Cutoffvalues of serum IgG4 and histopathological IgG41 plasma cells for diagnosis ofpatients with IgG4 related disease Int J Rheumatol “ Okubo T Oyamada Y Kawada M Kawarada Y Kitashiro S Okushiba SImmunoglobulinG4related disease presenting as a pulmonary nodule withan irregular margin Respirol Case Rep 201651e00208 Odaka M Mori S Asano H Yamashita M Kamiya N Morikawa TThoracoscopic resection for a pulmonary nodule with the infiltrate of IgG4positive plasma cells Asian J Endosc Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"Sintilimab blocks the interaction between programmed death1 PD1 and its ligands The safety andefficacy of sintilimab combined with oxaliplatincapecitabine CapeOx as firstline treatment were evaluated inpatients with gastric Ggastroesophageal junction GEJ adenocarcinoma in a phase Ib clinical trialMethods Patients with locally advanced or metastatic GGEJ adenocarcinoma without previous systemic treatmentwere enrolled as one cohort of a multicohort study Sintilimab was administered at a dose of mg intravenously IV incombination with CapeOx mgm2 capecitabine orally bid D1“ and mgm2 oxaliplatin IV D1 every daysfor up to cycles After combination treatment patients continued to receive sintilimab mg at weekly intervals asmaintenance therapy until progressive disease PD unacceptable toxicity withdrawal of informed consent or for up to months Adverse events AEs were monitored to assess safety in terms of their frequency intensity and causality Theefficacy endpoints included the objective response rate ORR disease control rate DCR progressionfree survival PFSand overall survival OS Tumor mutation burden TMB was evaluated for its association with clinical responseResults A total of patients were enrolled and received sintilimab plus CapeOx All patients reported treatmentrelatedAEs TRAEs Grade “ TRAEs were found in patients Seventeen patients obtained partial response and theORR was CI “ Three had stable disease and DCR was CI “ Asdata cutoff of May the median followup was months The median PFS was months CI “ andmedian OS had not been reached The OS rates at months and months were and No association wasobserved between TMB and efficacyContinued on next page Correspondence Nongxuzhjphoutlookcom1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou 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 0cJiang BMC Cancer Page of Continued from previous pageConclusions Sintilimab combined with CapeOx as firstline treatment demonstrated acceptable safety andpromising efficacyTrial registration ClinicalTrialsgov NCT02937116 Registered October Keywords Sintilimab Capecitabine Oxaliplatin Gastricgastroesophageal junction adenocarcinoma Tumormutation burdenBackgroundThe fifth most commonly diagnosed cancer worldwide isgastric cancerGC accounting for about ofcancerrelated deaths globally and the third most common cancer in China with almost half of worldwide newGC cases occurring in China annually [ ] The standard treatments exhibit regional differences among western countriesJapanKorea and China which areconsidered to be associated with different screening andearly detection methods as well as different biologicalbehaviors disease characteristics and ethnicity [“]treatment mainlySurgical resection is the only radical therapy for gastricgastroesophageal junction GGEJ cancer Howeversystemic chemotherapy is an alternative main therapyfor GGEJ cancer because of the high relapse rate afterpostresection surgery and for the many patients diagnosed at an advancedstage For advanced GGEJ cancerinvolves platinumbasedfirstlinechemotherapy using a combination oftwo or threedrugs trastuzumab is given to patients whose tumor ishuman epidermal growth factorreceptor2 HER2positive but the overall survival OS is disappointingsince the maximum OS time has been reported to be months [“] Any potential novel drug that willincrease patient survival times is urgently neededinparticular for firstline treatmentinhibitor treatmentImmune checkpointis a newapproach for tumor immunotherapy [ ] The treatment diminishes the immune system tolerance to tumorcells and improves the effective identification and eradication of tumor cells by blocking T cell inhibition [] Theprogrammed death1 PD1 antibody specifically binds toPD1 thereby inhibiting apoptosis of antigenspecific Tcells and thus reducing regulatory T cell Treg apoptosisby inhibiting the activation of PDL1 [ ]patients with recurrent or advanced GC In the ATTRACTION2 study nivolumab monotherapy improvedOS from to months hazard ratio CI“ P compared with a placebo in advanced GC that was refractory or intolerant to previoustreatment regimens []However between and of patients exhibit noresponse to PD1 blockade which is considered to beassociated with T cell exclusion or exhaustion or inadequate T cell trafficking and many immunosuppressivefactors accumulate in the tumor microenvironment []New therapy regimens that improve the response andlongterm efficacy are desperately needed The efficacy ofantiPD1 therapy in combination with chemotherapy hasbeen confirmed in nonsmallcell lung cancer [ ] Inaddition to direct tumor killing conventional cytotoxicchemotherapy has demonstrated immunoregulatory properties by enhancing tumor antigenicity disturbing immunesuppressive pathways inducing immunogenic cell deathand increasing effector Tcell reactions [] It is safe tohypothesize that antiPD1 antibodies in combination withchemotherapy may further improve the clinical outcomesof patients with advanced GC Sintilimab is a highly selective monoclonal IgG4 antibody that inhibits interactionsbetween PD1 and its ligands with strong antitumorresponse [] A phase 1a study for dose escalation hasdemonstrated the tolerance and pharmacological activity ofsintilimab in patients with advancedstage solid tumors butthere is limited evidence for the efficacy of antibodiesagainst PD1 plus chemotherapy in Chinese GGEJ adenocarcinoma patients Thus the present trial was conductedto investigate the safety and efficacy of sintilimab combinedwith CapeOx as firstline therapy for a cohort of patientswith GGEJ adenocarcinomaThe efficacy of antiPD1 antibodies monotherapy inpatients who had prior chemotherapy for advanced GChas been demonstrated and supported by several trialsIn the KEYNOTE012 and KEYNOTE059 trials pembrolizumab monotherapy showed objective responserates ORR of n [] and n []respectively in PDL1 positive advanced GCpatients after at least two prior systemic therapies Basedon such results the Food and Drug Administration approved pembrolizumab forthirdline treatment ofMethodsStudy design and patientsThe present study was an label multicenter phase Ibstudy to evaluate the safety and efficacy of sintilimab in cohorts of patients with solid tumors Patients age range“ years with cytologically or histologically confirmedunresectable GGEJ adenocarcinoma were enrolled in theGGEJ cohort Tumor nodes and metastases TNM staging has been evaluated according to the Union for International Cancer Control UICC TNM classification 8th 0cJiang BMC Cancer Page of edition [] The patients had received no previoussystemic treatment for advanced disease or had disease progression PD more than months after systemic adjuvant therapy Other major inclusion criteriawere at least one measurable lesion as defined by theResponse Evaluation Criteria in Solid Tumor RECISTversion criteria score or for Eastern TumorCollaborative Group Performance Status ECOGPSadequate an and bone marrow functions and lifeexpectancy ‰¥ weeks Patients with amplification oroverexpression of the HER2 gene were excluded fromthe trial Appendix contains a complete list of allinclusion and exclusion criteriaThe institutional review boards of all centers approvedthe protocols and the study was carried out in strictaccordance with the declaration of Helsinki principlesall participating patients signed consent forms beforetaking partProceduresAccording to NCCN guideline the preferred firstlinechemotherapy regimens for advanced gastric cancer arefluorouracil or capecitabine combined with cisplationor oxaliplatin [] Howeverthe results from theREAL2 study [] revealed significant clinical benefitof the oxaliplatincapecitabine CapeOx regimen whichled to the longest OS time of months comparedwith other regimens Oxaliplatin produces less renaltoxicity there is no requirement for hydration and ithas a lower emetic potential compared to cisplatinwhile capecitabine has no requirement for continuousintravenous IV infusion and is administered orallywhich should ensure an improved quality of life for patients in their homes Therefore a CapeOx regimen hasbeen chosen During the combination phase enrolledpatients were given sintilimab in combination withCapeOx for up to cycles every weeks Each cycleconsisted of intravenous sintilimab mg plus oxaliplatin mgm2 on day and capecitabine mgm2 twice daily orally from day to day Aftercombination treatment patients without PD continuedto receive sintilimab mg at weekly intervals asmaintenance therapy until PD unacceptable toxiceffects withdrawal of informed consent or for up to monthsStudy assessmentsAdverse events AEs were monitored for days after thelast administration of a treatment dose Responses wereassessed by computed tomography CT or magnetic resonance imaging MRI every weeks until PD new treatment initiation withdrawal of informed consent or deathEndpointsSafety was assessed as collected AEs according to theirtype frequency causality and severity grading defined bythe National Cancer Institute Common TerminologyCriteria CTCAE ver The efficacy endpoints werethe ORR disease control rate DCR time to responseTTR duration of response DOR progression freesurvival PFS and OS Efficacy was determined by an investigator according to RECIST v11 guidelinesExploratory endpoints were to evaluate the correlationof tumor mutation burden TMB with clinical efficacyTumor mutation burden analysisThe tumor biopsies and blood samples were collected atbaseline DNA sequences were extracted from biopsiesof tumors with matched blood samples and submittedfor next generation sequencing using a designed gene panel Genecast Beijing China TMB was determined by analysis of the quantity of somatic mutationsper megabase Mb Median TMB was used as a cutoffto define a tumor as highTMB HTMB and lowTMBLTMBStatistical analysisAll patients who received at least one study treatmentwere included in the safety and efficacy analyses AEswere coded following the Medical Dictionary for Regulatory Activity and tabulated by system an class andpreferred terms Causality between AEs and the studytreatment was assessed by the investigator ORR was calculated as the proportion of patients who had achieved acomplete response CR or partial response PR and the CIs were evaluated by the binomial distributionDCR was calculated as the proportion of patients whoobtained PR CR and stable disease SD and data arepresented with the CIs Median DOR TTR PFSOS and the PFS and OS rates at and months weredetermined using the KaplanMeier methodology Fisher™s test was used to compare the ORRs between patientswith HTMB and LTMBResultsFrom Dec to Oct patients werescreened and were enrolled in the GGEJ adenocarcinoma cohort Fig The median interval between initial diagnosis and screening was days range “Most patients had metastatic disease status and had ECOG scores of Table The TNMstage summary is shown in Table and the staging ofeach patient in Supplementary Table At data cutoff on May the median followuptime was months range “ The median treatment duration was months range “ Allpatients received more than cycles of treatment with 0cJiang BMC Cancer Page of Fig Flowchart of the studythe median doses of received sintilimab being range“SafetyAll of the patients reported at least one treatmentrelated adverse event TRAE and the most commonTRAE was platelet count decreased n Grade or treatmentrelated AEs TRAEs occurredin patients the most common also being aplatelet count decreased n Table NoTRAE was fatal and patient discontinued the treatment due to treatmentrelated Grade hepatic functionabnormal Sintilimabrelated AEs occurred in patients Grade “ sintilimabrelated AEs occurred in patients the most common being platelet countdecreased n Supplementary Table Chemotherapyrelated AEs were found in all patientsGrade “ chemotherapyinduced AEs were found in patients the most common being platelet countdecreased n Supplementary Table Fivepatientsadverseevents platelet count decreased n abnormal hepatic function n hypothyroidism n pneumonitis n and autoimmune colitis n reported treatmentrelated seriousEfficacyAll patients experienced a decrease in the sum oftheir target lesions Fig 2a and in the majority the lesions kept smaller than at baseline Fig 2b The medianTTR was months CI “ and the medianDOR was months CI “ According tothe best tumor response following RECIST guidelines patients reached a PR CI “ and patients obtained a confirmedobjective response ie by two continuous PRs at intervalsof weeks In addition patients had SD and DCR was CI “ Table One patient achieved a CR after the primary analysisby May This patient began the study treatmenton October and completed cycles of treatment before CRThe median PFS time was months CI “ and the month PFS rate was Median OSwas not reached and the 6month and 12month OSrates were and respectively Fig 2c dTumor mutation burdenValid results were obtained from patients The median TMB value was Mb The ORR was CI “ in patients with HTMBand CI “ in patients with LTMB No significant difference in clinical responseswere found between HTMB and LTMB patientsP Fig 2eDiscussionIn the present study the results from the GGEJ adenocarcinoma cohort in a Phase Ib study demonstratedmanageable safety and favorable antitumor activity ofsintilimab combined with a CapeOx regimen as firstlinetreatment for unresectable advanced metastatic GGEJadenocarcinoma 0cJiang BMC Cancer Page of Table Demographics and disease characteristicsTable Treatmentrelated adverse events TRAEsAge median range in yearsAll patients N to Gender n MaleFemaleECOG PS n Time since initial diagnosismedian range in daysDisease status n Locally advancedMetastaticLocation of the primary tumor n UpperMiddleLowerTNM staging n T3T4TxM0M1N1N2N3NxHistology n Poorly differentiated adenocarcinomaModerately differentiated adenocarcinomaUnknown differentiated adenocarcinoma “ ECOG Eastern Cooperative Oncology Group T tumor N node M metastasisIn terms of safety the incidence and severity of TRAEswith sintilimab and CapeOx were generally consistentwith those of known toxic effects of conventional chemotherapy [“] and previously reported side effects ofother antiPD1 antibody combined with chemotherapyregimens [ ] Platelet count white blood cell countand neutrophil count decreases were most commonly andmostly grade to reported TRAEs and are expected AEsassociated with CapeOx [“] Only patient reporteddiscontinuation of investigational drug application due toa TRAE abnormal hepatic function No treatmentrelated death occurred in this study and in general theaddition of sintilimab to CapeOx showed a manageablesafety profile and did not bear extra safety risksAll TRAEs nPlatelet count decreasedWhite blood cell count decreasedNeutrophil count decreasedHypothyroidismRashAlanine aminotransferase increasedAspartate aminotransferase increasedAnemiaHepatic function abnormalVomitingNauseaHyperchlorhydriaThyroid function test abnormalHypokalemiaHypesthesiaPyrexiaProteinuriaγglutamyl transferase increasedDiarrheaAutoimmune colitisAll graden Grade “n Pneumonitis Listed are any grade TRAE found in ‰¥ patients and all grade “ TRAEs In the present study after treatment with sintilimabplus CapeOx patients with unresectable GGEJ adenocarcinoma obtained an ORR of CI “ which is higher than that of conventional firstline chemotherapy For GGEJ adenocarcinoma firstline treatment mainly involves platinumbased chemotherapy and fluoropyrimidine[] The ORR ofcapecitabinebased or oxaliplatinbased therapies fGEJ adenocarcinoma was about “ [ ]The ORR for antiPD1 antibodies with a chemotherapy regimen were variableIn the KEYNOTE059study the ORR was CI “ forpembrolizumab plus cisplatin5fluorouracilm 5FUas firstline treatmentIn the KEYNOTE062study ORRs were and in patients with a ‰¥ and ‰¥ combined positive score CPS respectivelyafter they received pembrolizumab plus cisplatin5FUor capecitabine regimen as firstline therapy [] InATTRACTION04 the ORR for nivolumab with S1oxaliplatin was CI “ and theORR for nivolumab with CapeOx was CI“the ORR wasIn another study[][] 0cJiang BMC Cancer Page of Fig Evaluation of efficacy and tumor responses a Maximum change in tumor size from baseline Seventeen of patients obtained PR basedon the percentage changes of the sum of the maximum diameter of the tumor lesion range ˆ’ to ˆ’ b the change of lesiondiameters over time from baseline each line represents the changes in one patient c PFS KaplanMeier curve d OS KaplanMeier curve e theobjective response rate in low and high TMB groups OS overall survival PFS progressionfree survival PR partial response SD stable diseaseTMB tumor mutation burdenreported to be for an antiPD1 antibody toripalimab plus CapeOx treatment []Sintilimab plus CapeOx also showed favorable longterm efficacy Median PFS was months CI “ and the 6month PFS rate was Median OS wasnot reached and the 6month and month OS rateswere and respectively which was higherthan for conventional treatments with a median PFS of months CI “ for capecitabinecisplatinregimen [] and a median OS of months CI“epirubicinoxaliplatincapecitabineregimen [] The median PFS times for antiPD1foran 0cJiang BMC Cancer Page of Table Efficacy evaluation of sintilimabEfficacy evaluationCRnSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1288502007251zAdditional file Table S1 TNM stages of each patient Table S2Sintilimab related adverse events Table S3 Chemotherapyrelated adverse events Appendix Inclusion and exclusion criteriaPRSDPDORR CI CI “ CI “DCR CICI confidence interval CR complete response DCR disease control rate ORRoverall response rate PD progressive disease PR partial response SDstable diseaseAbbreviationsAEs Adverse events CapeOx Oxaliplatincapecitabine CPS Combinedpositive score CR Complete response CT Computed tomographyDCR Disease control rate DOR Duration of response ECOGPS EasternTumor Collaborative Group Performance Status G Gastric GC Gastric cancerGEJ Gastroesophageal junction HER2 Human epidermal growth factorreceptor2 HTMB High tumor mutation burden LTMB Low tumormutation burden Mb Megabase MRI Magnetic resonance imagingNGS Nextgeneration sequencing ORR Objective response rate OS Overallsurvival PD Progressive disease PD1 Programmed death1 PDL1 Proteinprogrammed deathligand PFS Progressionfree survival PR Partialresponse SD Stable disease TMB Tumor mutation burden TNM Tumornodes and metastases TRAEs Treatmentrelated AEs TTR Time to responseUICC Union for International Cancer ControlAcknowledgementsWe thank the patients and their families and the participating study teamsfor making this study possible and Yuan Fang Innovent Biologics IncSuzhou China for assistance with drafting the manuscriptAuthors™ contributionsHPJ NX LS and HZ were responsible for the design of the study HPJ YLZJQ CM XX NL CX HW LST and LS were responsible for acquisition of datafurthermore NX LS HPJ SYW and DLZ were in charge of analysis andinterpretation of data HPJ drafted the manuscript NX LS HZ SYW and DLZrevised and commented the draft BP conducted the biomarker analysis Allauthors read and approved the final manuscriptFundingThe study was sponsored by Innovent Biologics Inc and cofunded by EliLilly and company The study was also supported by the National Health andFamily Planning Commission Research Fund Zhejiang Provincial Medicaland Health major Science and Technology Plan Project Grant No KWJZJ and the Public Welfare Technology Application Research Project ofZhejiang Province Grant No LGF20E030004 Innovent Biologics Inc involved in the design of the study and collection analysis and interpretationof data and in writing the manuscript All remaining funding bodies had norole in the design of the study and collection analysis and interpretation ofdata and in writing the manuscriptAvailability of data and materialsThe datasets generated andor analyzed during the current study are notpublicly available since the new drug is being submitted to the NationalMedical Products Administration for approval but are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe trial protocol was approved by the Institutional Review Boards andEthical Committee of The First Hospital Affiliated to Zhejiang UniversitySchool of Medicine and Beijing Cancer Hospital and the study was carriedout strictly following the declaration of Helsinki principles all participatingpatients signed consent forms before taking part in the trial The trialnational registration number is NCT02937116Consent for publicationNot applicableCompeting interestsHui Zhou Shuyan Wang Donglei Zhu Bo Peng are the staff of InnoventBiologics Inc Suzhou China Suzhou China Lin Shen is the associate editorof BMC Cancer All remaining authors declare that they have no competinginterestsantibodies with a chemotherapy regimen were variableranging from to months a finding which mightbe associated with different populations and diseasestatus [“]Nextgeneration sequencing NGS hasresearcherenabled to perform target capture sequencing which hasbeen proposed as a reliable technique to identifymutated driver genes and for the estimation of TMBsIts use has led to the detection of actionable alterationsin various cancer related genes [] Regarding highTMB and the efficacy of PD1 treatments inconsistentresults have been reported in previous studies Wang suggested that TMB might be associatedwith better efficacy for PD1 monotherapy [] whereasMishima [] did not find a significant relationshipbetween TMB and the response of gastric cancers toPD1 therapy [] The latter data is in accordance withour finding that after treatment with sintilimab in combination with CapeOx no significant difference in theclinical responses was found between HTMB and LTMB patients However using the median TMB as acutoff is difficult to extrapolate to the real world clinicand bias due to the small sample size could not beexcluded in the present study In addition it has beennoted that up to now there is no uniform standard forHTMB [] and further investigations are urgentlyrequiredConclusionsOur results strongly indicate that sintilimab combined withCapeOx is an option for the firstline treatment of patientswith advanced or metastatic GGEJ adenocarcinoma However the sample size was small and it was a singlearmstudy without a comparator The large scale doubleblinded and randomized Phase III clinical trial ORIENT16for previously untreated advanced GGEJ adenocarcinomapatients is being conducted to evaluate the efficacy andsafety of sintilimab combined with CapeOx vs CapeOxalone ClinicalTrialsgov Identifier NCT03745170 0cJiang BMC Cancer Page of Author details1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou China 2Department of Surgical Oncology The First Affiliated Hospital Schoolof Medicine Zhejiang University Hangzhou China 3Department of MedicalScience and Strategy Oncology Innovent Biologics Inc Suzhou China4Department of Translational Medicine Innovent Biologics Inc SuzhouChina 5Department of Medical Oncology Beijing Cancer Hospital BeijingChinaReceived March 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 “Chen W Zheng R Baade PD Zhang S Zeng H Bray F Cancer statisticsin China CA Cancer J Clin “Bickenbach K Strong VE Comparisons of gastric Cancer treatments east vsWest J Gastric Cancer “Lui FH Tuan B Swenson SL Wong RJ Ethnic disparities in gastric cancerincidence and survival in the USA an updated analysis of SEERdata Dig Dis Sci “Ye XS Yu C Aggarwal A Reinhard C Genomic alterations and molecularsubtypes of gastric cancers in Asians Chin J Cancer Van Cutsem E Moiseyenko VM Tjulandin S Majlis A Constenla M Boni C Phase III study of docetaxel and cisplatin plus fluorouracil comparedwith cisplatin and fluorouracil as firstline therapy for advanced gastriccancer a report of the V325 study group J Clin Oncol “Shah MA Janjigian YY Stoller R Shibata S Kemeny M Krishnamurthi S et alRandomized multicenter phase II study of modified Docetaxel Cisplatinand fluorouracil DCF versus DCF plus growth factor support in patientswith metastatic gastric adenocarcinoma a study of the US gastric Cancerconsortium J Clin Oncol “AlBatran SE Hartmann JT Probst S Schmalenberg H Hollerbach SHofheinz R Phase III trial in metastatic gastroesophagealadenocarcinoma with fluorouracil leucovorin plus either oxaliplatin orcisplatin a study of the Arbeitsgemeinschaft Internistische Onkologie J ClinOncol “Kang YK Kang WK Shin DB Chen J Xiong J Wang J Capecitabinecisplatin versus 5fluorouracilcisplatin as firstline therapy in patients withadvanced gastric cancer a randomised phase III noninferiority trial AnnOncol “ Bang YJ Van Cutsem E Feyereislova A Chung HC Shen L Sawaki A et alTrastuzumab in combination with chemotherapy versus chemotherapyalone for treatment of HER2positive advanced gastric or gastrooesophageal junction cancer ToGA a phase label randomisedcontrolled trial Lancet “ Wei SC Duffy CR Allison JP Fundamental mechanisms of immunecheckpoint blockade therapy Cancer Discov “ Park YJ Kuen DS Chung Y Future prospects of immune checkpointblockade in cancer from response prediction to overcoming resistance ExpMol Med MarinAcevedo JA Dholaria B Soyano AE Knutson KL Chumsri S Lou YNext generation of immune checkpoint therapy in cancer newdevelopments and challenges J Hematol Oncol Lowther DE Goods BA Lucca LE Lerner BA Raddassi K van Dijk D et alPD1 marks dysfunctional regulatory T cells in malignant gliomas JCIInsight 201615e85935 Dong H Strome SE Salomao DR Tamura H Hirano F Flies DB Tumorassociated B7H1 promotes Tcell apoptosis a potential mechanism ofimmune evasion Nat Med “ Muro K Chung HC Shankaran V Geva R Catenacci D Gupta S et alPembrolizumab for patients with PDL1positive advanced gastric cancerKEYNOTE012 a multicentre label phase 1b trial Lancet Oncol “Fuchs CS Doi T Jang RW Muro K Satoh T Machado M Safety andefficacy of Pembrolizumab Monotherapy in patients with previously treatedadvanced gastric and Gastroesophageal junction Cancer phase clinicalKEYNOTE059 trial JAMA Oncol 201845e180013Kang YK Boku N Satoh T Ryu MH Chao Y Kato K Nivolumab inpatients with advanced gastric or gastrooesophageal junction cancerrefractory to or intolerant of at least two previous chemotherapy regimensONO453812 ATTRACTION2 a randomised doubleblind placebocontrolled phase trial Lancet “Song M Chen X Wang L Zhang Y Future of antiPD1PDL1 applicationscombinations with other therapeutic regimens Chin J Cancer Res Gandhi L RodriguezAbreu D Gadgeel S Esteban E Felip E De Angelis F Pembrolizumab plus chemotherapy in metastatic nonsmallcell lungCancer N Engl J Med “ PazAres L Luft A Vicente D Tafreshi A Gumus M Mazieres J et alPembrolizumab plus chemotherapy for squamous nonsmallcell lungCancer N Engl J Med “ Yan Y Kumar AB Finnes H Markovic SN Park S Dronca RS Combiningimmune checkpoint inhibitors with conventional Cancer therapy FrontImmunol Wang J Fei K Jing H Wu Z Wu W Zhou S Durable blockade of PD1signaling links preclinical efficacy of sintilimab to its clinical benefit MAbs“ Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th ed Oxford Wiley National Comprehensive Cancer Network Guidelines for Gastric CancerVersion p Cunningham D Starling N Rao S Iveson T Nicolson M Coxon F et alCapecitabine and oxaliplatin for advanced esophagogastric cancer N Engl JMed “Lordick F Kang YK Chung HC Salman P Oh SC Bodoky G et alCapecitabine and cisplatin with or without cetuximab for patients withpreviously untreated advanced gastric cancer EXPAND a randomised label phase trial Lancet Oncol “ Waddell T Chau I Cunningham D Gonzalez D Okines AF Okines C et alEpirubicin oxaliplatin and capecitabine with or without panitumumab forpatients with previously untreated advanced oesophagogastric cancer REAL3a randomised label phase trial Lancet Oncol “ Bang YJ Kang YK Catenacci DV Muro K Fuchs CS Geva R et alPembrolizumab alone or in combination with chemotherapy as firstlinetherapy for patients with advanced gastric or gastroesophageal junctionadenocarcinoma results from the phase II nonrandomized KEYNOTE059study Gastric Cancer “ Boku N Ryu MH Kato K Chung HC Minashi K Lee KW Safety andefficacy of nivolumab in combination with S1capecitabine plus oxaliplatinin patients with previously untreated unresectable advanced or recurrentgastricgastroesophageal junction cancer interim results of a randomizedphase II trial ATTRACTION4 Ann Oncol “Tabernero J Cutsem EV Bang YJ Fuchs CS Wyrwicz L Lee KW et alPembrolizumab with or without chemotherapy versus chemotherapy inadvanced GGEJ adenocarcinoma the phase keynote062 study J ClinOncol 20193718supplLBA4007 Wang F Wei XL Wang FH Xu N Shen L Dai GH Safety efficacy andtumor mutational burden as a biomarker of overall survival benefit inchemorefractory gastric cancer treated with toripalimab a PD1 antibody inphase IbII clinical trial NCT02915432 Ann Oncol “ Cai H Jing C Chang X Ding D Han T Yang J Mutational landscape ofgastric cancer and clinical application of genomic profiling based on targetnextgeneration sequencing J Transl Med Mishima S Kawazoe A Nakamura Y Sasaki A Kotani D Kuboki Y et alClinicopathological and molecular features of responders to nivolumab forpatients with advanced gastric cancer J Immunother Cancer Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Mukonal is an active member of carbazole alkaloids isolated from Murraya koenigii It has been shown to possess remarkable biological and pharmacological activities including anticancer activity Therefore the aim of current investigation was to explore antibreast cancer activity of mukonal and to explore the underlying mechanism Results indicate that mukonal has potential to induce antiproliferative effects against MDAMB231 and SKBR3 cells with an IC50 of µM No significant toxicity of mukonal was observed in case of normal breast cells The antiproliferative effects of mukonal were found to proceed via apoptosis which was further supported by increased cleavage of PARP and caspase3 and reduced expression of Bcl2 Mukonal induced autophagic cells death in breast cancer cells as was evidenced by formation of autophagosomes and enhanced expressions of Beclin1 LC3BI and LC3BII proteins In vivo examination of antibreast cancer property of mukonal indicated that it could potentially reduce tumor weight and volume in xenografted mice models In mukonal has a remarkable potential of inhibiting breast cancer via induction of apoptosis and autophagy Mukonal also inhibited xenografted tumors models in a dosedependent manner Therefore mukonal may prove lead molecule in breast cancer drug discovery and treatment provided further investigations are recommendedKeywords Breast cancer Alkaloids Carbazole alkaloids Mukonal Autophagy ApoptosisIntroductionBreast cancer is the most frequent and devastating disease prevailing in females worldwide Alone in the year of more than a0million deaths were recorded globally due to this lethal malignancy in women Sun et a0 al Every year approximately of all cancers over a0million diagnosed in women are breast cancer United States registered over of all cancers in women were due breast cancer in the year of Siegel et a0al Metastatic nature of breast cancer leads to easy transfer of disease to distant places in the body wherein it develops Correspondence wangcqy2020163comDepartment of Thyroid Breast Surgery Gong An County People™s Hospital Gong An Jingzhou Hubei Chinaindividually like brain lung liver and bones Early detection of the disease may lead with better overall survival chances and prognosis Screening of breast cancer is widely performed by mammography which has been proved fruitful in lowering the mortality rate effectually Several risk factors have been linked to enhance the possibility of breast cancer development like that of genetic mutations family history estrogen levels aging sex and poor lifestyle Majeed et a0al Primary development of breast cancer takes place from ductal hyperproliferation and then maturing into metastatic or benign tumors Thus far significant advances have been achieved in theoretical and clinical investigations of breast cancer The current strategies for effective management of breast cancer incorporate biologicalprevention chemoprevention 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 0cWang a0et a0al AMB Expr Page of and screening Smith and Henderson Besides recent advances and effective management breast cancer remains major cause of cancer related deaths in females of age “ a0 years Therefore there is a pressing need for novel and capable chemopreventive drugs that can assists us with better results in overcoming this disastrous malignancy Since time immemorial natural products have revealed health promoting effects in human beings Natural products are mostly the secondary metabolites synthesized by the plants to survive and adapt to harsh environmental conditions Williams et a0 al Alkaloids are a major class of naturally occurring secondary metabolites in plants with huge medicinal and biological properties including antidiabetic analgesic antiinflammatory antihypertensive antimalarial and anticancer Mukonal molecule is an active member of carbazole alkaloids found in Murraya koenigii Bhattacharyya and Chakraborty This molecule has shown significant antioxidant antimicrobial and anticancer activity Samanta et a0al Mukonal has been reported to show anticancer activities against different human cancer cell lines in a0vitro including laryngeal AMCHN8 cancer cells and nasopharyngeal CNE1 carcinoma cells It has shown a remarkable potential of autophagy initiation apoptosis induction modification of mitochondrial membrane potential cell cycle arrest blocking of MEKERK and PI3KAKT signalling pathways Li et a0al Guo et a0al Therefore current investigation was designed to unveil the antibreast cancer potential of mukonal along with its effects of autophagy and apoptosis inductionMaterials and a0methodsReagents chemicals and a0cell culturesMukonal with of purity by HPLC and other chemicals were bought from SigmaAldrich Darmstadt Germany unless otherwise mentioned Breast cancer cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and normal breast cell line MB157 were procured from Type Culture Collection of Chinese Academy of Sciences Shanghai China All cell cultures were grown in RPMI1640 media maintaining fetal bovine serum Thermo Fisher Scientific Inc Waltham United States and penicillin G a0Uml and streptomycin a0µgml as suitable antibiotics Allinclusive cell cultures were placed and maintained within a humid environment of CO2 concentration and a temperature of a0°CDetermination of a0cellular proliferationThe cellular proliferation of MDAMB231 CAMA MDAMB436 and SKBR3 and a normal breast MB157 cell line were estimated after mukonal exposure by 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT assay In brief both cancer and noncancer cells were placed with a concentration of — cells per well of 96well plates and precultured for a0h in a humid environment of CO2 concentration and a temperature of a0 °C Thereafter each well plate was supplemented with different mukonal doses viz and a0µM and left untouched on incubation for a0h Mukonal treated cancer and noncancer breast cells were washed twice with phosphate buffered saline PBS prior to staining with a0µl of MTT stock solution of concentration a0 mgml for a0 h The formazan crystals then evolved were dissolved with dimethyl sulphoxide followed by colorimetric analysis Finally absorbance was taken at a0nm to determine the optical density using microplate spectrophotometer BioRad Laboratories Inc Hercules United States Experiments for individual mukonal concentrations were repeated thriceColony formation assayMDAMB231 and SKBR3 were plated onto 6well plates with cells per well After a0h of preculturing the cells treatment with variant mukonal doses of and a0µM was instigated Thereafter cancer breast cells were left on incubation for days devoid of any physical and chemical disturbance After days of incubation cell colonies were stained with crystal violet Finally MDAMB231 and SKBR3 cell colonies were totaled under a light microscope OLYMPUS Japan and only colonies with cells were considered for countingAnnexin VPI staining assayAnnexin VPI dual staining assay was performed to monitor and quantify apoptosis in mukonal treated cancer MDAMB231 and SKBR3 breast cells These cancer cells were placed onto 6well plates and subjected to variant mukonal doses viz and a0µM for a0h After that cells were washed in PBS fixed in of formaldehyde and yet again washed in PBS Finally these cells were stained with annexin VPI dual staining solution and eventually analyzed through flow cytometryTransmission electron microscopyMukonal treated MDAMB231 and SKBR3 cells at variant doses and a0µM were subjected to electron microscopy for autophagy assessment Mukonal treated cancer cells were fixed in the solution of glutaraldehyde bearing a0m of sodium cacodylate Afterwards post fixation of treated cells was carried out by of osmium tetroxide followed by moisture removal using alcohol Then cells were prepared for implantation over Epon for sectioning and finally investigated under Zeiss CEM electron microscope Oberkochen Germany 0cWang a0et a0al AMB Expr Page of Assessment of a0protein expressions by a0western blottingThe expressions levels of apoptosis and autophagy allied proteins were evaluated by western blotting Mukonal treated and a0 µM and untreated controls tumerous breast cells MDAMB231 and SKBR3 were lysed with RIPA buffer for protein collection Each lysate was subjected to bicinchoninic acid assay for quantification of proteins and a0µg of proteins from each sample were run on SDSPAGE Thereafter proteins were transferred to PVDF membranes and these membranes were blocked with nonfat milk at room temperature for a0h blocked membranes were subjected to suitable primary antibodies of dilution anticaspase3 antiPARP antiBAX antiBcl2 antiLC3BI antiLC3BII and antiBeclin1 Santa Cruz Biotechnology Inc Dallas United States overnight at a0°C Afterwards secondary antibody treatment was instigated with dilutions of horse radish peroxidaseconjugated antirabbit secondary antibody Santa Cruz Biotechnology Inc Dallas United States for a0h at room temperature Finally the protein bands were visualized using ECL Advanced Western Blot Detection kit GE Healthcare Life Sciences SwedenXenograft studyXenograft studies were carried out by strictly obeying the ethical guidelines permitted by animal ethics committee Gong An County People™s Hospital Gong An Jingzhou Hubei China Immunodeficient nude mice of 6weeks weighing “ a0 g were placed in sterile steel cages These cages were placed in an environment of a0 h cycle of lightdark relative humidity of and a moderate temperature of °C These mice were given subcutaneous injections of SKBR3 cells — on their left flank As the tumor became superficially apparent treatment with mukonal was instigated by intraperitoneal insertion with of DMS dissolved mukonal and diluted normal saline at doses of and a0mgkg body weight This treatment procedure was repeated each second of a week and only of DMSO was injected to the control mice The tumor volume and weight was monitored after every week and the procedure lasted for weeks The mice were then sacrificed for this noble cause by inhaling of deep anesthesia with isoflurane The tumor volume was determined using the following formula V W — W — L2 where ˜V™ is the volume ˜W™ is the width and ˜L™ is the length of the tumor The study was approved by the animal ethics committee of Gong An County People™s Hospital Gong An Jingzhou Hubei China under approval number GACPH022019Statistical analysisStatistical analysis of data collected by execution of each individual experiment in triplicates was performed via analysis of variance ANOVA and followed by Tukey™s posthoc test Entire data were represented as mean ± SE standard error considering p as statistically significantResultsMukonal inhibited proliferation of a0breast cancer cellsThe proliferation rate of four different cancer breast cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and a normal breast MB157 cell line was determined using MTT assay after treatment with variant Mukonal a0µM Results revealed that mukonal significantly retarded the proliferation of these cancer breast cells with an IC50 value ranging from a0 µM to a0µM Table a0 Higher efficiency with lower IC50 value was recorded in case of MDAMB231 and SKBR3 cells Fig a0 1a Therefore rest of the studies was carried on these two cell lines The viability in case of MDAMB231 cells reduced from to almost Fig a01b and that of SKBR3 cells from to nearly with enhanced doses of mukonal from to a0µM Fig a01c The mukonal induced no significant cytotoxicity against normal MB157 breast cells and a high IC50 of a0µM was obtained Fig a0 1d Enhanced cytotoxicity was observed in case of normal MB157 cells at higher mukonal doses Clonogenic assay was used to monitor the impact of mukonal treatment over colony generation propensity of MDAMB231 and SKBR3 cells On treatment of MDAMB231 and SKBR3 cells with mukonal remarkable suppression of their colonies was observed in comparison to controls The numbers of MDAMB231 colonies left over were nearly and those of SKBR3 cells were nearly after days long treatment at a0µM of mukonal concentration Fig a0 Therefore mukonal induced remarkable toxicity against MDAMB231 and SKBR3 cells in comparison to MB157 cells which indicates specificity in anticancer activity of mukonalTable Effects of a0 Mukonal on a0 the a0 viability of a0 breast cancer cells as a0 depicted by a0 MTT assay and a0 presented as a0 IC50S noBreast cancer cell linesIC50 µMMDAMB231CAMA1MDAMB436SKBR3MB157The experiments were performed in triplicate 0cWang a0et a0al AMB Expr Page of Fig Mukonal inhibits the growth of breast cancer cells a Chemical structure of mukonal molecule b The viability of SKBR3 breast tumor cells after being subjected varying mukonal concentration as indicated SKBR3 cells were exposed to mukonal for h and then subsequently stained with MTT solution for calorimetric analysis c The viability of MDAMB231 breast tumor cells after being subjected varying mukonal concentration as indicated d The viability of normal MB157 breast cells after being subjected varying mukonal concentration as indicated All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureFig Clonogenic analysis of MDAMB231 and SKBR3 cells after being exposed to indicated mukonal doses MDAMB231 and SKBR3 breast tumor cell lines were treated with mukonal left untouched for days and stained with crystal violet to calculate the number of colonies generated provided considering colonies with number of cells for calculation All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Mukonal induced apoptosis in a0breast cancer cellsThe annexin VPI assay was employed to determine the percentage of apoptosis in MDAMB231 and SKBR3 cells Results indicated that mukonal could potentially exhibit proapoptotic effects against both MDAMB231 as well as SKBR3 cells in a dosereliant fashion In comparison to control group the number of early apoptotic late apoptotic and necrotic cell percentage enhanced significantly in treated groups The percentage of apoptotic cells was raised to about in case of treated SKBR3 cells Fig a03a The apoptotic cell percentage of MDAMB231 cells at controls was but it enhanced to nearly at a0 µM of mukonal concentration Fig a03b Further western blotting analysis indicated that Mukonal enhanced the cleavage of caspase3 and PARP levels Moreover the expression of Bcl2 decreased and Bax increased with increase in the dosage of mukonal in case of SKBR3 cells Fig a03c The expressions of cleaved caspase3 and cleaved PARP enhanced significantly and Bcl2 PARP and Bax decreased with enhancing doses of mukonal in case of MDAMB231 cells Fig a03d Therefore the results from annexin VPI staining and western blotting analysis indicated that antiproliferative effects of mukonal could be mediated via its apoptosis inducing propensityMukonal induced autophagy in a0breast cancer cellsThe impact of mukonal exposure on cellular autophagy in MDAMB231 and SKBR3 cells was analyzed through transmission electron microscopy and western blotting assay Results indicated that mukonal potentially induce proautophagic effects in both of these cancer breast cell lines In comparison to control groups autophagosomes are clearly visible in both mukonal treated SKBR3 Fig a04a and MDAMB231 Fig a04b cells After exposure to variant doses “ a0µM of mukonal MDAMB231 and SKBR3 cell lines showed enhanced expression levels of Beclin1 LC3BI and LC3BII proteins Which indicated molecular features of autophagic cell death in mukonal treated MDAMB231 and SKBR3 cancer cells Fig a04c d Therefore it may be concluded that mukonal exhibits antiproliferative effects mediated via its autophagy inducing potentialIn vivo inhibition of a0tumor growth by a0mukonalNude mice xenografts were used to determine in a0 vivo anticancer effects of mukonal The results showed that SKBR3 tumor growth was remarkably retarded by mukonal administration in comparison to control group At the end of 6weeks the average tumor volume and weight in untreated control group was substantially advanced than mukonal treated groups Moreover the in a0 vivo anticancer effects of mukonal were dose and timedependent manner Fig a05a bDiscussionDespite significant advancements in cancer research management it still remains the most prevalently diagnosed cancer and cause of death in women globally Better approaches are needed to understand and recognize this disease at molecular levels Due to timely diagnoses of the disease North America has registered over 5year survival rate among the breast cancer patients DeSantis et a0al High frequency of occurrence late diagnosis and disastrous side effects of present day chemopreventives creates an emergency for novel therapeutic drugs that can deliver better results with higher efficacy Waks and Winer Collignon et a0 al Mukonal is a carbazole alkaloid and has revealed to possess remarkable pharmacological and biological propensities It has been reported to induce anticancer effects against different human cancer cell lines Li et a0al Guo et a0al Therefore the current study was undertaken due to accumulative evidences suggesting that mukonal has a great propensity to act as an anticancer agent The results revealed that mukonal decreased the proliferation rate of five variant breast cancer cell lines but remarkable results with IC50 of a0µM was recorded against breast cancer MDAMB231 and SKBR3 cancer cell lines Mukonal showed a minuscule toxicity against normal breast MB157 cell line indicating some specificity of inducing toxicity against cancerous breast cell lines Mukonal induced potential inhibition of colony generation by MDAMB231 and SKBR3 cells Further chemopreventive drugs target several cellular processes to induce cytotoxicity against cancer cells Apoptosis has been a focal target of chemopreventives and is often termed as typeI PCD programmed cell death Khursheed et a0 al Apoptosis remains dormant in normal cells but in case of injury malfunction aging and macromolecule accumulation in cells it plays a vital role of elimination Bonofiglio et a0al Herein mukonal induced apoptotic cell death in MDAMB231 and SKBR3 cell lines in a dosereliant fashion as suggested by annexin VFITC assay Similar results have been reported previously wherein mukonal induced apoptosis in laryngeal cancer cells Mukonal stimulated apoptosis was further supported by enhanced expression levels of cleaved PARP cleaved caspase3 and Bax and retarded expression levels of caspase3 PARP and Bcl2 proteins in both the cancerous cell lines after mukonal exposure Autophagy is another process that remains conserved in multicellular anisms activated under stressful conditions like starvation Levy et a0 al Autophagy is often termed as typeII PCD and 0cWang a0et a0al AMB Expr Page of Fig a Flow cytometric analysis of mukonal treated SKBR3 cells SKBR3 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically b Flow cytometric analysis of mukonal treated MDAMB231 cells MDAMB231 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically c Western blotting analysis was performed to assess the activity of apoptosis allied proteins in SKBR3 cells Results indicated that mukonal enhanced activity of proapoptosis proteins wherein blocking of antiapoptotic protein expression d Western blotting analysis was performed to assess the activity of apoptosis allied proteins in MDAMB231 cells Results indicating that mukonal enhanced activity of proapoptosis proteins in MDAMB231 cells wherein blocking of antiapoptotic protein expression All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Fig a TEM analysis of SKBR3 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture b TEM analysis of MDAMB231 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture c Western blotting results of SKBR3 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonal d Western blotting results of MDAMB231 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonalalso remains as focal target of chemopreventives PoilletPerez et a0al Autophagy plays a key role in the degradation of marcomolecules and damaged anelle This process is completely hallmarked by the formation of autophagosomes which on maturation turns into autolysosomes Garc­aPrat et a0 al Mukonal was observed to induce autophagic cell death in both MDAMB231 and SKBR3 cell lines Similar results have been reported previous wherein mukonal induced autophagic cell death in human nasopharyngeal cancer cells Both of these cancerous cell lines showed enhanced expressions levels of LC3BI LC3BII and Beclin1 proteins indicating autophagy initiation at molecular levelsThe in a0 vivo investigation of mukonal revealed that it noticeably retarded growth of MDAMB231 and SKBR3 breast tumor growth in comparison to untreated control group and no apparent toxicity was detected The tumor weight and volume in mice models were observed to decline comparative to increased doses of mukonalIn the results of this investigation revealed that mukonal could potentially induce antibreast cancer effects both in a0 vitro and in a0 vivo against MDAMB231 and SKBR3 cell lines The antibreast cancer effects of mukonal were observed to mediate through induction of autophagy and apoptosis These studies point towards the potential of Mukonal in the treatment of breast cancer However further in a0vitro and in a0vivo studies are required to fully establish it as a lead molecule in the development of breast cancer chemotherapy 0cWang a0et a0al AMB Expr Page of Received June Accepted July ReferencesBhattacharyya P Chakraborty A Mukonal a probable biogenetic intermediate of pyranocarbazole alkaloids from Murraya koenigii Phytochemistry “Bonofiglio D Giordano C De Amicis F Lanzino M Ando S Natural products as promising antitumoral agents in breast cancer mechanisms of action and molecular targets MiniRev Med Chem “Collignon J Lousberg L Schroeder H Jerusalem G Triplenegative breast cancer treatment challenges and solutions Breast Cancer Targets Therapy DeSantis CE Fedewa SA Goding Sauer A Kramer JL Smith RA Jemal A Breast cancer statistics convergence of incidence rates between black and white women CA Cancer J Clin “Garc­aPrat L Mart­nezVicente M Perdiguero E Ortet L Rodr­guezUbreva J Rebollo E RuizBonilla V Gutarra S Ballestar E Serrano AL Sandri M Autophagy maintains stemness by preventing senescence Nature “Guo Y Hao Y Guan G Ma S Zhu Z Guo F Bai J Mukonal inhibits cell proliferation alters mitochondrial membrane potential and induces apoptosis and autophagy in human CNE1 nasopharyngeal carcinoma cells Med Sci Mon Int Med J Exp Clin Res Khursheed A Rather MA Rashid R Plantbased natural compounds and herbal extracts as promising apoptotic agents their implications for cancer prevention and treatment Adv Biomed Pharma “Levy JM Towers CG Thorburn A Targeting autophagy in cancer Nat Rev Cancer Li L Huizhi L Binu W Xinxin D Longjun W Liping Y Yingying Z Anticancer activity of mukonal against human laryngeal cancer cells involves apoptosis cell cycle arrest and inhibition of PI3KAKT and MEKERK signalling pathways Med Sci Mon Int Med J Exp Clin Res Majeed W Aslam B Javed I Khaliq T Muhammad F Ali A Raza A Breast cancer major risk factors and recent developments in treatment Asian Pac J Cancer Prev “PoilletPerez L Xie X Zhan L Yang Y Sharp DW Hu ZS Su X Maganti A Jiang C Lu W Zheng H Autophagy maintains tumour growth through circulating arginine Nature “Samanta SK Kandimalla R Gogoi B Dutta KN Choudhury P Deb PK Devi R Pal BC Talukdar NC Phytochemical portfolio and anticancer activity of Murraya koenigii and its primary active component mahanine Pharmacol Res “Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “Smith G Henderson IC New treatments for breast cancer Semin Oncol “Sun YS Zhao Z Yang ZN Xu F Lu HJ Zhu ZY Shi W Jiang J Yao PP Zhu HP Risk factors and preventions of breast cancer Int J Biol Sci Waks AG Winer EP Breast cancer treatment a review JAMA “Williams DH Stone MJ Hauck PR Rahman SK Why are secondary metabolites natural products biosynthesized J Nat Prod “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsFig Mukonal inhibits MDAMB231 and SKBR3 tumor growth in vivo a Tumor volume and b tumor weight were measured at indicated time intervals and doses of mukonal molecule All the experiments for separate drug concentration were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureAcknowledgementsAll the author of this manuscript is thankful to Gong An County People™s Hospital Gong An Jingzhou Hubei China to conduct the presented protocolAuthors™ contributionsWW and ZJ designed the protocol of the study WW ZZ XZ LC and SB performed the experimental work and collect the data for presented study WW and ZJ involve in the statistical analysis ZJ supervised the work and drafted the manuscript although all author contributed for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateThe study was approved by the animal ethics committee by Gong An County People™s Hospital Gong An Jingzhou Hubei China under approval number 721GACPH022019Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0c'

Thyroid_Cancer

"Vitamin D is a fatsoluble vitamin vitamin D is essential to sustain health and it protects againstosteoporosis It is crucial to the human body™s physiology in terms of muscular movement and neurological signaltransmission and to the immune system in defense against invading pathogensCase presentation This was a case of a 26yearold Sudanese woman who presented with a 2year history ofanosmia recurrent nasal polyps back pain and chronic fatigue She was diagnosed as having a case of vitamin Ddeficiency and responded well to treatmentConclusion There is an association between vitamin D deficiency and recurrent allergic nasal conditionsKeywords Vitamin D deficiency Allergy Nasal polyps Backache Chronic fatigabilityBackgroundVitamin D is a fatsoluble vitamin it is naturally presentin some foods and as dietary supplements It is also produced endogenously through exposure to ultraviolet raysfrom sunlight Vitamin D obtained from sun exposurefood and supplements is biologically inert and mustundergo two hydroxylations in the body for activationTheand produces hydroxyvitamin D 25OHD also known as calcidiolThe second occurs in the kidney and forms the physiologically active 125dihydroxy vitamin D 125OH2D alsoknown as calcitriol []first occursin theliverVitamin D is found in cells throughout the body vitamin D is essential to sustain health and it protectsagainst osteoporosis It is crucial to the human body™sphysiology in terms of muscular movement and neurological signal transmission and to the immune system indefense against invading pathogens []Although there are different methods and criteria fordefining vitamin D levels the criteria Holick proposed Correspondence drmuhanadkamalhotmailcom1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum SudanFull list of author information is available at the end of the have been widely accepted In this proposal vitamin Ddeficiency is defined as blood level of less than ngmlinsufficiency of vitamin D is defined as blood levels ranging between and ngml and sufficiency if greaterthan or equal to ngml [] About one billion peopleglobally have vitamin D deficiency and of the population has vitamin D insufficiency The majority ofaffected people with vitamin D deficiency are the elderlyobese patients nursing home residents and hospitalizedpatients Vitamin D deficiency arises from multiplecauses including inadequate dietary intake and inadequate exposure to sunlight Certain malabsorption syndromes such as celiac disease short bowel syndromegastric bypass some medications and cystic fibrosis mayalso lead to vitamin D deficiency []Vitamin D deficiency is now more prevalent than everand should be screened in highrisk populations Manyconflicting studies now show an association betweenvitamin D deficiency and cancer cardiovascular diseasediabetes autoimmune diseases and neuropsychiatricdisorders [ ] 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 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of two functional endoscopic sinusCase presentationThis was a case of a 26yearold Sudanese woman married who has a 3yearold boy This woman presentedto our ear nose and throat ENT department complaining of anosmia for the past years She had a history ofsurgeriesFESSs for nasal polyps the first one was years agoand the second one was years prior to presentationShe complained of being highly sensitive to different irritants including dust weather change perfumes andpetsShe also stated that she attended more than three different physicians due to generalized fatigue and gettingtired easily after simple daily activity in addition to sleeping for more than hours a dayShe attended an orthopedic clinic for unspecified lower back pain that was notrelated to any type of trauma or physical activity a lumbosacral magnetic resonance imaging MRI was done andrevealed no abnormal findingsShe mentioned that she isknown to be anxious most of the time and aggressive toward simple reactions from her family members She hadno psychiatric history and was not using any medicationsShe was not known to be diabetic or hypertensive orto have any chronic illnesses she was not on any regularmedication She is a housewife of high socioeconomicstatus she is well educated graduated from dentalschool with a bachelor™s degree but currently notemployed She has never consumed tobacco or alcoholshe practiced regular cardio exercisesOn examinationshe looked healthy well not pale or jaundiced Herpulse rate was 74minute and her blood pressure was Her body mass index BMI was All systems examinations were normal except for bilateral nasalpolyps Complete blood count CBC renal function testREF electrolyte liver function test LFT thyroid function test TFT urine analysis general urine test antinuclear antibody ANA and rheumatoid factor RFwere all normal An imaging profile included lumbosacral MRI a computed tomography CT scan of her sinuses and electrocardiogram ECG which were normalexcept for bilateral nasal polyps and severe sinusitis thatlooked allergic to fungi in natureShe underwent FESSsurgery to remove the polyps and clean out her sinusesup to weeks after surgery she used nasal steroidsmometasone furoate two times a day but hersymptoms regarding anosmia were not improved MRIof her brain and a CT scan of her sinuses were done andboth revealed normal features A vitamin D deficiencywas suggested and the laboratory results revealed a lowvitamin D level of ngml Treatment with vitamin Dsupplement was prescribed at international unitsIU weekly for weeks and then IU maintenancedose daily she was advised to take food rich in vitaminD and get exposed to sunlight for minutes three timesa week after the loading dose of supplement She was atregular followup for months at rates of weekly for thefirst month every weeks for the second month andmonthly for the rest of the followup period At eachvisit she was assessed with clinical history and examination It was noticed that the symptoms of tirednesssleeping anosmia and back pain were dramatically improving during that period At the months followupher blood level of vitamin D was normal she describedher condition as free from all symptoms and shereturned back to normal physical activityDiscussion and sThis was a nonclassical case of vitamin D deficiency ofa 26yearold woman who presented with chronic anosmia and recurrent nasal polyps She was diagnosed ashaving a case of vitamin D deficiency and respondedwell to vitamin D replacement therapy This case correlated an association between decreased levels of vitaminD and recurrent nasal polyps that led in time to chronicanosmia as a result of chronic high sensitivity reactionstriggered by our patient™s autoimmune system The literature links chronic rhinosinusitis with nasal polypsCRSwNP with asthma and allergic rhinitis but the cellular and molecular mechanisms that contribute to theclinical symptoms are not fully understood Sinonasalepithelial cell barrier defectsincreased exposure topathogenic and colonized bacteria and dysregulation ofthe host immune system are all thought to play prominent roles in disease pathogenesis []Despite all the previous surgical and medical interventions over the past years our patient™s condition did notimprove and she still complained of anosmia A study revealed that this patient was experiencing excessive allergicreactions that led to recurrent nasal polyps It is wellknown that classical clinical effects of vitamin D deficiencyare bones and musculoskeletalrelated disorders severallines of evidence demonstrate the effects of vitamin D onproinflammatory cytokines regulatory T cells and immune responses with a conflicting interpretation of theeffects of vitamin D on allergic diseases []The working diagnosis was suggested in relation tosome musculoskeletal symptoms and chronic fatigue especially when the imaging profile for her lower back andall routine investigations were normal It has been suggested that clinicians should routinely test for hypovitaminosis D in patients with musculoskeletal symptomssuch as bone pain myalgias and generalized weaknesswhich might be misdiagnosed asfibromyalgia andchronic fatigue [] The most common causes of anosmia were assessed as well and they were negative theseincluded sinonasal diseases post infectious disorder andposttraumatic disorder and congenital defects and disorders caused by neurodegenerative disease [] 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of Authors™ contributionsMI analyzed and interpreted the findings of the case report and was themajor contributor in writing the manuscript ME reviewed the report andadded valuable comments All authors read and approved the finalmanuscriptFundingNoneAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from Albasar Institutional Review BoardConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum Sudan 2Preventive Medicine and EpidemiologyAlneelain University Khartoum SudanReceived March Accepted July Thus blood level for vitamin D was requested and theresults were of low D levelIn the past history of the previous nasal polyps surgeries our patient noted that there was no anosmia and hermain complaints were classic complaints of sinusitis including sneezing nasal blockage and headache Soonafter surgery her symptoms improved except for theallergyrelated symptoms despite usage of inhaled steroids spray She stated that at the last time the presentation was different since it was only anosmia indicatingthat there was significant inflammation that affected thesmell receptors around the olfactory epithelium Afterthe last nasal polyps and sinuses drainage surgery thesymptoms related to allergic reactions including chronicsneezing did not improve for up to weeks and she wasstill suffering from hyposmia although that was a fairpostoperative period for recoveryThe symptoms of anosmia and sneezing and othersystematic symptoms gradually started to improve aftervitamin D supplements indicating that the main reasonbehind her symptoms was vitamin D deficiency She wasfollowed up for up to months after establishment ofvitamin D supplements and at the last followup she hada normal sense of smell and she was free from backpain fatigue and allergyrelated symptomsThis was a nonclassical presentation as our patientwas young and she did not have alkaline phosphatasecalcium and phosphorus abnormalities [] that are expected in cases of vitamin D deficiencyThis case revealed an association between decreasedlevels of vitamin D and recurrent nasal polyps that ledto anosmia as a result of hypersensitive reactions produced by the body™s systemsAlthough vitamin D deficiency is prevalent measurement of serum 25OHD level is expensive and universalscreening is not supported However vitamin D testingmay benefit those at risk for severe deficiencyIt is highly recommended to consider vitamin D deficiency among all patients with unspecified symptoms orin cases of nondiagnosed disorder regardless of the presenting complaintIn there is an association between vitaminD deficiency and recurrent allergic nasal conditionsAbbreviationsCRSwNP Chronic rhinosinusitis with nasal polyps CT Computedtomography ECG Electrocardiogram ENT Ear nose and throatFESS Functional endoscopic sinus surgery BMI Body mass indexCBC Complete blood count RFT Renal function test LFT Liver function testTFT Thyroid function test ANA Antinuclear antibody RF Rheumatoid factorIU International unitReferencesNational Institutes of Health Vitamin D fact sheet for health professionals httpsodsodnihgovfactsheetsVitaminDHealthProfessionalen2Accessed Apr National Institutes for Health NIH Vitamin D fact sheet for consumers httpsodsodnihgovfactsheetsVitaminDConsumer Accessed Dec Kuriacose R Olive KE Vitamin D insufficiencydeficiency managementSouth Med J “ httpsdoi101097SMJSizar O Khare S Givler A Vitamin D deficiency Treasure Island Stat PearlsPublishing httpswwwncbinlmnihgovbooksNBK532266 PMID Accessed Dec Wlliam B Fatme A Meis M Targeted 25hydroxyvitamin D concentrationmeasurements and vitamin D3 supplementation can have important patientand public health benefits Eur J Clin Nutr “ httpsdoi101038s4143002005640Hanmin W Weiwen C Dongqing L Xiaoe Y Xiaode Z Nancy O et alVitamin D and chronic diseases Aging Dis “ httpsdoi1014336AD20161021 Whitney W Ropert P Robert C Chronic rhinosinusitis with nasal polyps JAllergy Clin Immunol Pract “ httpsdoi101016jjaipThacher TD Clarke BL Vitamin D insufficiency Mayo Clin Proc “ httpsdoi104065mcp20100567Kennel KA Drake MT Hurley DL Vitamin D deficiency in adults when totest and how to treat Mayo Clin Proc “ httpsdoi104065mcp20100138Sanne B Elbrich M Duncan B Antje W Veronika S Joel D et al Anosmia aclinical review Chem Senses “ httpsdoi101093chemsebjx025Shikino K Ikusaka M Yamashita T Vitamin Ddeficient osteomalacia due toexcessive selfrestrictions for atopic dermatitis BMJ Case Rep httpsdoi101136bcr2014204558AcknowledgementsNot applicablePublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

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

"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha People™s Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha People™s Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into 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 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [“] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chen™s group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[“]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in œheadtohead alignment nextto the DNA [] and thus form stable œladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [“] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[“]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[“]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ “]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcoma“associated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGAS“STING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem cell“like CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING

Thyroid_Cancer

American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ‰¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was “ mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research “ Fu 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 the work 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 For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ‰¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundary“lower hyoid margin lower boundary“superior sternal fossa upper margin of the unknown artery and external“lateral margin of the carotid sheath and lower boundary“anterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ‰¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients™ complete medical records were available All of the surgeons had years™ experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the œat least  principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using χ2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients™ age range was “ years old with a median of ± years The maximum diameter range of the cancer lesion was “ mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ‰¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients™ gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimoto™s thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimoto™s thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1“ According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about “ On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8“ These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japan™s Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ‰¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ‰¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 œ American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimoto™s thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter‰¤ mm mm x ‰¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMχ2PYes n No n “““Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβˆ’ˆ’SEWaldPOR CI OR““““““““Abbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ‰¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The χ2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions χ2 P nodule morphology χ2 P and extra thyroidal expansion of cancer focus χ2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimoto™s thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol “ doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg “ doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence “ Cancer Causes Control “ doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid “ doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA “ United States “ doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid “ doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery “ discussion “ doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin “ doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery “ doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid “ doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid “ doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary “ doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol “ doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid “ doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res “ doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid “ doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c'

Thyroid_Cancer

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

Thyroid_Cancer

"deltalike protein DLL3 may be related with prognosis inpatients with small cell lung cancer SCLC However this finding remains controversial in small cell lung cancerThis metaanalysis was systematically performed to evaluate the prognostic value of DLL3 in SCLCMethods The PubMed EMBASE and Web of Science databases were retrieved to collect the eligible referencesThrough Stata software we pooled hazard ratios HR with confidence intervals CI by using random orfixedeffects models to evaluate the association between DLL3 and SCLC survival resultsResults A total of interrelated studies including patients were qualified After we removed study theremaining studies including patients were pooled to testify that high expression of DLL3 was an inferiorprognostic for patients with SCLC in Asian populations HR CI I2 p Thepooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations RR CI I2 p But the expression of DLL3 was not correlated with sex RR CI I2 p smoking history RR CI I2 p and tumour stage RR CI I2 p Conclusions Our metaanalysis confirms that in Asian populations high expression of DLL3 was a potential poorprognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populationsKeywords Deltalike protein DLL3 Prognostic Small cell lung cancer SCLC MetaanalysisIntroductionLung cancer has the highest morbidity and mortality ofall malignant tumours which is one of the most common cancers worldwide [] The major histologic subtypes oflung cancerNSCLC and small cell lung cancer SCLC [] SCLC is an invasive highgrade malignancy withearly development and a bad prognosis Most patientswith SCLC are advanced with widespread metastasiswhen they are diagnosed and systemic chemotherapy islung cancer are nonsmall cell Correspondence ligaofenghl126comDepartment of Thoracic Surgery The Third Affiliated Hospital of KunmingMedical University Yunnan Cancer Hospital Kunming Yunnan Chinathe most effective therapy [] Few therapeutic methodsare available after SCLC relapse and the prognosis is especially poor Therapeutic treatments for patients withSCLC have essentially remained unchanged in recentyears [] Thus further research into the mechanism ofSCLC and the exploration of new therapeutic targets forSCLC is imperativeDeltalike protein DLL3 is a transmembrane protein that promotes the development of neuroendocrinetumours through its reciprocity with the Notch pathway[] It is expressed in cancer tissues in approximately of patients with SCLC and other neuroendocrineis not expressed in nonneuroendocrinecancer but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen World Journal of Surgical Oncology Page of tumours or normal tissues [] Most studies have shownthat DLL3 is a latent treatment target point for SCLC[“] Recently many researches initiate to focus on theprognostic value of DLL3 in SCLC Some of the researchers demonstrated that DLL3 had no correlationwith prognosis in SCLC patients [ ] but other investigators showed that DLL3 was associated with thesurvival of patients with SCLC [] So previous studiesof the prognostic value of DLL3 in SCLC remain controversial we performed this systematic metaanalysis forresolving this controversialPublished s discussing the prognostic value ofDLL3 in SCLC were systematically reviewed in ourmetaanalysis We aimed to include all correlationalstudies to assess the prognostic value of DLL3 andattempted to identify an accurate biomarker to guideprognosis and treatment for SCLC in the futureMethodsSearch strategyThe PRISMA guidelines were followed for our metaanalysis see Additional file [] The literatures werecollected through retrieving the PubMed EMBASE andWeb of Science databases from the initial date to February The search strategy was œDLL3 OR deltalike protein  all fields AND œnonsmall cell lung cancer OR NSCLC OR small cell lung cancer OR SCLC ORLung Adenocarcinoma OR Lung Squamous cell carcinoma OR lung cancer OR lung tumour OR lung neoplasm OR lung carcinoma all fields There was nolimitation on the publication status All eligible studieswere retrieved and the headlines and s of all thereference lists of the reviews or studies were independently filtrated through three authors based on the criteria Differences between two authors were resolved bythe third author™s opinionsInclusion criteriaResearches were included by following the standards studies reported the prognostic value of DLL3 in SCLC studies were published as original s studiesreported the data of HR and CI or provided survivalcurve and studies in which the prognostic value wasinvestigated by survival analysis with overall survivalOS diseasefree survival DFS progressionfree survivalRFS ordiseasespecific survival DSS We excluded the animalresearches and other review literaturesPFS relapserecurrencefree survivalData extractionTwo researchers independently extracted the data included the author country edition year sample capacitypatients™ sex smoking history distant metastasis tumourstage histologic subtype biomarkers scoring methodsboth univariateand multivariatecutoffs value and the data of HR and CI We extracted the multivariate analysis outcome if one study includedanalysisoutcomes When we could not extract the data of HRand CI directly in the but the survival curveswere reported we extracted and digitized the survivalcurves by operating the Engauge Digitizer softwarehttpdigitizersourcefenet and we estimated thedata of HR and CI by the Excel programme files asexploited by Tierney [] When the data of HRand survival curves were not reported we contacted thecorresponding authors of eligible s by email to obtain the original data these s were excluded ifthere was no responseAssessment of study qualityTwo researchers independently used the Quality InPrognosis Studies QUIPS tool to assess risk of bias ofall the publications [] According to the criteria every was evaluated as low risk moderate risk or highrisk by following six different areas study participationstudy attrition prognostic factor measurement outcomemeasurement study confounding and statistical analysisand reporting [] Differences were settled throughdiscussionStatistical analysisTwo authors independently extracted the HR and CI from the original s The HR was used to describe the high DLL3 expression versus low DLL3 expression We took the reciprocal of the HR if the sshowed the low DLL3 expression vs high DLL3 expression We observed that high expression of DLL3 portended a worse prognosis when HR and that HR indicated preferable prognosis For the analysis results p was considered statistically significant Statisticalheterogeneity was assessed by calculating the I2 statistic[] The presence of heterogeneity was indicated whenI2 and then a randomeffects model was appliedto poolthe results [] a fixedeffects model wasemployed to pool the results when I2 [] Weperformed further subgroup or sensitivity analysis toanalyse the heterogeneity The publication bias was estimated by Begg™s and Egger™s tests when p indicates no publication bias [] Through Stata software we performed this metaanalysis and acquiredthe forest plotsResultsSearch results and study characteristicsUsing the searching strategy described above a total of original documents were identified from the databases with approximately studies remaining after excluding duplicates Afterandscreening thetitles 0cChen World Journal of Surgical Oncology Page of s of the publications publications werenot related to evaluating the prognosis role of DLL3in SCLC Finally we were left with eligible studiesafter screening the full text among which swere included in our final analysis Fig [“]Ten s were excluded for the following reasonsseven s were review researches and commentaries one did notreport hazard ratios andKaplanMeier curves and s were s anddid not report relevant outcomes The eligible s™ characteristics are shown in Table All theses came out from the initial date to February and the patients were diagnosed as SCLC byhistopathology Among these studies all studies investigated DLL3 byinparaffinembedded tissueimmunohistochemistryIHCQuality assessment of the included studiesWe performed quality evaluations of the s following the QUIPS tool and two authors independently evaluated allthe literature Differences were resolved bydiscussion After screening all included s we foundthat there were no researches that reported study attritionand there were no standardization of cutoffs value forevaluating DLL3 expression And then studies were evaluated as low risk were evaluated as moderate risk and study was evaluated as high risk Table This outcomeindicated that most of the studies we included were of amedium or high qualityPrognostic value of DLL3 in SCLCAs shown in Fig eligible s were pooled foranalysing the prognostic value of DLL3 in SCLC TheFig The flow diagram of studies selection 0cChen World Journal of Surgical Oncology Page of Table Study characteristics of the eligible sStudyYear Country No ofsamplesSexmalefemaleSmokinghistorynonyesYan [] ChinaDistantmetastasisnegativepositiveXie [] USATanaka [] JapanRegzedmaa [] ChinaHuang [] ChinaFuruta [] JapanNANANANANANANATumourstageIIIIIIIVHistologicsubtypeMethod Outcomes BiomarkersCutoffvalueNANASCLCSCLCSCLCSCLCSCLCSCLCIHCIHCIHCIHCIHCIHCOSOSOSOSDLL3TTF1DLL3DLL3DLL3CTLA4MSTNscore ‰¥ ‰¥ ‰¥ score ‰¥ OSPFSDLL3OSDLL3ASCL1score ‰¥ ‰¥ SCLC small cell lung cancer IHC immunohistochemistry OS overall survival PFS progressionfree survival DLL3 deltalike protein CTLA4 cytotoxic T lymphocyteassociated protein MSTN mesothelin ASCL1 achaetescute homologue1 NA not applicableresults showed that high expression of DLL3 indicatedno prognostic value in patients with SCLC HR CI I2 p Howeverprominent heterogeneity existed in the pooled results sowe conducted further subgroup analysis Among the sixstudies three s were from China two were fromJapan and one was from America We divided the studies into the Asian group and the American group forsubgroup analysis As shown in Additional file FigA1we found that the heterogeneity was in the Asiangroup and we believed that the main cause of the heterogeneity was the study from American So we decidedto eliminate the study from American and only analysedthe remained studies After we removed the Americanstudy the outcomes indicated that high DLL3 expressionwas a poor prognosis marker in SCLC HR CI I2 p Fig Then wedivided the studies into two groups according to thesize of the sample and the final outcomes indicated thathigh DLL3 expression was a poor prognosis marker inthe group with sample size greater than HR CI p Fig But there was nosignificant relationship between DLL3 expression andprognosis in the group with sample size less than HR CI I2 p Fig Begg™s test p and Egger™s test p there was nodemonstratedresultsthatpublication bias in our metaanalysis Fig Finallysensitivity analysis showed that allthe studies werestable see Additional file FigA6The clinical characteristics of patients with DLL3expression in SCLCThe pooled results showed that the expression of DLL3was not correlated with sex RR CI I2 p Table Additional file FigA2 smoking history RR CI I2 p Table Additional file FigA3 and tumour stage RR CI I2 p Table Additional file FigA4 But DLL3 was highly expressed in patients withdistant metastasis RR CI I2 p Table Additional file FigA5DiscussionMany studies have shown that DLL3 played a significantprognosis value in patients with cancer For examplehigh DLL3 expression was associated with shorter OSand PFS in small cell bladder cancer [] High DLL3expression was associated with bad OS and usuallyexpressed in older patients and advanced stage in endometrial cancer [] Xie reported that high DLL3expression predicted a better OS in patients with SCLC[] and other studiesforeboded that high DLL3Table Quality assessment of included studiesStudyStudyparticipation—‹Yan []Xie []Tanaka []Regzedmaa []Huang []—‹—‹—‹Furuta []œ—Ž indicates low risk of bias œStudyattrition——————Prognostic factormeasurement—‹—‹—‹—‹—‹—‹Outcomemeasurement—‹—‹—‹—‹—‹Study confounding—‹—‹—‹—Statistical analysisand reporting—‹—‹—‹—‹—‹—‹Total riskof biasLowModerateLowLowHighModerate moderate risk of bias and œ— high risk of bias 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in SCLC patients DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneityexpression was an inferior prognostic marker for SCLC[ ] Thus the results of different studies remainedcontroversial We included a total of studies with patients with SCLC to assess the prognostic value ofDLL3 in SCLC by pooling the data of HR and CIFirst we performed this metaanalysis using studiesBut heterogeneity was observed in our pooled resultsThrough further subgroup analysis we found that astudy from the American was the main cause of heterogeneity As we removed the American study we foundFig Forest plots of prognostic value of DLL3 in Asia patients with SCLCDLL3 deltalike protein USA the United States of America HR hazardratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneity 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in different studies with different sample sizes DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneitythat high expression of DLL3 is a marker of poor prognosis in SCLC But we also noticed that the results ofthe American study were contrary to our conclusionsthey reported that high expression of DLL3 is a markerof good prognosis in SCLC One interpretation of thisresult was that DLL3 expression varies between differentpopulations The American study indicated that the highexpression of DLL3 was a marker of good prognosis[] and there was no significant correlation betweenDLL3 expression and prognosis in several Japan studies[ ] while the high expression of DLL3 is a markerof poor prognosis in China studies [ ] These results showed that the expression of DLL3 might be different in different populations However only one studyFig Funnel plot of DLL3 present on overall survival DLL3 deltalike protein HR hazard ratios 0cChen World Journal of Surgical Oncology Page of Table Summary of the correlation between DLL3 expression and the clinical characteristics of patients with SCLCClinical characteristicsSex malefemaleRR CI No of studiesI2 p Smoking historynonyesDistant metastasis negativepositive P valueTumour stage IIIIIIIVSCLC small cell lung cancer RR relative ratios CI confidence intervals DLL3 deltalike protein I2 percentage heterogeneity between studies p testfor heterogeneity researched the correlation with DLL3 expression andprognosis in SCLC outside of Asia so the results needto be treated with caution In addition more studieswere suggested for the future to further verify the existence of such differencesOur other explanation of this result was that the expression of DLL3 was the same in different populationsAmong the included studies the sample size of studieswas less than including the America and Japanstudies Therefore we speculated that insufficient sample size might cause bias in the results We conducted asubgroup analysis according to the sample size and theresults showed that the high expression of DLL3 in thestudies with large sample sizes N ‰¥ was associatedwith poor prognosis while the pooled results of thestudies with low sample sizes N showed no significant correlation between the expression of DLL3 andprognosis Therefore the sample size may be one of thereasons for the differences in DLL3 expression in eachstudy Moreover immunohistochemistry was used to detect DLL3 expression in all of the studies Immunohistochemical staining is a semiquantitative method and isevaluated with great subjectivity [] Different antibodies and different cutoff values for DLL3 expressionwere employed in all the included studies and thus couldalso be another cause of the differences in results TheAmerican study also explained their different results byclaiming that many of the other studies were performedusing mRNA expression instead of protein expression orused different cutoffs value of DLL3 expression []Researches havereported that DLL3 is highlyexpressed in SCLC [ ] which suggested that DLL3might promote the development of SCLC Therefore wealso discussed the correlation between DLL3 expressionand the clinical characteristics of patients with SCLCThe pooled results showed that DLL3 expression had nosignificant correlation with patients™ sex smoking statusand stage while DLL3 often highly expressed in metastasis patients of SCLC Our survival analysis outcomeswere consistent with this result which suggested thathigh expression of DLL3 might be one of the factorscontributing to poor prognosis in patients with advancedmetastatic SCLC in Asia However only a few studiesreported the correlation between the expression of DLL3and clinical characteristics and the methods and cutoffs values used to detect the DLL3 expression in eachstudy were not uniform Therefore more reliable studiesare needed to further verify our outcomes Our resultssuggest that it is valuable to further investigate the correlation between DLL3 and the clinical characteristics ofpatients with SCLCOur metaanalysis is the first to focus on the prognostic value of DLL3 in SCLC The significance of thismetaanalysis lies in providing a basic direction and evidence for further research into the mechanism of DLL3in SCLC For SCLC Notch1 over expression could induce G1 cell cycle arrest [] Previous studies reportedthat DLL3 downregulated the Notch receptor expression thereby the Notch signalling pathway was inhibitedwithin the cell [] Therefore high expression of DLL3can promote the development of SCLC by inhibiting theNotch signalling pathway Studies also have shown thatthe high expression of DLL3 may reduce the sensitivityof chemotherapy drugs [] These studies have demonstrated that DLL3 may be associated with the prognosisof SCLC and also consistent with our metaanalysis results Thus studies of the corresponding targeted drugsof DLL3 can effectively inhibit the expression of DLL3and thus improve the survival of SCLC Rovalpituzumabtesirine RovaT is a new antibodydrug conjugate directed against DLL3 in SCLC [] A phase I trial foundthat patients with high DLL3 expression in SCLCshowed a better response to RovaT than those with alow DLL3 expression [] However disappointingly thephase III TAHOE trial has been stopped because theRovaT group showed a worse OS compared to the control group [] But more clinical trials are recruitingparticipator to investigate RovaT as maintenance therapy in advanced stage SCLC The lack of progress withthis drug does not prevent us from making a breakthrough with other similar drugs Some researches foundthat the intratumoural and intertumoural distributionof DLL3 protein in SCLC is homogeneous [] supporting the conclusion that biopsy specimens are a reliablesource for DLL3 evaluation for targeted therapy Inaddition most studies have demonstrated that DLL3 is 0cChen World Journal of Surgical Oncology Page of highly expressed in SCLC while it is not or is lessexpressed in other types of lung cancer and normal tissues [] Therefore the expression of DLL3 can be detected by biopsy as an indicator for diagnosis predictingtherapeutic efficacy and monitoring recurrence or metastasis of SCLC in the futureAlthough our study fully explains the prognostic valueof DLL3 in SCLC our analysis still has several limitations Firstlarge heterogeneity was observed in thepooled results This is explained by the observation thatthe evaluation criteria for the expression of DLL3 areparticularly mixed and there are no international standards for cutoffs values to determine the expression ofDLL3 Thus the scoring methods and cutoffs values ofDLL3 should be unified to strengthen our conclusionsOtherwise the detection method of DLL3 in most studies is mainly immunohistochemistry at present which isa semiquantitative subjective and inaccurate detectionmethod Different studies show different prognosticvalues of DLL3 Therefore we need other more precisedetection methods to evaluate the expression of DLL3 inSCLC in the futureSecond the therapy method is also a key limitationThe current studies only focus on tissue specimens frompatients with SCLC after surgery or biopsy and fewstudies reported the treatment methods in their researches However the prognostic value of DLL3 may liein the therapeutic method Therefore every study shouldpay attention to the impact of patienttreatmentmethods on prognosis in the futureThird some of the original studies did not report thedata of HR and CI The HR and CI resultswere measured from survival curves an evaluationmethod with certain deviation and subjectivity whichmight influence the authenticity of the resultsConclusionIn summary our metaanalysis confirmed that high expression of DLL3 was a potential poor prognostic biomarker for SCLC in Asian populations moreover DLL3expression was correlated with advanced metastasisSCLC in Asian populations However the relationshipbetween DLL3 expression and the prognostic or clinicalcharacteristics of patients with SCLC in European andAmerican populations need to be further verified Thusdetecting the expression of DLL3 in tumour tissue willbe helpful to guide therapy in Asian patients of SCLCFor our research other highquality studies especiallyfrom European and American countries are required toconfirm our findings about the prognosis value of DLL3in SCLC in the future In view of the limitations of ouranalysisthe conclusions should be interpreted withcautionSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12957020020045Additional file The PRISMA checklistAdditional file FigA1 Forest plots of prognostic value of DLL3 inSCLC SCLCsmall cell lung cancer HRhazard ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA2 Forestplots of the correlation between DLL3 expression and sex of patientswith SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA3 Forestplots of the correlation between DLL3 expression and smoking history ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA4 Forestplots of the correlation between DLL3 expression and tumour stage ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA5 Forestplots of the correlation between DLL3 expression and metastasis ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA6Sensitivity analysis of all the studiesAbbreviationsCI Confidence intervals DLL3 Deltalike protein DFS Diseasefree survivalDSS Diseasespecific survival HR Hazard ratios IHC ImmunohistochemistryOS Overall survival PFS Progressionfree survival QUIPS Quality In PrognosisStudies RFS Relapserecurrencefree survival RR Risk ratio SCLC Small celllung cancerAcknowledgementsNot applicableAuthors™ contributionsAll authors contributed to the study conception and design Materialpreparation data collection and analysis were performed by Benchao ChenHeng Li and Chao Liu The first draft of the manuscript was written byBenchao Chen and all authors commented on previous versions of themanuscript All authors read and approved the final manuscriptAuthors™ informationNot applicableFundingThis work was supported by the National Natural Science Foundation ofChina [No ]Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived July Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwide 0cChen World Journal of Surgical Oncology Page of Regzedmaa O Li Y Li Y Zhang H Wang J Gong H Prevalence ofDLL3 CTLA4 and MSTN expression in patients with small cell lung cancerOnco Targets Ther “ httpsdoi102147OTTS216362 Huang J Cao D Sha J Zhu X Han S DLL3 is regulated by LIN28B and miR518d5p and regulates cell proliferation migration and chemotherapyresponse in advanced small cell lung cancer Biochem Biophys ResCommun “ httpsdoi101016jbbrc201904130Furuta M SakakibaraKonishi J Kikuchi H Yokouchi H Nishihara HMinemura H Analysis of DLL3 and ASCL1 in surgically resected smallcell lung cancer HOT1702 Oncologist 20192411e1172“ httpsdoi101634theoncologist20180676Koshkin VS Elson P MagiGalluzzi C McKenney J Smith KS Shadrach B Prognostic value of DLL3 expression and clinicopathologic features insmall cell bladder cancer SCBC J Clin Oncol Juan Wang Kaishuo Zhang Zi Liu Upregulated deltalike protein expression is a diagnostic and prognostic marker in endometrial cancer aretrospective study Medicine Baltimore 20189751e13442 httpsdoi101097MD0000000000013442 Matos LLD Stabenow E Tavares MR Ferraz AR Capelozzi VL Pinhal MADSImmunohistochemistry quantification by a digital computerassistedmethod compared to semiquantitative analysis Clinics “httpsdoi101590S180759322006000500008 Hann CL Mensztern D Dowlati A Burns TF Jotte RM Pennell NA et alA study of rovalpituzumab tesirine in frontline treatment of patients withDLL3 expressing extensive small cell lung cancer J Clin Oncol httpsdoi101200JCO20173515_supplTPS2598Furuta M Sakakibara JK Shoji T Takashima Y Kikuchi H Kikuchi E et alDLL3 regulates migration and invasion of small cell lung cancer Cancer Res httpsdoi10115815387445Am20183158Sriuranpong V Bes MW Ravi RK Arnold DR Nelkin BD Baylin SB et alNotch signaling induces cell cycle arrest in small cell lung cancer cells CancerRes httpsdoi1010970000282020010400000012 Deng SM Yan XC Liang L Wang L Liu Y Duan JL The Notch liganddeltalike promotes tumor growth and inhibits Notch signaling in lungcancer cells in mice Biochem Biophys Res Commun “httpsdoi101016jbbrc201612117Komarnitsky P Lee H Shah M Wong S Gulbranson S Dziubinski J et alRovalpituzumab tesirine vs topotecan in patients with advanced small celllung cancer following 1st line chemotherapy J Thorac Oncol S1974“ Udagawa H Akamatsu H Tanaka K Takeda M Kanda S Kirita K Phase Isafety and pharmacokinetics study of rovalpituzumab tesirine in Japanesepatients with advanced recurrent small cell lung cancer Lung Cancer “ httpsdoi101016jlungcan201907025Komarnitsky P Lee HJ Shah M Wong S Gulbranson S Dziubinski J Aphase trial of rovalpituzumab tesirine vs topotecan in patients withadvanced small cell lung cancer following frontline platinumbasedchemotherapy Ann Oncol 201728v542 httpsdoi101093annoncmdx386009 Odashiro P Tomarchio G Gagne A Orain M Desmeules P Joubert P DLL3expression in small cell lung carcinomas SCLC Mod Pathol 202033SUPPL“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsfor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Amini A Byers LA Welsh JW Komaki RU Progress in the management oflimitedstage small cell lung cancer Cancer httpsdoi101002cncr28505 Mead GM Arnold AM Green JA Williams CJ Whitehouse JM SMALLCELLLUNG CANCER[J] Cancer Chemother Pharmacol httpsdoi101016S0140673680907333Powell HA Tata LJ Baldwin DR Potter VA Stanley RA Khakwani A et alTreatment decisions and survival for people with smallcell lung cancer Br JCancer “ httpsdoi101038bjc2013812Huang RSP Holmes BF Powell C Marati RV Tyree D Admire B Deltalike protein prevalence in small cell lung cancer and DLL3 SP347 assaycharacteristics Arch Pathol Lab Med “ httpsdoi105858arpa20180497OA Mensztern D Besse B Greillier L SantanaDavila R Ready N Hann CL Efficacy and safety of rovalpituzumab tesirine in thirdline and beyondpatients with DLL3expressing relapsedrefractory smallcell lung cancerresults from the phase II TRINITY study Clin Cancer Res “ httpsdoi10115810780432CCR191133Isobe Y Sato K Nishinaga Y Takahashi K Taki S Yasui H Near infraredphotoimmunotherapy targeting DLL3 for small cell lung cancerEBioMedicine httpsdoi101016jebiom2020102632Rossi A Rovalpituzumab tesirine and DLL3 a new challenge for smallcelllung cancer Lancet Oncol “ httpsdoi101016s1470Rudin CM Pietanza MC Bauer TM Ready N Mensztern D Glisson BS Rovalpituzumab tesirine a DLL3targeted antibodydrug conjugate inrecurrent smallcell lung cancer a firstinhuman firstinclass labelphase study Lancet Oncol “ httpsdoi101016s1470204516305654Fu X Tian T Liu M Ruan Z Liang X Nan K The expression andprognostic roles of PDL1 PAPR1 and DLL3 in small cell lung cancer JThorac Oncol 20191410S1024“ httpsdoi101016jjtho2019082267Lim S Hong M Kim SP Chung SH P2 Prevalence of DLL3 Expressionand its prognostic role in extensive stage small cell lung cancer J ThoracOncol 20191410S820 httpsdoi101016jjtho2019081763 Poirier JT Targeting DLL3 in smallcell lung cancer with novel modalities JThorac Oncol 2020152S8 httpsdoi101016jjtho201912024Liberati A Altman DG Tetzlaff J Mulrow C Gøtzsche PC Ioannidis JPA et alThe PRISMA statement for reporting systematic reviews and metaanalysesof studies that evaluate health care interventions explanation andelaboration J Clin Epidemiol 20096210e1“e34 httpsdoi101016jjclinepi200906006Tierney JF Stewart LA Ghersi D Burdett S Sydes MR Practical methods forincorporating summary timetoevent data into metaanalysis Trials httpsdoi10118617456215816Jill A Hayden D van der Windt DA Cartwright JL Côté P Bombardier CAssessing bias in studies of prognostic factors Ann Int Med “httpsdoi10732600034819158420130219000009 Higgins TJP Measuring inconsistency in metaanalyses BMJ “ httpsdoi101136bmj3277414557 Dersimonian R Nan L Metaanalysis in clinical trials Control Clin Trials “ httpsdoi1010160197245686900462 Mengersen KL Tweedie RL Biggerstaff B The impact of method choice onmetaanalysis Aust N Z J Stat “ httpsdoi101111j1467842X1995tb00869x Ahmed I Sutton AJ Riley RD Assessment o

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

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

Thyroid_Cancer

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

Thyroid_Cancer

Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A case“controlstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction Hardy“Weinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 — ˆ’ oddsratio OR confidence interval CI “ however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI “ The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [“] Graves ophthalmopathyGO is one common anspecific complication affecting “ of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [“] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online 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 Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO [“]Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [“] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acase“control study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range “ y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbecco™s modified Eagle™s medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at —¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturer™s protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day “Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3ˆ’Cˆ’Mycˆ’NR3C1 pCMV3ˆ’Cˆ’Mycˆ’FOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturer™s protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturer™s protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNA“IP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM Tris“HCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydI“dC and glycerol] for min at —¦C in a final volume of μl FOXP3“ or NR3C1“nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean ˆ’ SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ [ˆ’] ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbSignificance of age were evaluated by t testP005P00010039a — 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the Hardy“Weinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI “ Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 — ˆ’ OR CI ˆ’ which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI ˆ’ Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG ˆ’0929bAbbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with significanceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from “ days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAGˆ’2495cGG — ˆ’ bˆ’0154bˆ’2467bˆ’Abbreviations CI confidence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using — or — contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with significanceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding affinities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearson™s correlation ˆ’ Spearman™s correlation ˆ’ Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearson™s correlation Spearman™s correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3C“E The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [“] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding affinities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 B“E Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GO”atherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via œDrug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE Hardy“Weinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves™ Disease N Engl J Med “ 101056NEJMra1510030 Brent GA Clinical practice Graves™ disease N Engl J Med “ 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves™ disease CMAJ Canadian Med Assoc J J de l™Assoc Med Canadienne “ 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 BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves™ disease Endocrinol Metab Clin North Am 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Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves™ andHashimoto™s diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab “ Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet “101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity “ 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics “101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves™ ophthalmopathy in a genderdependent manner Ophthalmology “101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves™ disease in Chinese patients Endocrine “ 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Graves™ophthalmopathy patients Ophthalmology “ 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves™ ophthalmopathy Hum Immunol “ 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves™ ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol “ Siegmund T Usadel KH Donner H Braun J Walfish PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves™ disease Thyroid Official J Am Thyroid Assoc “ 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with Graves™Ophthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem “ 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med “ 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi

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

generate a map of the copy number variations CNV segregating in a population of Murciano‘Granadina goats the most important dairy breed in Spain and to ascertain the main biologi‘cal functions of the genes that map to copy number variable regionsResults Using a dataset that comprised Murciano‘Granadina goats genotyped with the Goat SNP50 Bead‘Chip we were able to detect and autosomal CNV with the PennCNV and QuantiSNP software respec‘tively By applying the EnsembleCNV algorithm these CNV were assembled into CNV regions CNVR of which of the total CNVR count were consistently called by PennCNV and QuantiSNP and used in subsequent analyses In this set of CNVR we identified gain loss and gainloss events The total length of all the CNVR Mb represented of the goat autosomal genome Mb whereas their size ranged from kb to Mb with an average size of kb Functional annotation of the genes that overlapped with the CNVR revealed an enrichment of pathways related with olfactory transduction fold‘enrichment q‘value — ˆ’ ABC transporters fold‘enrichment q‘value — ˆ’ and bile secretion fold‘enrichment q‘value — ˆ’Conclusions A previous study reported that the average number of CNVR per goat breed was CNVR50 breeds which is much smaller than the number we found here CNVR We attribute this difference to the fact that the previous study included multiple caprine breeds that were represented by small to moderate numbers of individuals Given the low frequencies of CNV in our study the average frequency of CNV is such a design would probably underestimate the levels of the diversity of CNV at the within‘breed level We also observed that functions related with sensory perception metabolism and embryo development are overrepresented in the set of genes that overlapped with CNV and that these loci often belong to large multigene families with tens hundreds or thousands of paralogous members a feature that could favor the occurrence of duplications or deletions by non‘allelic homologous recombinationCorrespondence marcelamillsuabcat Centre for Research in Agricultural Genomics CRAG CSIC‘IRTA‘UAB‘UB Universitat Aut²noma de Barcelona Bellaterra SpainFull list of author information is available at the end of the BackgroundCopy number variations CNV encompass genomic deletions or duplications with sizes ranging from base pairs bp to several megabases Mb and which display polymorphisms in terms of copy number among individuals of a particular species [“] In livestock a broad array of phenotypes related with among others morphology [ ] pigmentation [“] sexual development 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 0cGuan a0et a0al Genet Sel Evol Page of [] and susceptibility to disease [] is caused by the segregation of CNV Genome scans to detect structural variations in cattle have revealed that CNV regions CNVR are often enriched in genes that are involved in immunity [“] metabolism [ ] embryo development [ ] and sensory perception [ ] There is evidence that the dNdS ratios of genes that map to taurine CNV are generally higher than those of genes that do not overlap with CNV which indicates that CNV genes probably evolve under reduced selective constraint [] The analysis of gene networks has also shown that genes that colocalize with duplications tend to have fewer interactions with other genes than loci that do not overlap with CNV reinforcing the idea that genes mapping to duplicated regions have fewer essential housekeeping functions than nonCNV genes and also have reduced pleiotropy []Although structural chromosomal variations can have strong effects on gene expression and phenotypic variability technical limitations and the moderate quality of genome assemblies have hampered CNV mapping in livestock [] Until recently this has been particularly true for goats In Fontanesi et a0al [] published the first caprine CNV map by identifying with the Bovine a0k aCGH array CNVR including and copy loss and gain variants respectively Later on resequencing the genome of individuals from several caprine breeds made it possible to identify CNV that overlap with pigmentation genes and to detect an association between increased ASIP copy number and light pigmentation [] The first worldwide survey of copy number variation in goats was performed within the Goat ADAPTmap Project httpwwwgoata daptm ap and involved the genomewide genotyping of goats from breeds [] This study resulted in the identification of CNVR among which several overlapped with genes that are functionally related with local adaptation such as coat color muscle development metabolic processes and embryonic development [] Moreover the patterns of the diversity of CNV differed according to geographic origin which indicates that they have been influenced by population history [] In another study on individuals from East African goat breeds Nandolo et a0al [] detected CNVR More recently Henkel et a0al [] demonstrated the existence of complex patterns of structural variation in the regions containing the caprine ASIP and KIT genes with potential causal effects on pigmentation In spite of these efforts the description of structural chromosomal variation in goats is still lagging behind that of other domestic species Most of the CNV surveys in goats have analyzed large populations that represent a mixture of different breeds each with a limited number of individuals [ ] thus making it difficult to assess the magnitude of the CNV diversity at the withinbreed level Our goal was to fill this gap by analyzing a population of individuals from a single Spanish breed MurcianoGranadina and to investigate the functional roles of genes that map to CNVR and compare these results with data obtained in composite goat populationsMethodsGenomic DNA extraction and a0high‘throughput genotypingBlood samples from MurcianoGranadina female goats from farms that are connected through the use of artificial insemination were collected in EDTA K3 coated vacuum tubes and stored at ˆ’ a0 °C before processing Genomic DNA was isolated by a modified saltingout procedure [] Four volumes of red cell lysis solution Tris“HCl a0 mmolL pH EDTA a0mmolL Tween were added to a0mL of whole blood and this mixture was centrifuged at —g Pelleted cells were resuspended in a0mL lysis buffer Tris“HCl a0 mmolL pH EDTA a0 mmolL SDS NaCl a0 mmolL plus µL proteinase K a0 mgmL The resulting mixture was incubated at a0°C for h followed by centrifugation at —g in the presence of a0mL of ammonium acetate a0molL The supernatant a0mL was mixed with a0mL of isopropanol which was subsequently centrifuged at —g for a0min The supernatant was removed and the DNA pellet was washed with a0 mL of ethanol After centrifuging at —g for a0min the DNA precipitate was dried at room temperature and resuspended in a0mL of TE buffer a0mmolL Tris pH a0mmolL EDTA pH Highthroughput genotyping of the MurcianoGranadina DNA samples was carried out with the Goat SNP50 BeadChip [] according to the manufacturer™s instructions Illumina Signal intensity ratios ie log R Ratio or LRR the total probe intensity of a SNP referred to a canonical set of normal controls [] and B allele frequencies or BAF relative quantity of one allele compared to the other one [] were exported for each single nucleotide polymorphism SNP with the GenomeStudio software Illumina https emeaillum inacom Then SNP coordinates were converted to the latest version of the goat reference genome ARS1 [] After filtering out unmapped and nonautosomal SNPs and those with a call rate lower than a set of SNPs remained for CNV mappingCopy number variant calling with a0PennCNV and a0QuantiSNPBased on their excellent performance in comparative studies we selected two software packages PennCNV v105 [] and QuantiSNP v2 [] to call CNV in the MurcianoGranadina population [ ] The PennCNV software [] detects CNV by applying the default 0cGuan a0et a0al Genet Sel Evol Page of parameters of the HiddenMarkov model Population frequencies of B alleles were compiled based on the BAF of each SNP in the population We used the œ“gcmodelfile option to adjust œgenomic waves [] The number of goat chromosomes was set with the œ“lastchr  instruction The QuantiSNP analysis [] assumes an objective Bayes hiddenMarkov model to improve the accuracy of segmental aneuploidy identification and mapping This CNV calling software was run under default parameters by modifying the œ“chr  option The CNV that were supported by less than three SNPs were removed from the filtered set used hereDefinition and a0functional annotation of a0copy number variant regionsWe used the EnsembleCNV algorithm beta version [] to assemble CNVR All CNV called by PennCNV andor QuantiSNP were combined to generate a set of initial CNVR by using the heuristic algorithm threshold of minimum overlap described in [] Subsequently CNVR boundaries were refined by considering the local correlation structure of the LRR values of the SNPs mapping to CNVR [] Then we reassigned the CNV calls that were initially obtained with PennCNV and QuantiSNP to each refined CNVR so that the final set of CNVR comprised only those that were simultaneously detected by both callers The resulting CNVR were matched to gene features that are annotated in the National Center for Biotechnology Information NCBI https wwwncbinlmnihgov by using BEDTools v2250 [] In addition we performed gene ontology GO enrichment and pathway analyses using the DAVID Bioinformatics Resources [ ] based on human and goat background gene sets The statistical significance was set to a qvalue ‰¤ Confirmation of a0copy number variant regions by a0quantitative real‘time PCRIn order to evaluate the rate of false positives in our experiment we conducted quantitative realtime PCR qPCR experiments to obtain an independent estimate of the copy number of four putative CNVR CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 Primers were designed with the Primer Express software Applied Biosystems to amplify specific regions of the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes see Additional file a0 Table a0 S1 As reference genes we used the melanocortin receptor MC1R and glucagon GCG genes see Additional file a0 Table a0S1 loci [ “] Quantitative PCR reactions contained a0ng genomic DNA µL — SybrSelect Master mix Applied Biosystems a0pmol of each forward and reverse primer and ultrapure water to a maximum final volume of µL Each sample was analyzed in triplicate in order to obtain averaged copy number estimates Reactions were loaded onto 384well plates and run in a QuantStudio a0K Flex RealTime PCR System instrument Applied Biosystems The specificity of the PCR reactions was evaluated with a melting curve analysis procedure and the efficiency was assessed with standard curves Thus relative copy number was inferred with the qbase software Biogazelle Ghent Belgium by using the ˆ’ΔΔCt approach [] Copy number values were calibrated by taking as a reference four samples which according to Goat SNP50 BeadChip data had two copies of the investigated genomic lociResultsDetection of a0copy number variation in a0Murciano‘Granadina goatsThe initial calling with PennCNV and QuantiSNP yielded and autosomal CNV respectively By using the EnsembleCNV tool [] we assigned these CNV into CNVR with refined boundaries of which of the total CNVR count were detected simultaneously by PennCNV and QuantiSNP The resulting CNVR included copy gain copy loss and copy gainloss variants Fig a0 and Table a0 and see Additional file a0 Table a0 S2 The total length of the CNVR covered a0Mb of the goat autosomal genome a0Mb whereas their individual size ranged from a0kb to a0Mb with an average of a0kb Fig a02a and Table a0 Moreover we found that of the CNVR showed minimum allele frequencies lower than with an average frequency of Fig a02b In addition CNVR with frequencies higher than were distributed over seven caprine chromosomes With a frequency of CNVR_1229_chr21 was the CNVR with the highest frequency in the whole dataset see Additional file a0 Table a0S2 By using the BEDTools v2250 program [] of the CNVR that we detected overlapped with unique CNVR published by Liu et a0al [] Fig a0 and see Additional file a0 Table a0S2 The CNVR that were detected in both studies are referred to as œshared CNVR whereas those that were identified in our study only are referred to as œnonshared CNVR Fig a0 Six of the ten œshared CNVR with frequencies higher than show positional concordance with six CNVR detected by Liu et a0al [] see Additional file a0 Table a0S2Functional annotation of a0the a0genes that a0are located in a0copy number variable regionsWithin the CNVR defined in our study we detected proteincoding genes according to the goat reference genome annotation ARS1 [] from the NCBI database see Additional file a0 Table a0S2 and Additional file a0 0cGuan a0et a0al Genet Sel Evol Page of Fig Genomic distribution of CNVR detected with the PennCNV and QuantiSNP software on the caprine autosomes Squares triangles and circles represent copy number gain loss and gainloss events respectively Red and black colors represent shared and non‘shared CNVR respectively Shared CNVR are those detected both in our study and in Liu et al [] while non‘shared CNVR are those identified only in our studyTable a0S3 In a survey of the diversity of CNV in goats with a worldwide distribution Liu et a0 al [] detected copy number variable genes of which were also identified in our study and are referred to as œshared copy number variable genes see Additional file a0 Table a0S3 Among the œshared copy number variable genes the ASIP and ADAMTS20 genes are particularly relevant they are involved in pigmentation [ “] and colocalize with selection signals detected in a worldwide sample of goats [] In addition we found that about of the annotated genes that colocalize with CNVR are olfactory receptors or olfactory receptorlike genes see Additional file a0 Table a0S3 Consistently the most significantly enriched pathway was œOlfactory transduction qvalue — ˆ’ Table a0 followed by œABC transporter qvalue — ˆ’ Table a0 A significant pathway related with immunity ie Fc epsilon RI signaling qvalue was also identified based on a human background gene set Table a0 Several overrepresented GO terms were related with embryonic skeletal system morphogenesis qvalue — ˆ’ and Gprotein coupled purinergic nucleotide receptor activity 0cGuan a0et a0al Genet Sel Evol Page of Table Main features of a0 copy number variation regions CNVR detected in a0 MurcianoGranadina goatsSummary statisticsTotalGainLossGainlossTotal length MbTotal number of CNVRNumber of CNVR kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR kb“ MbNumber of CNVR ‰¥ MbAverage number of SNPs per CNVRMinimum size of CNVR kbMaximum size of CNVR kbAverage CNVR size kbStandard deviation of CNVR size kb qvalue — ˆ’ Table a0 Interestingly the copy number variable genes were also enriched in pathways with metabolic significance such as prolactin signaling and insulin signaling as well as GO terms related with feeding behavior but none of these pathways reached the significance threshold qvalue ‰¤ after correction for multiple testing see Additional file a0 Table a0S4 Several of the pathways outlined in Additional file a0 Table a0S4 play important roles in immunity eg chemokine signaling B cell receptor signaling and T cell receptor signaling cancer eg endometrial cancer proteoglycans in cancer thyroid cancer as well as in oncogenic signaling eg Ras and ErbB signaling see Additional file a0 Table a0S4 but most of them are not significant after correction for multiple testingFig Histograms displaying the distribution of CNVR according to their size a and frequency b CNVR that were longer than kb were included in the ‘kb bin whereas those with frequencies above were grouped in the bin The histograms were drawn by using the ggplot2 package httpggplo t2tidyv erse implemented in R https wwwr‘proje ct 0cGuan a0et a0al Genet Sel Evol Page of Table Functional enrichment of a0genes colocalizing with a0CNVR detected in a0 MurcianoGranadina goatsBackground gene setCategory IDTermNumber of a0genesFold enrichment P valueq‘valueGoatGoatGoatHumanHumanHumanHumanHumanHumanHumanKEGGKEGGKEGGKEGGGOBPGOBPGOBPGOCCGOMFGOMFOlfactory transductionABC transportersBile secretionFc epsilon RI signaling pathwaychx04740chx02010chx04976hsa04664GO0009952 Anteriorposterior pattern specificationGO0048704 Embryonic skeletal system morphogenesisGO0035589 G‘protein coupled purinergic nucleotide receptor signaling pathwayGO0016020 MembraneGO0003677 DNA bindingGO0045028 G‘protein coupled purinergic nucleotide receptor activity126Eˆ’ 161Eˆ’333Eˆ’ 427Eˆ’446Eˆ’ 570Eˆ’140Eˆ’ 176Eˆ’936Eˆ’ 161Eˆ’713Eˆ’ 122Eˆ’418Eˆ’ 716Eˆ’145Eˆ’ 198Eˆ’110Eˆ’ 160Eˆ’424Eˆ’ 622Eˆ’KEGG Kyoto Encyclopedia of Genes and Genomes pathway GOMF gene ontology GO term related with molecular function GOBP GO term related with biological process GOCC GO term related with cellular componentValidation of a0four copy number variants by a0real‘time quantitative polymerase chain reactionIn order to confirm our results we selected four CNVR ie CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 that colocalized with the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes respectively the primers used to amplify these CNVR are listed in Additional file a0 Table a0 S1 As shown in Fig a0 the estimated copy numbers obtained by qPCR analysis of MurcianoGranadina goat samples were to copies relative to the calibrator ADAMTS20 to copies BST1 to copies NCKAP5 and to copies TNFAIP2 According to D™haene et a0 al [] copy number estimates between and most likely correspond to a normal copy number of whereas any number below or above these thresholds could represent a deletion or a duplication respectively Thus based on these values evidence of copy number variation was inferred for three of the four genes analyzed by qPCRFig Relative quantification of four copy number variation regions by real‘time quantitative polymerase chain reaction analysis a CNVR_371_chr5 ADAMTS20 b CNVR_506_chr6 BST1 c CNVR_160_chr2 NCKAP5 d CNVR_1229_chr21 TNFAIP2 The x and y axes represent sample ID and relative quantification of CNVR mean ± standard error with each sample analyzed in triplicate respectively As calibrator we used the average of four samples estimated to have two copies diploid status based on the Goat SNP50 BeadChip analysis 0cGuan a0et a0al Genet Sel Evol Page of DiscussionIn this work our aim was to characterize copy number variation in MurcianoGranadina goats a native Spanish breed used for milk production By genotyping MurcianoGranadina goats with a SNP array we were able to identify CNVR covering of the goat genome whereas Liu et a0 al [] identified CNVR that covered of the goat genome The latter higher percentage reported by Liu et a0al [] can be explained by the fact that they analyzed breeds with different geographical origins ie a composite population that is probably much more diverse than that used in our work Besides the pipeline that we used to identify CNVR is more stringent than that employed by Liu et a0al [] removing CNVR that were not consistently detected by PennCNV and QuantiSNP In the literature estimates of to for CNVR coverage in the human genome are reported [] Our results and those obtained by Liu et a0al [] are consistent with these valuesIndeed when Liu et a0 al [] calculated the CNVR length for each breed normalized by the goat genome size their results agreed well with our estimate of For instance this parameter reached values of in goats from Southeastern Africa and in goats from Northwestern Africa and Eastern Mediterranean whereas it was lowest for individuals from West Asia [] The number of CNV detected at the withinbreed level by Liu et a0 al [] was on average CNV per breed and ranged from to whereas the average number of CNVR was only per breed [] Since the number of detected CNVR is proportional to population size for most of the breeds investigated in [] the level of withinbreed CNV variation is probably underestimated In summary one important conclusion from our study is that the magnitude of CNV diversity at the withinbreed level is likely to be much larger than that previously reported in studies that analyzed multiple populations each represented by a small or moderate number of individualsMost of the CNVR that we report here ranged in size from to a0kb with a mean size of a0kb Similarly the average CNVR size reported by Liu et a0al [] was a0kb Both estimates are quite large and reflect that mediumdensity SNP arrays are not well suited to detect small CNVR in spite of their high abundance In cattle the average sizes of CNVR detected with the Illumina BovineHD Genotyping BeadChip a0K SNPs [] Illumina wholegenome sequencing and PacBio sequencing [] were and a0 kb respectively Another consistent feature of CNVR is that in general their frequencies are low or very low In our study approximately of the CNVR had frequencies lower than and the average frequency was Liu et a0al [] reported lower CNVR frequencies ranging from Alpine and Northern European goats to Northwestern African goats This decreased average CNVR frequency is not very significant and probably reflects differences in sampling size and the use of composite populations with multiple breeds each one with its specific CNVR frequenciesThe CNVR detected in our study covered proteincoding genes Pathway analyses reflected a substantial enrichment of genes that are involved in olfactory perception which is consistent with previous reports in cattle [ ] In this regard there is an important difference between our results and those by Liu et a0al [] Whereas in the study of Liu et a0al [] the term œsensory perception was underrepresented among the CNV genes fold enrichment in our work the terms œolfactory transduction fold enrichment and œGprotein coupled purinergic nucleotide receptor activity fold enrichment were overrepresented and many CNV genes were olfactory receptors The two terms mentioned before are closely related because a broad array of purinergic receptors are differentially expressed in the olfactory receptor neurons that modulate odor responsiveness [] Moreover purinergic nucleotides are important neuromodulators of peripheral auditory and visual sensory systems [] In cattle Keel et a0al [] reported that œsensory perception of smell and œGprotein coupled receptor signaling pathway were significantly overrepresented in the proteincoding genes that overlapped with CNVR Similarly Upadhyay et a0 al [] showed that œsensory perceptions of smell and œchemical stimuli are enriched in their set of CNV genes A potential explanation for the underrepresentation of the œsensory perception functional category among the genes overlapping CNV reported by Liu et a0al [] could be that in goats these genes are not well annotated yet so the majority of them are identified with a LOC prefix and a number and as a consequence of this they are not correctly detected by PANTHER [] thus biasing the results obtained in the gene ontology enrichment analysisLoci belonging to large multigene families might be more prone to colocalize with CNV because paralogous genes can act as templates in nonallelic homologous recombination events which promote increases or reductions in copy number [] It should be noted that olfactory receptor genes constitute the largest gene superfamily and in humans more than genes and pseudogenes have been identified [] In cattle olfactory receptor genes and pseudogenes are distributed in clusters across bovine chromosomes [] and similar numbers have been reported for pigs [] Moreover purifying selection against CNV is probably less 0cGuan a0et a0al Genet Sel Evol Page of intense in regions that contain olfactoryreceptor genes than in genomic regions that contain genes with essential functions [] Interestingly copy number changes in the olfactory receptor genes of wild and domestic mammals might have consequences on food foraging as well as on mate and predator recognition [ ]In the set of genes that colocalize with CNVR we also detected an enrichment of loci related with the multigene family of ATP binding cassette ABC transporters a result that agrees well with previous findings in humans [“] and cattle [ ] In mammals ABC transporters fulfill the mission of carrying a broad array of endogenous substrates such as amino acids peptides sugars anions and hydrophobic compounds and metabolites across lipid membranes At least ABC genes that belong to eight subfamilies have been identified in the human genome [] Copy number variation in the human ABCC4 and ABCC6 genes is associated with susceptibility to esophageal squamous cell carcinoma [] and to the rare autosomal recessive disease pseudoxanthoma elasticum [] respectively Moreover largescale deletions of the human ABCA1 gene are a causative factor for hypoalphalipoproteinemia [] a disease that is characterized by the complete absence of the apolipoprotein AI and extremely low levels of plasma highdensity lipoprotein HDL cholesterol We also found a highly significant enrichment of pathways related with embryo development anteriorposterior pattern specification embryonic skeletal system morphogenesis as previously reported [] These pathways are featured by genes that belong to the Hox multigene family of transcription factors possibly reflecting the genomic instability of certain homeobox gene clusters as evidenced by the existence of many syntenyparalogy breakpoints and assembly gaps as outlined in comparative studies []Although not significant after correction for multiple testing we detected an enrichment of pathways with metabolic significance such as prolactin and insulin signaling which could have an impact on milk production and growth [“] Interestingly the comparison of our work with that of Liu et a0al [] revealed proteincoding genes that colocalize with the set of shared CNVR One of the most relevant shared genes encodes ASIP a protein that increases the ratio of pheomelanin to eumelanin by binding to the melanocortin receptor and delivering an antagonist signal that blocks the downstream expression of eumelanogenic enzymes [] Mutations in the ASIP gene play critical roles in animal pigmentation [] For instance the causal factor of the white color typical of many sheep breeds is the ubiquitous expression of a duplicated copy of the ASIP coding sequence which is regulated by a duplicated promoter corresponding to the itchy E3 ubiquitin protein ligase gene [ ] Although some studies proposed that the ASIP CNV might be associated with different pigmentation patterns in goats [ ] no functional assay has verified an association of ASIP copy number with ASIP mRNA levels Another interesting shared copy number variable gene is ADAMTS20 which was also identified in two previous CNV surveys [ ] This gene encodes a metalloproteinase with an important role in melanoblast survival by mediating Kit signaling [] and in palatogenesis [] Bertolini et a0al [] performed a selection scan in white vs colored black and red goats and detected a selective sweep in the ADAMTS20 gene In the light of these results the potential involvement of a structural variation in ADAMTS20 in goat pigmentation should be explored further Moreover it is worthwhile to mention that several CNVR genes have functions related with production and reproduction traits For instance the NCKAP5 gene which colocalizes with CNVR_160_chr2 frequency is associated with milk fat percentage in cattle [] Taking the above evidence into account the implication of structural chromosomal variations in the genetic determinism of traits of economic interest with a complex inheritance deserves further exploration by designing tools that allow inferring CNVR genotypes with high confidenceConclusionsWith the PennCNV and QuantiSNP software we detected CNVR in the genome of the MurcianoGranadina breed In a a0 previous study [] that used a less stringent pipeline only PennCNV was used and included multiple populations with small to moderate sample sizes the average number of CNVR events per breed was One conclusion of our study is that CNV surveys which are based on a broad array of breeds represented by only a few individuals underestimate the true levels of the CNV diversity at the withinbreed level The main reason for this outcome is that since the majority of CNV have very low frequencies they cannot be detected efficiently when sample size is small and in consequence much of the existing variation is missed We have also found that genes that overlap with CNV are functionally related with olfactory transduction embryo development ABC transporters and Gprotein coupled purinergic nucleotide receptor activity Most of these genes belong to large multigene families encompassing tens hundreds or thousands of paralogous genes that could act as substrates in nonallelic homologous recombination events which is one of the main mechanisms generating duplications and deletions in humans and other species Finally we detected CNV that colocalize with the ASIP and ADAMTS20 pigmentation genes 0cGuan a0et a0al Genet Sel Evol Page of which according to previous studies have been subjected to positive selection for coat color in goatsSupplementary informationSupplementary information accompanies this paper at https doi101186s1271 ‘‘ ‘ Additional file a0 Table a0S1 List of primers used in the real‘time quanti

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

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

Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a “ increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy × magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415× patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Children™s Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM“ µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers × cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415× treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control × magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE “ BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415× patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and × cells were injected into the flank of NOD scid gamma mice at “ weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415× tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPad™s Prism software was used for the statistical analysisAbbreviationsCOG Children™s Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr “ httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am “ httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther “ httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics “ httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget “ httpsdoiorg1018632oncotarget3732 [PubMed] O™Connor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma “ httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol “ httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol “ httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer “ httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia “ httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov “ httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c'

Thyroid_Cancer

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

Thyroid_Cancer

Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a “ increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM“ µM measured by Cell Titer Glo„¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy × magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415× patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Children™s Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM“ µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers × cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415× treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control × magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE “ BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415× patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and × cells were injected into the flank of NOD scid gamma mice at “ weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415× tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPad™s Prism software was used for the statistical analysisAbbreviationsCOG Children™s Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr “ httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am “ httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther “ httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics “ httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget “ httpsdoiorg1018632oncotarget3732 [PubMed] O™Connor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma “ httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol “ httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol “ httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer “ httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia “ httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov “ httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c'

Thyroid_Cancer

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

Thyroid_Cancer

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

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

Thyroid_Cancer

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

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

Thyroid_Cancer

prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early‘onset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patients”with a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the œsilent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2“ Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance ˜RCspecific™ genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15“ A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10“ To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n ‰ a0years at diagnosis specimens collected between July “December All genetic test results from germline testing of individuals diagnosed with RC at ‰ during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of ‰ a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions “ and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PV”see below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN cˆ’ to cˆ’ MLH1 cˆ’ to cˆ’ and MSH2 cˆ’ to cˆ’ Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as ˜not reported™ Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term œrenal cancer was queried and the results were noted as œassociated with ClinVar search term ˜Renal Cancer™Statistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histology™s were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ‰¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fisher™s exact tests were used and a0pvalues ‰ were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between “ a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between “ a0years of age as compared to patients ‰ diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed ‰ Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in “ of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women ‰ in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434“After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI “ ExAc OR CI “ We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC ‰ a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC ‰ a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at ‰ a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported ‰ a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset œother based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see œMethods The groups were compared to median age at first RC diagnosis of or ‰¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1

Thyroid_Cancer

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

Thyroid_Cancer

"Postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in COVID19 patientsMethods We evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans Complete macroscopic and microscopicautopsies were performed on different ans in COVID19 nonsurvivors Presence of SARSCoV2 was evaluatedwith immunohistochemistry IHC in lung samples and with realtime reversetranscription polymerase chainreaction RTPCR test in the lung and other ansResults Pulmonary findings revealed earlystage diffuse alveolar damage DAD in out of patients andmicrothrombi in small lung arteries in patients Latestage DAD atypical pneumocytes andor acute pneumoniawere also observed Four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedThere was no evidence of myocarditis hepatitis or encephalitis Kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients Spongiosis and vascular congestion were the mostfrequently encountered brain lesions No specific SARSCoV2 lesions were observed in any an IHC revealedpositive cells with a heterogeneous distribution in the lungs of of the patients RTPCR yielded a widedistribution of SARSCoV2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brainConclusions In autopsies revealed a great heterogeneity of COVID19associated an injury and theremarkable absence of any specific viral lesions even when RTPCR identified the presence of the virus in many ansKeywords COVID19 SARSCoV2 Autopsy RTPCR Immunohistochemistry Correspondence IsabelleSalmonerasmeulbacbe1Department of Pathology Erasme Hospital Universit Libre de BruxellesULB Route de Lennik Brussels Belgium2Centre Universitaire inter Rgional d™expertise en Anatomie PathologiqueHospitali¨re CurePath CHIREC CHU Tivoli ULB Rue de Borfilet 12A Jumet BelgiumFull 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 0cRemmelink Critical Care Page of severeacuteincludingBackgroundCoronavirusesrespiratorysyndrome coronavirus SARSCoV and Middle Eastrespiratory syndrome coronavirus MERSCoV causesevere acute respiratory failure which is associated withhigh mortality rates [] The novel SARSCoV2 strainexhibits phylogenetic similaritiesto SARSCoV andcauses coronavirus disease COVID19 which hasresulted in more than deaths worldwide so farAs the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that SARSCoV2 can directly alter cellfunction by a link to the angiotensin converting enzyme ACE2 receptor which is almost ubiquitous in thehuman body []Nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with COVID19remain poorly understood Controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease []In this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of COVID19 Most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] The aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from COVID19MethodsStudy designIn this postmortem study we included the first adultpatients years who died in our hospital either in aCOVID19 unit or an intensive care unit from March with confirmed SARSCoV2 infection ie positiveRTPCR assay on nasopharyngeal swab andor bronchoalveolar lavage specimen Exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination The study protocol wasapproved by the local ethics committee P2020218Data collectionrelevantWe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport Acute respiratory distress syndrome ARDSand acute kidney injury AKI were defined accordingto standard definitions [ ]Postmortem procedureThe Belgian Public Health Institute Sciensano guidelines were integrated into our postmortem procedure[] The cadavers were kept in the refrigerator at °Cand autopsies were performed to h after death toensure the safety of the autopsy team Personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves FFP3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots In the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationAll analyses were performed at normal pressurespleen bone marrow kidney bladderUsing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking The material was biobanked by BiobanqueH´pital ErasmeULB BE_BERA1 CUB H´pital ErasmeBBMRIERIC The banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain For the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken For safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe Additional file Additional Material Formalinfixed paraffinembedded FFPEtissues underwentstandard processing to provide hematoxylin and eosinHEstained sections Special stains and immunohistochemistry IHC were used for lung Masson™s trichromeperiodic acidSchiff [PAS] GomoriGrocott antiCMVIHC antiHSV IHC antiPneumocystis J IHC and kidneyPAS Masson™s trichromeJones methenamine silversamplesMorphological analysisMorphological analysis was performed on HE stainedglass slides using the SecundOs digital platform TribVnHealth Care Chatillon Francefor digital diagnosisafter the acquisition of whole slide digital scans — magnification using a Nanozoomer HT slide scanner Hamamatsu Hamamatsu City JapanSARSCoV2 detection by immunohistochemistrySince no antibody against SARSCoV2 has been validatedfor IHC on FFPE tissues we selected an antiSARSnucleocapsid protein antibody Standard IHC was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display SARSnucleocapsid protein Invitrogen PA141098dilution on Dako Omnis Agilent Technologies 0cRemmelink Critical Care Page of Santa Clara CA USA using the Envision Flexdetection system according to the manufacturer™sprotocol [] The sections were counterstained withhematoxylin Negative tissue controls were obtainedfrom patients who had an autopsy before the COVID pandemic Semiquantitative IHC evaluation wasperformed by two senior pathologists ND MR as follows negative ˆ’ between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one — field SARSCoV2 detection by rRTPCRTotal nucleic acid was extracted from FFPE tissues usingthe Maxwell RSC DNA FFPE Kit reference AS1450Promega Corporation Madison WI USA and thePromega Maxwell extractor following the protocol described by the manufacturer Onestep RTPCR assaysspecific for the amplification of SARSCoV2 E envelopeprotein gene were adapted from a published protocol[] Briefly μL of RNA ng was amplified in μL reaction mixture containing μL of TaqMan FastVirus 1step master mix Life Technologies μM ofeach forward ACAGGTACGTTAATAGTTAATAGCGT and reverse ATATTGCAGCAGTACGCACACAprimers and μM of probe FAMACACTAGCCATCCTTACTGCGCTTCGBBQ The amplification condition was °C for min for reverse transcriptionfollowed by °C for s and then cycles of °C for s and °C for s A clinical sample highly positivefor SARSCoV2 was diluted and used as a positive control in each analysis A clinical sample obtainedfrom a patient who was autopsied before the COVID19pandemic was used as a negative control The quality ofthe RNA from the samples showing negative results wasassessed by amplification of the human MET RNA according to a validated ISO15189 accredited methodused as a routine diagnostic method in our laboratoryStatistical analysisData are reported as counts percentage or medians[interquartile ranges IQRs] All data were analyzedusing GraphPad Prism Version GraphPad Software San Diego CA USAResultsStudy cohortThe main characteristics of the study cohort malesout of median age [“] years are given inTable The time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days All except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n None ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza A and B virusesEleven of the patients were treated with mechanicalventilation Eleven patients died in the ICU and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n Laboratory data are reported in Additional file Table S1Macroscopic findingsOne patient had had a left pneumonectomy and onepatient a right bilobectomy The lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage Thrombi were found in the large pulmonary arteries in patients and lung infarction in patientsPleural adhesions associated with pleural effusions were observed in cases We observed cardiomegaly in and hepatomegaly in patients The kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct The gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis In the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhageMicroscopic findingsfile As shown in Figs and and AdditionalTable S2the main pulmonary findings includedearlystage diffuse alveolar damage DAD which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryII pneumocytehyperplasia Microthrombi were noted in the smallpulmonary arteries in patients Ten of the patientsalso had advanced DAD lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells We observed no viralinclusions or squamous metaplasiaand typeinfiltrateAll the patients who survived more than weeksn had late DAD lesions There was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct In of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi The other patients had longer 0cRemmelink Critical Care Page of Table Characteristics of the study populationIDCT scanComorbiditiesAgeSexrRTPCRPOSTime todeathAntemorteman failureARDSAKIPOSNEGNEGMFMFMCADCVDDiabetesHypertensionCADCRFLiver cirrhosisCOPDCancerHypertensionCancerCVDCOPDCancerGGOPOSMAGGOPOSPOSTreatmentsCause of deathMechanicalventilationAntibioticsHydroxychloroquineAntibioticsCorticosteroidsMechanical ventilationHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMOHydroxychloroquineRemdesivirCorticosteroidsAntiobioticsHydroxychloroquineAntibioticsCardiogenicshockMOFRespiratory failureRespiratory failureRespiratory failureMesentericischemiaMOFRespiratory failureRespiratory failureAntibioticsSeptic shockMOFMechanical ventilationRRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMORRTHydroxychloroquineOseltamivirAntibioticsHydroxychloroquineAntibioticsRespiratory failureRespiratory failureSudden deathMechanical ventilationHydroxychloroquineAntibioticsMOFHydroxychloroquineRespiratory failureHydroxychloroquineAntibioticsRespiratory failureARDSAKIHypoxichepatitisARDSAKIARDSARDSAKIHypoxichepatitisARDSAKIARDSAKIHypoxichepatitisAKIARDSAKIARDSAKIARDSARDSAKIHypoxichepatitisARDSARDSAKIHypoxichepatitisMHypertensionCRFBCPOSMNoneBCPOSMFHypertensionCADCVDCRFDiabetesHypertensionDiabetesEmphysemaPOSGGOPOSMHypertensionDiabetesGGOBCPOSMMMFDiabetesLiver cirrhosisCancerDiabetesHypertensionCADDiabetesDiabetesHypertensionDiabetesBCPOSGGOPOSGGOBCGGOBCPOSPOS 0cRemmelink Critical Care Page of Table Characteristics of the study population ContinuedIDComorbiditiesCT scanAgeSexMGGOLPPOSrRTPCRTime todeathFMHypertensionDiabetesLiver transplantHypertensionCVDGGOBCPOSGGOBCLPPOSAntemorteman failureARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismTreatmentsCause of deathMechanical ventilationRRTHydroxychloroquineRemdesivirAntibioticsMechanical ventilationRRTHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineAntibioticsSeptic shockMOFSeptic shockMOFSeptic shockMOFTime to death time from admission to death days Cause of death was reported by the attending physician M male F female rRTPCR reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test NEG negative POS positive CAD coronary artery disease CVD cerebrovascular disease LP lobarpneumonia GGO groundglass opacity MA minor abnormalities BC bilateral consolidation COPD chronic obstructive pulmonary disease CRF chronic renal failureARDS acute respiratory distress syndrome AKI acute kidney injury ECMO extracorporeal membrane oxygenation RRT renal replacement therapy MOF multiplean failuredelays between hospitalization and death daysthey had no microthrombiFifteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis Histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in In the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients Chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisFig Main histological findings Green finding present gray finding absent black unavailable 0cRemmelink Critical Care Page of Fig Pulmonary histological findings a Earlystage diffuse alveolar damage DAD hyaline membrane HE — magnification with a zoom ona giant cell — magnification b Fibrin thrombi in a pulmonary artery HE — magnification c Latestage DAD fibroblastic proliferationHE — magnification d Latestage DAD fibroblastic proliferation Trichrome staining — magnification e Acute pneumonia HE — magnification f AntiSARSCoV immunohistochemistry IHCpositive cells — magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryLiver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate The histological changes in theabdominal ans including the esophagus stomachand colon are reported in Additional file Table S2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisBrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n We found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalSARSCoV2 detection in the lungs by IHCSARSCoV2 was identified by IHC in the lungs of ofthe patients Fig Howeverthere was largevariability in the distribution of SARSCoV2positivecells in the lung parenchymaSARSCoV2 detection by RTPCRSARSCoV2 RNA was detected in at least one anfrom every patient Fig In the lung RTPCR waspositive in patients with threshold cycle Ct valuesvarying from to Viral RNA was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples Ct valuesfor nonpulmonary ans ranged from to Eight patients had positive RTPCR in all tested ansabnormalitiesDiscussionThis postmortem study showed several histopathologicalin COVID19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though SARSCoV2 was detected in all examined ans using RTPCR We decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe This approach enabled us to 0cRemmelink Critical Care Page of Fig Detection of SARSCoV2 by immunohistochemistry IHC in FFPE post mortem lung samples of patients Semiquantitative evaluationœˆ’ negative result œ scattered positive cells between and positive cellswhole slide œ positive isolated cells cellswhole slide butno foci œ foci of positive cells more than positive cells in one — field NA not availabledocument the considerable heterogeneity of histologicallesions and of SARSCoV2 spread through the bodyThe diagnosis of SARSCoV2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases In a previous autopsy study in COVID19patients [] the authors reported that DAD associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia No obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report [] Desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ARDS of many different causes especially inearlyphase ARDS [] The presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice As in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of SARSCoV2 infection [] Finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to SARSCoV2In the absence of typical postmortem viral featuresour results show that RTPCR is feasible on FFPE blocksand could be used in postmortem analyses to identifythe presence of SARSCoV2 in multiple ans and tounderstand the spread of the virus within the humanbody The discordant RTPCR and IHC results fordetection of SARSCoV2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the RTPCR whereas lowlevel viral replication might not be detected by IHC Moreover IHC wasbased on the only available antibodies which aretargeted against SARSCoV New antibodies againstSARSCoV2 need to be developed to improve theaccuracy of IHC in the analysis of tissue samples fromsuspected or confirmed COVID19 patientsMost of the previous postmortem studies in COVID19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofSARSCoV2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations As for previous coronavirusdiseases [ ] the lungs are the most affected ans inCOVID19 However DAD findings werehighly 0cRemmelink Critical Care Page of Fig Molecular detection of SARSCov2 RNA in postmortem samples Detection of SARSCoV2 by reverse transcription realtime polymerasechain reaction RTPCR in FFPE postmortem tissues of patients œ positive result œˆ’ negative result œNA tissue not available NC noninformative test result due to lowquality RNAheterogeneous including both earlyonset and additionallate lesions This finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase [] As some patients died outsidethe ICU without receiving mechanical ventilation we couldnot estimate lung compliance before death The heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the DADreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections In a recent report needle postmortem biopsies suggested that COVID19 is not associated withDAD but rather with an acute fibrinous and anizingpneumonia AFOP consequently requiring corticoid treatment [] A diagnosis of AFOP is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with DAD and complete lunganalysis not just biopsies are necessary to exclude theirpresence Moreover AFOP may be a fibrinous variant ofDAD [] The limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for SARSCoV2 using RTPCR [] when compared to almost in our series In addition we did notfind specific œendothelitis as previously reported in a smallcase series [] Considering the heterogeneity of postmortem COVID19 associated lesions molecular and IHCassessments are mandatory in the histological analysis ofCOVID19 tissue samplesPatients with COVID19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism PE [] In ourstudy three patients had PE already diagnosed beforedeath Four patients had pulmonary infarction In a previous study acute PE was considered as the main causeof death in four patients [] however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of PEwhen compared to our study Although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofARDS regardless of etiology [ ] As such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in COVID19 0cRemmelink Critical Care Page of patients remains to be further studied In a systematicreview of pathologicalfindings in COVID19 Polak [] identified a timeline in the histopathologicalfindings in the lung with epithelial DAD denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms Few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease Our resultsare consistent with those of Polak [] except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients We did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis The cases ofœacute cardiac injury reported in COVID19 clinicalstudies [] do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia [] However using RTPCR we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theACE2 receptor which is ubiquitous but may notdirectly cause an injury As extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatSARSCoV2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in COVID19associated an dysfunction AsRTPCR mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance []just detect residual viral genomeThis study has several limitations i we only includedpatients who had had a positive RTPCR on nasopharyngeal swab andor bronchoalveolar lavage To ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic CTscan findings suggestive of COVID19 but had negativeRTPCR results This limitation in our study reflects thedifficulty of diagnosing COVID19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death This delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow SARSCoV2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related toCOVID19 Finally we did not evaluate specific mechanisms involved in the pathogenesis of an injuryConclusionThese results underline the heterogeneity of an injuriesduring COVID19 disease and the absence of specificSARSCoV2 lesions Using RTPCR SARSCoV2 couldbe detected in all ans even those without evidentmicroscopic lesionsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s13054020032185Additional file Critical careautopsyCovid Additional materialProcedure to obtain brain samplesAdditional file Critical careautopsyCovid Additional Table S1Laboratory findings on the day of admissionAdditional file Critical careautopsyCovid Additional Table S2Detailed histological findings in all patientsAbbreviationsACE2 Angiotensin converting enzyme AFOP Acute fibrinous andanizing pneumonia AKI Acute kidney injury ARDS Acute respiratorydistress syndrome COVID19 Coronavirus disease Ct Threshold cycleDAD Diffuse alveolar damage FFPE Formalinfixed paraffinembeddedHE Hematoxylin and eosin IHC Immunohistochemistry IQRs Interquartileranges MERSCoV Middle East respiratory syndrome coronavirusPAS Periodic acidSchiff PE Pulmonary embolism RTPCR Realtime reversetranscription polymerase chain reaction SARSCoV Severe acute respiratorysyndrome coronavirusAcknowledgmentsThe authors thank Nathalie Lijsen Christophe Valleys Gees LacroixBarbara Alexiou Dominique Penninck Nicole Haye and Audrey Verrellen fortechnical and logistic supports Prof Frdric Schuind and Dr DjamelEddineYahiaCherif for neurosurgical procedure Egor Zindy DIAPath ULB forproofreading the paper and Dr MariePaule Van Craynest for trainees™supervisionAuthors™ contributionsIS had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis IS FT JLV and CD drafted the paper MRCV LL PL MLR CM ALT JCG LP RDM SD SR ND LP and OD collected thedata MR ND and RDM did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published All authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedFundingThis study received financial support from Fonds Y Bo«l Brussels BelgiumFonds Erasme pour la Recherche Mdicale Brussels Belgium and œAppel  projet Spcial COVID19 ULB Brussels Belgium The CMMI is supported bythe European Regional Development Fund and the Walloon Region ofBelgium Walloniabiomed grant no project œCMMIULBsupport the Center for Microscopy and Molecular Imaging and its DIAPathdepartment CD is a Senior Research Associate with the FNRS BelgianNational Fund for Scientific Research 0cRemmelink Critical Care Page of Availability of data and materialsThe data that support the findings of this study are available from thecorresponding author on reasonable request Participant data without namesand identifiers will be made available after approval from the correspondingauthor and local Ethics Committee The research team will provide an emailaddress for communication once the data are approved to be shared withothers The proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data Additional materials may also be required during theprocess D'Haene N Melndez B Blanchard O De N¨ve N Lebrun L VanCampenhout C Design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas CancersBasel Corman VM Landt O Kaiser M Molenkamp R Meijer A Chu DK et alDetection of novel coronavirus 2019nCoV by realtime RTPCR EuroSurveill de Hemptinne Q Remmelink M Brimioulle S Salmon I Vincent JL ARDS aclinicopathological confrontation Chest “ Menter T Haslbauer JD Nienhold R Savic S Hopfer H Deigendesch N et alEthics approval and consent to participateThe study protocol was approved by the local ethics committee ErasmeHospital P2020218 The ethical committee has waived the need for writteninformed consentPostmortem examination of COVID19 patients reveals diffuse alveolardamage with severe capillary congestion and var

Thyroid_Cancer

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

Thyroid_Cancer

"pharmacological therapies and treatments targeting pancreatic neuroendocrine tumorsPNETs have proven ineffective far too often Therefore there is an urgent need for alternative therapeuticapproaches Zyflamend a combination of antiinflammatory herbal extracts that has proven to be effective invarious in vitro and in vivo cancer platforms shows promise However its effects on pancreatic cancer in particularremain largely unexploredMethods In the current study we investigated the effects of Zyflamend on the survival of betaTC6 pancreaticinsulinoma cells TC6 and conducted a detailed analysis of the underlying molecular mechanismsResults Herein we demonstrate that Zyflamend treatment decreased cell proliferation in a dosedependent mannerconcomitant with increased apoptotic cell death and cell cycle arrest at the G2M phase At the molecular level treatmentwith Zyflamend led to the induction of ER stress autophagy and the activation of cJun Nterminal kinase JNK pathwayNotably pharmacological inhibition of JNK abrogated the proapoptotic effects of Zyflamend Furthermore Zyflamendexacerbated the effects of streptozotocin and adriamycininduced ER stress autophagy and apoptosisConclusion The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer viamodulation of the JNK pathwayKeywords Pancreatic neuroendocrine tumor cells Zyflamend JNK Apoptosis Autophagy ER stressPlain English summaryThrough investigating the effects of treating an experimental model of pancreatic neuroendocrine tumor cells withZyflamend we discovered a novel therapeutic potential ofthis polyherbal blend Findings from this study could helppioneer future advancements in our understanding of howphytochemicals and natural compounds could synergistically prove effective against pancreatic cancer by alteringcancer cell survival and proliferation Furthermore the evidence presented within promotes Zyflamend as an adjuvantprospect where it could enhance the effectiveness of standard cancer therapies In addition we believe that thesenovel findings will be of major interest to a broad spectrumof scientists and may pave the way towards more effectiveand translatable therapies Correspondence abettaieutkedu1Department of Nutrition University of Tennessee Knoxville CumberlandAvenue Jessie Harris Building Knoxville TN USA3Graduate School of Genome Science and Technology University ofTennessee Knoxville TN USAFull list of author information is available at the end of the BackgroundPancreatic cancer remains one of the deadliest types ofcancer in the United States with over new casesand deaths in accounting for of allcancer deaths [] Recent epidemiological studies predict 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 0cPuckett Cell Communication and Signaling Page of that in pancreatic cancer will be the third leadingcause of cancerrelated death [] Although advancements in science and health care have led to decreasedmortality from numerous forms of cancer pancreaticcancer survival rates have not improved significantlyover the past several decades leaving a desperate needfor more effective treatment options The risk of developing pancreatic cancer has been associated with numerous biological environmental pathological andgenetic factors These factors include variables such asfamilial history chronic pancreatitis smoking obesityand diabetes reviewed in [ ] In addition hereditaryfamilial factors and germline mutations could contributeto increased risk of cancer onset However the survivaloutcomehighlydependent upon the time of diagnosis While pancreaticductal adenocarcinoma PDAC is the most commonlycontracted and investigated subtype the pathological nature ofthe rarer pancreatic neuroendocrine tumorsPNETSs remains elusive []effectivenesstreatmentandarePNETs account for less than of all pancreatic cancers and are often diagnosed at a late stage in patientswith advanced metastasis making surgery a nonviabletreatment option [ ] Additionally because of theirheterogeneous clinical presentation and responses tochemotherapeutic agents current pharmacological therapies and treatment options targeting PNETs have toooften proven ineffective [] PNETs treatment optionsoften include the use of chemotherapeutic compoundssuch as streptozocin 5fluorouracil doxorubicin andcisplatin both alone or in combination reviewed in [] The effectiveness of these compounds often increases at higher doses but this directly exacerbates therisk for cytotoxicity and collateral side effects [] Inaddition adjunct therapy involving the combination ofvarious treatment approaches such as surgery and radiotherapy is often implemented [] In pursuit of survivaland improved quality of life patients often seek to enhancetherapiesthrough dietary and supplemental means [ ]effectiveness ofconventionaltheNew Chapter Brattleboro VT first launched Zyflamend based on the idea of combining extracts of ten different herbs to effectively reduce inflammation throughcyclooxygenase COX inhibition [] A large volume ofresearch has emerged over the last two decades that supports the antiinflammatory properties of Zyflamend andits ability to inhibit COX in various types of cancer including prostate [] melanoma [] and oral cancer[] Individually many of the extracted components ofZyflamend have proven to exhibit anticancer activity[ ] However the high doses required to optimizeeffectiveness against cancer could prove infeasible forthe majority In theory the combined effects generatedthrough integrating these unique and powerful herbscould grant superior benefit over their isolated form[] Additionally Zyflamend has shown the capabilityto interact with a variety of integral cellular signalingpathways beyond COX These signaling pathways andmechanisms of interaction include AMPactivated protein kinase AMPK [ ] nuclear factor kappalightchainenhancer of activated B cells NFκB [ ]mammalian target of rapamycin mTORC1 [] apoptosis cell growth [ ] endoplasmic reticulum ERstress [ ] and finally autophagy [] Whilethese studies show that Zyflamend could exhibit profound potential in the therapeutic application more research is required to elucidate the molecular basisunderlying its anticancer effects In the current studywe investigated the effects of Zyflamend on the survivalof betaTC6 pancreatic insulinoma cells TC6 anddeciphered the underlying molecular mechanismsMethodsChemicals and reagentsMedia sera and trypsin for cell culture were purchasedfrom Gibco Thermo Fisher Scientific Waltham MAPrimary antibodies and secondary antibodies were acquired from varying sources Supplementary Table General caspases inhibitor ZVADfmk was obtainedfrom Calbiochem La Jolla CA Zyflamend„¢whole bodywas purchased from New Chapter New Chapter IncBrattleboro VT Zyflamend composition is indicated inSupplementary Table Quality assurance is in full compliance with Good Manufacturing Practicing Standardsas mandated by CRF Part Additionally full description and characterization of Zyflamend and itspreparation have been previously described in detail[] Chemical reagents such as dithiothreitol DTTpercoll digitonin phenylmethylsulfonyl fluoride PMSFprotease inhibitors cocktail sodium deoxycholate Tritonglycolbis2aminoethylNNN€²NX100€²tetraacetic acid EGTA sodium fluoride NaF sodium phenylbutyrate 4PBA Hoechst propidium iodide streptozotocin STZ adriamycin ADRautophagy inhibitor 3methyladenine 3MA and JNKinhibitor SP600125 were acquired from MilliporeSigma Burlington MA Finally AMPK inhibitor BML aka compound C was purchased from Santa CruzBiotechnology Santa Cruz CAethyleneCell cultureMouse betaTC6 pancreatic insulinoma TC6 ATCC®CRL11506„¢ and rat pancreatic insulinoma RIN5FATCC® CRL2058„¢ cells were cultured as monolayersin Eagle€™s modified Dulbecco medium plus Lglutamine mM sodium pyruvate mM and fetal bovineserum FBS GibcoThermo Fisher Scientific WalthamMA Cells were maintained in tissue culture plates 0cPuckett Cell Communication and Signaling Page of Thermo Fisher Scientific Waltham MA at °C in ahumidified atmosphere of CO2 Medium was replaced with fresh medium h before experimentsZyflamend treatmentZyflamend was dissolved in dimethyl sulfoxide DMSOat a concentration of mgml Cells were treated withZyflamend at the indicated concentrations and for theindicated durations Treatments were terminated by twowashes with icecold phosphate buffer saline PBSPlates were then flashfrozen in liquid nitrogen andstored at ˆ’ °C until further analysesProliferation assayCell proliferation assay was performed using the sulforhodamine B SRB MilliporeSigma method as previously described [] with modification Briefly an equalnumber of TC6 cells X cells were seeded in well plates Six h later cells were treated with the indicated concentrations of Zyflamend and incubated at °C in an atmosphere of CO2 for the indicatedtime Treatment was stopped by two washes with icecold PBS and cells were fixed with trichloroaceticacid in PBS Intracellular proteins were stained for min at room temperature using SRB dissolved in acetic acid Excess SRB stain was removed by rinsingthe plates thoroughly with running tap water Plateswere airdried for at least h prior to dissolving the stainin mM Tris pH Intracellular proteins werequantified using the Synergy„¢ HTX MultiMode microplate reader BioTek Instruments Inc Winooski VT ata wavelength of nm The relative survival rates ofcells were determined by dividing the absorbance observed for a given treatment by the absorbance detectedin control cells treated with DMSO and expressed as afold changeCytotoxicity assayThe MTT [45dimethylthiazol2yl]25diphenyltetrazolium bromide cytotoxicity assay was performed aspreviously described with modification [] Briefly cells were plated in a 96well plate for h Then afreshly prepared solution of Zyflamend alone or in combination with ZVADfmk 10uM 4PBA 250uMSP600125 10uM STZ mM or adriamycin μMfor an additional h The experiment was terminatedby adding μl of the MTT solution mgml to eachwell for h then the cell culture medium was removedand the dye was dissolved in μl SDS solution overnight at °C Relative cytotoxicity was determinedby measuring the absorbance at nm using the Synergy„¢ HTX MultiMode microplate readerColognic testColonie formation assay was performed as previously described [ ] with modification Cells were seeded inthe presence of DMSO control Zyflamend alone or incombination with ZVADfmk uM 4PBA uM SP600125 uM STZ mM or adriamycin μM After h media was replaced with a freshlyprepared new cell culture media and plates were incubated for days at °C in an atmosphere of CO2After incubation the colonies were washed with icecoldPBS fixed and stained with a mixture of glutaraldehyde and crystal violet for min The plates werewashed with water dried and colonies with morethan cellscolony were counted The relative number of colonies in each condition was determined bydividing the number of colonies for a given treatmentby the total number of colonies in DMSO treatedcells control and expressed as a percentage relativeto DMSOtreated cells CtrlWestern blotting analysisCells were lysed in radioimmunoprecipitation assayRIPA buffer as previously described [] Lysates wereclarified by centrifugation at g for min andprotein concentrations was determined using bicinchoninic acid assay kit Pierce Chemical Dallas TX Proteins €“ μg were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresisSDSPAGE andtransferred to polyvinylidene fluoride PVDF membranes Immunoblotting of lysates was performed withprimary antibodies Supplementary Table and afterincubation with secondary antibodies proteins were visualized using Luminata„¢ Forte Western Chemiluminescent HRP Substrate MilliporeSigma Pixel intensitiesofusingFluorChem Q Imaging software Alpha Innotech CorpSan Leandro CA Data for phosphorylated proteins arepresented as the intensity of phosphorylation normalizedto total protein expression while total protein expression was normalized to the loading control actinimmunoreactivebands werequantifiedMorphological analysis of apoptosisTC6 cells were exposed to Zyflamend for the indicated duration then washed with PBS and labeledwith Hoechst μgmlbluegreenfluorescence Hoechst binds to condensed nuclearchromatin [] and was used to visualize apoptoticcells green fluorescence by fluorescence microscopyLeica DMI8 Leica Microsystems Inc Buffalo GroveIL For each condition atleast cells werecounted Percentages of apoptotic cells were calculated relative to total cellsin PBS 0cPuckett Cell Communication and Signaling Page of MilliporeSigmaAnnexin V stainingQuantification of externalized phosphatidylserine anearly event in the apoptotic cascade was performedusing flow cytometry as previously described [] withmodification Briefly €“ confluent TC6 cellswere exposed to Zyflamend for h then washed withPBS and resuspended in μl of PBS containing FBS Immediately after an equal volume ofthe 2XGuava Nexin reagentcontainingAnnexin V Fluorescein isothiocyanate FITC and aminoactinomycin D 7AAD was added to each treatment and incubated for min at room temperatureunder lightprotected conditions Intensities of fluorescence emitted by Annexin V FITC and 7AAD weremeasured using the Guava® easyCyte Flow CytometerMilliporeSigma on PM1 and PM2 channels respectively Viable negative for both Annexin V and 7AADstaining and apoptotic cells both at early Annexin Vpositive 7AAD negative and late positive for bothAnnexin V and 7AAD stages were quantified using theInCyte„¢ and GuavaSuite Software package LuminexCorp Austin TXCell cycle analysisCell cycle analysis was conducted through assessing theDNA content of cells stained with propidium iodide aspreviously described [] with modification Briefly €“ confluent TC6 cells were starved in serummedia for h then complete growth media was addedto the cells along with various freshly prepared concentrations of Zyflamend h later cells were harvestedwashed twice with icecold with PBS and fixed overnight in ethanol at °C Next cells were washedtwice with icecold PBS and incubated in a freshly prepared RNase solution [ mM TrisHCl pH containing Uml of DNasefree RNase A AppliedBiosystems Austin TX] for min at °C Cells werewashed twice with icecold PBS and incubated in a solution of propidium iodide PI μgml in PBS overnightat °C under light protected conditions Fluorescenceintensity of PI was measured using the Guava® easyCyteflow cytometer on PM2 channel DNA histogram analysis was performed on cells using the InCyte„¢ andGuavaSuite Software package and the proportions ofcells with one or two copies of their chromosomal DNAwere calculatedStatistical analysisData were analyzed using JMP Pro program SASInstitute NC and presented as means standard errorof the mean SEM Unpaired heteroscedastic twotailStudent€™s t test was used for all statistical analyses anddifferences were considered significant at p Singlesymbol such as or €  was used to indicate a p valuethat is less than while double symbol such as or€ €  corresponds to a p value that is less than ResultsZyflamend decreases cell proliferation causes G2M cellcycle arrest and induces apoptotic cell death inpancreatic cancer cellsWe first examined the effects of varying doses of Zyflamend on the proliferation of pancreatic insulinoma TC6 cells Zyflamend caused a significant dose andtimedependent decrease in cell growth Fig 1a Additionally a Zyflamend dose of μgml was sufficient toinhibit cell proliferation by after h of treatmentwhile a dose of μgml completely abolished cell proliferation Fig 1a In line with these findings cell cycleanalysis demonstrated that Zyflamend alters cell cycledistribution in a dosedependent manner Indeed Zyflamend treatment resulted in the enrichment of the G2Mfraction with N DNA content which was accompaniedby a reduction in cell cycle progression through the G0G1 and S phases Fig 1bc These results suggest thatZyflamendinduced inhibition of cell proliferation is mediated at least in part through cell cycle arrest in theG2M phaseIn order to determine whether Zyflamendinduced inhibition of cell proliferation was associated with apoptotic cell death we determined changes in apoptosis inTC6 cells treated with increasing doses of Zyflamend and μgml for h usingtwo approaches the Guava Nexin Annexin V assay andHoechst stain Using the Annexin V assay thepercentages of both Annexin V positive7AAD negativecells reflective of early apoptotic cells and Annexin Vpositive7AAD positive cells reflective oflate apoptosis exhibited a dosedependent and significant increase in response to Zyflamend treatment Fig 1deConsistent with this observationthe number ofHoechstpositive cells was also higher in Zyflamendtreated cells compared to control cells Fig 1fgHoechst is a nucleic acid dye that binds to condensed chromatin in the nucleus of apoptotic cells thusgiving an assessment of overall apoptotic cell death []At a dose of and μgml the percentages ofapoptotic cells were ± ± and ± respectively further emphasizing the proapoptotic effects of Zyflamend on these cells Similarfindings were obtained using the MTT assay Fig 1hA human equivalent dose of Zyflamend induces apoptoticcell death in TC6 cellsTo further characterize the proapoptotic properties ofZyflamend we conducted a time course analysis using aphysiological relevant fixed dose of Zyflamend μgml [] This dose is representative of the maximum 0cPuckett Cell Communication and Signaling Page of Fig Zyflamend Reduces Cell Survival and Induces Cell Death of Pancreatic Cancer Cells in a Dose Dependent Manner a Effects of Zyflamendon cell survival and proliferation cells were treated with increasing doses of Zyflamend for h Line graphs represent the intensity of SRBstaining reflective of the cell number and presented as means SEM bc Cell cycle analysis and assessment of DNA content in TC6 cellstreated with DMSO control or the indicated concentration of Zyflamend for h Representative histogram distributions for each treatment areshown c Bar graphs represent the percentages of cells in each phase of the cell cycle which were estimated using the GuavaSuite Softwarepackage and are presented as means SEM from three independent experiments p p indicate significant difference betweenthe indicated concentration and control cells treated with the vehicle DMSO de Zyflamend treatment induces apoptosis in TC6 Cells confluent cells were treated with increasing concentrations of Zyflamend and then labeled with Annexin VFITC and 7AAD Representative dotplots are shown Annexin V positive and 7AAD negative cells lower right quadrants represent early stages of apoptosis whereas cells that arepositive for both Annexin V and 7AAD upper right quadrants are in late stages of apoptosis e Bar graphs represent live early and lateapoptotic cells are presented as means SEM of at least three independent experiments p p indicate significant differencebetween the indicated concentration of Zyflamend and control cells treated with the vehicle DMSO fg Chromatin condensation in cells treatedwith increasing doses of Zyflamend for h Representative images are shown Scale bar μm g Bar graphs represent the number of apoptoticcells Hoechst positive as means SEM of at least three independent experiments h Cell toxicity assay using the MTT method Bar graphsrepresent the intensity of formazan produced from MTT by viable cells staining reflective of the cell number and presented as means SEM ofat least three independent experiments In g and h p p indicate a significant difference between cells treated with Zyflamendand nontreated cellstreatmentplasma concentration of a primary ingredient of Zyflamend curcumin that was reported in humans after oraladministration [] At this dose a marked increase inchromatin condensation and apoptotic cell number wasobserved after h ofFig 2ab Subsequently markers of apoptosis and cell survival were investigated using Western blotting Zyflamend inducedcleavage of caspase3 and its downstream target polyADPribose polymerase PARP Fig 2cd In additionwe examined changes in the mitogenactivated proteinkinases MAP kinases pathways in response to Zyflamend Our data revealed that TC6 cells treated withZyflamend exhibited a marked decrease in the phosphorylation of protein kinase B AKT and extracellularsignalregulated kinases ERK particularly after h oftreatmentTo determine whether Zyflamendinduced cell deathwas associated with the caspase dependent pathwaysof apoptosis we tested whether blocking caspases€™activity usingcarbobenzoxyvalylalanylaspartyl[Omethyl]fluoromethylketone ZVADfmk could inhibit Zyflamendinduced chromatin condensation andapoptosis ZVADfmk is a potent cell permeable pancaspase inhibitor which acts by irreversibly bindingto the catalytic site of the caspase proteases and thusinhibiting their activities Our study shows that pretreatment with ZVADfmk caused a significant decreased in the levels of chromatin condensation inZyflamendtreated cells Fig 2ef Additionally ZVADfmk treatment alleviated Zyflamendinduced celltoxicity as judged by the MTT Fig 2g and the colony formation Fig 2hi assays Taken together ourfindings indicate that Zyflamend treatment reducescell viability and induces cell death through the induction of the apoptotic machinery in TC6 cellsZyflamend induces ER stress apoptosis and autophagyresponses in TC6 cellsA plethora of intrinsic and extrinsic pathways can leadto apoptosis in response to stressors including ER stressand autophagy among many more Therefore in orderto dissect the precise molecular mechanism mediatingthe proapoptotic effects of Zyflamend we examined theactivation of key signaling molecules related to thesepathways Zyflamend μgml significantly inducedER stressactivation of ERjudged byasthe 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Apoptotic Cell Death in TC6 Cells ab Effects of Zyflamend on chromatin condensation Cells were treated withZyflamend μgml for the indicated time and chromatin condensation was evaluated by fluorescence microscopy using Hoechst Representative images are shown Scale bar μm b Bar graphs represent the number of apoptotic cells Hoechst positive as means SEMp indicates a significant difference between cells treated with Zyflamend and nontreated cells cd Immunoblots of key proteins in cellsurvival and apoptosis markers in cells treated with μgml of Zyflamend for the indicated time d Bar graphs represent cleaved caspase3 CCasp 3actin cleaved PARP CPARPactin pAKTAKT and pERKERK as means SEM p p indicates a significant differencebetween cells treated with Zyflamend and nontreated cells ef Chromatin condensation in TC6 cells treated with μgml Zyflamend withand without the pancaspase inhibitor ZVADfmk Representative images are shown Scale bar μm f Bar graphs represent the number ofapoptotic cells Hoechst positive as means SEM g Cell toxicity assay using the MTT method Bar graphs represent the intensity of formazanstaining reflective of the cell number and presented as means SEM hi Colony formation assay i Bar graphs represent the relative number ofcolonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSOtreated cells Ctrl and expressed as a percentage In g and i p p indicate a significant difference between cells treated withZyflamend and nontreated cells € p € € p indicate a significant difference between cells treated with ZVADfmk and nontreatedproteinCHOPby Westerntransmembrane sensors protein kinase RNAlike endoplasmic reticulum kinase PERK and inositolrequiringtransmembrane kinaseendoribonuclease 1α IRE1αalong with downstream targets such as eukaryotic translation initiation factor alpha EIF2α and CEBP homologousblottingZyflamend induced ER stress as evidenced by increasedPERK Thr980 EIF2α Ser51 and IRE1α Ser724 phosphorylation Fig 3a Furthermore the level of CHOPexpression was elevated a direct downstream target ofboth the PERK and IRE1 pathways The activation ofCHOP a potent inducer of apoptotic cell death [ ]in response to ER stress Fig 3ab strengthens our conclusions of Zyflamendinduced apoptosis in these cellsMoreover Zyflamend has been shown to activateAMPK and our results recapitulate these previous findings []The AMPK signaling pathway has been shown toinregulate autophagy and cell death [] Thereforeorder to assess whether Zyflamend induces autophagy inTC6 cells we immunoblotted for autophagyrelatedproteins We observed a time dependent increase inbeclin microtubuleassociated proteins 1A1B lightchain LC3I II and autophagyrelated proteins and ATG57 Fig 3cd The increase in the expressionof these proteins is indicative of elevated autophagy inthese cells Because ER stress inflammation and autophagy can all lead to apoptosis we used the pancaspaseinhibitor ZVADfmk to determine which pathway mightbe responsible for the proapoptotic effects of Zyflamend Our data shows that while there was a significantattenuation of Zyflamendinduced cleavage of caspase3and its downstream target PARP treatment with ZVADfmk had no effects on Zyflamendinduced activation of the AMPK autophagy and ER stress signalingcascades Fig 3ef These findings suggestthat ERstress autophagy and MAP kinases pathways are upstream of the apoptotic signaling cascade that might bemediating the proapoptotic effects of Zyflamend in TC6 cellsZyflamendinduced cell death is mediated through the ERstressJNKautophagy pathwayThe exact molecular mechanisms leading to apoptosisby Zyflamend in cancer cells hashave not been revealedyet although recent studies have supported the role ofAMPK in the regulation of cancer cell growth bioenergetics autophagy and cell death To investigate the potential role of AMPK in Zyflamendinduced apoptosiswe pretreated cells with the AMPK inhibitor compoundC CC μM for h prior to Zyflamend treatment foran additional h The dose and duration of exposurewere determined based on the ability of compound C toreverse AMPKdependent inhibitory phosphorylation ofacetylCoA carboxylase ACC data not shown Cellswere then examined for AMPK activation as well as activation of inflammation ER stress autophagy and celldeath Fig 4a While the level of phosphorylated AMPKwas reduced in cotreated cells pretreatment with compound C had no effects on Zyflamendinduced JNKphosphorylation ER stress autophagy or cell death Fig4ab These data suggest that Zyflamendinduced apoptosis in TC6 cells is independent of AMPK activationNext we sought to examine whether blocking autophagyusing 3MA could protect cells against Zyflamendinduced apoptosis 3MA inhibits autophagy by blockingautophagosome formation via the inhibition of class Iand class III phosphatidylinositol 3kinases PI3K []Cells were preincubated with 3MA nM for hprior to Zyflamend treatment Cotreatment with Zyflamend and 3MA significantly decreased the expressionof beclin LC3 and cleaved caspase3 but had no effects on ER stress markers nor on AMPK phosphorylation Fig 4cd These data suggest that autophagymediates Zyflamendinduced apoptosis and that bothAMPK and ER stress activation by Zyflamend occur upstream of autophagy and apoptosisThe relationship between these two fundamental processes ER stress and autophagy is complex and poorlyunderstood Recent literature demonstrates that bothpathways display dual roles in cell survival in multiple 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Inflammatory ER Stress and Autophagy Responses in TC6 Cells ab Total cell lysates from control and Zyflamendtreated cells for and h were immunoblotted for ER stress markers pPERK pEIF2α pIRE1α their respective unphosphorylatedproteins sXBP1 CHOP and actin as a loading control Representative immunoblots are shown b Bar graphs represent pPERKPERK pEIF2αEIF2α pIRE1IRE1 sXBP1actin and CHOPactin as means SEM p p indicate a significant difference between cells treatedwith Zyflamend and nontreated cells cd Markers of autophagy were examined in the same lysates using antibodies against Beclin LC3 IIIATG5 ATG7 and actin as a loading control d Bar graphs represent Beclin 1actin LC3actin ATG5actin and ATG7actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells ef Immunoblots of keyproteins in autophagy AMPK ER stress and apoptosis signaling in TC6 cells treated with μgml Zyflamend with and without the pancaspase inhibitor ZVADfmk Representative immunoblots are shown f Bar graphs represent pAMPKAMPK pPERKPERK pEIF2αEIF2α pIRE1αIRE1α sXBP1actin CHOPactin pJNKJNK Beclin 1actin LC3IIIactin and cleaved caspase3actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells € p € € p indicate a significant differencebetween cells treated with ZVADfmk and nontreated cellscancer celllines Similar to ER stress autophagy hasbeen shown to promote cell survival by clearing unwanted components from the cells Nonetheless a considerable body of evidence also indicates that bothautophagy and ER stress can lead to apoptosis in tumorcells In addition to this a growing body of literaturesupports existing crosstalk between the two pathways[ ] However which pathway is upstream of theother is yet to be determined Our data suggest thatZyflamendinduced autophagy is likely to be downstream of ER stress To test this hypothesis we pretreated TC6 cells with an ER stress inhibitor phenylbutyrate 4PBA mM for h prior to Zyflamend treatment and we examined changes in inflammation ER stress autophagy and cell death Fig 4ef PBA is a cell permeant chemical chaperone that hasbeen shown to inhibit ER stress and ER stressinducedapoptosis in many cancer cell types including pancreaticcancer cells [ ] Our findings show a profound decrease in ER stress autophagy and cell death markers inresponse to Zyflamend Fig 4ef when cells were pretreated with 4PBA Additionally 4PBA treatment alleviated the decrease in cell proliferation Fig 4g and colonycaused by ZyflamendFurthermore pretreatment of TC6 cells with 4PBAreduced Zyflamendinduced chromatin condensationFig 4jk Conversely 4PBA did not alter the activationof AMPK by Zyflamend Fig 4ef suggesting that ERstress occurs upstream of autophagy and apoptotic celldeathformation Fig 4hiPrevious studies have shown that the ER stress sensorIRE1 may promote autophagy through the TRAF2ASK1JNK pathway [ ] To test this hypothesis wetreated TC6 cells with SP600125 a selective JNK inhibitor and investigated changes in inflammation ERstress and proliferation in response to Zyflamend treatmentFig As expected cells pretreated withSP600125 exhibited a significant reduction in the phosphorylation of JNK and reduced expression of autophagyand cell death markers in response to Zyflamend Fig5ab Likewise JNK inhibition protected TC6 cellsfrom Zyflamendinduced reduction in cell survival Fig5c colo

Thyroid_Cancer

Treatment Strategy for Metastatic Spinal Tumors A Narrative ReviewSam Yeol Chang Sujung Mok Sung Cheol Park Hyoungmin Kim BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Seoul Korea Metastatic spinal tumors are common and their rising incidence can be attributed to the expanding aging population and increased survival rates among cancer patients The decisionmaking process in the treatment of spinal metastasis requires a multidisciplinary approach that includes medical and radiation oncology surgery and rehabilitation Various decisionmaking systems have been proposed in the literature in order to estimate survival and suggest appropriate treatment options for patients experiencing spinal metastasis However recent advances in treatment modalities for spinal metastasis such as stereotactic radiosurgery and minimally invasive surgical techniques have reshaped clinical practices concerning patients with spinal metastasis making a demand for further improvements on current decisionmaking systems In this review recent improvements in treatment modalities and the evolution of decisionmaking systems for metastatic spinal tumors are discussedKeywords Spinal metastasis Decisionmaking system Radiosurgery Minimally invasive surgical procedures Separation surgeryIntroductionThe spine has been identified as the most common site for malignant metastasis in the musculoskeletal system and vice versa spinal metastasis is considered the most common malignant lesion in the spine [] The incidence of metastatic spinal tumors has seen an increasing trend due to the growing aging population worldwide and continued improvements in survival rates among cancer patients [] Symptomatic spinal metastasis is often the first clinical manifestation for “ of cancer patients [] whereas up to of cancer patients may experience spinal metastasis at some point during the course of their disease [] The objectives of surgical treatment in patients with metastatic spine tumors are mostly palliative Spine surgeons make an effort to maintain or improve the patient™s quality of life during the remainder of their survival by reducing pain and preserving ambulatory function via surgical treatment []Because clinical manifestations and treatment responses vary widely among cancer patients a multidisciplinary decisionmaking process that integrates medical and radiation oncology together with surgery along with assistance from pathology and diagnostic radiology deemed is essential when deciding to conduct surgical treatment for spinal metastasis [] In the literature various decisionmaking systems have been developed and introduced to date in an effort to aid in this decisionmaking process Received Jul Revised Jul Accepted Jul Corresponding author BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Daehakro Jongnogu Seoul KoreaTel Fax Email bschangsnuackrCopyright ’¸ by Korean Society of Spine SurgeryThis is an Access distributed under the terms of the Creative Commons Attribution NonCommercial License httpcreativecommonslicensesbync40which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly citedAsian Spine Journal ¢ pISSN eISSN ¢ wwwasianspinejournalAsian Spine JournalASJAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J [] However recent advancements in the treatment of metastatic spine tumors have injected more complexity into this decisionmaking process and demanded the evolution of the decisionmaking system itself these recent advances include the development of stereotactic spine radiosurgery SRS the introduction of minimally invasive surgical techniques and the evolution of various target therapies for individual primary cancers [] In this review we discussed the development and evolution of various decisionmaking systems in spinal metastasis treatment Current concepts and recent trends in radiotherapy and surgery for spinal metastasis are also includedDecisionMaking Systems for Managing Metastatic Spinal TumorsPrognostic factors for metastatic spinal tumorsWhen attempting to choose an appropriate treatment for a patient with spinal metastasis establishing an accurate estimation of the individual™s life expectancy is the most crucial To do this one must first identify prognostic factors associated with the survival of patients with spinal metastasis As such many authors have conducted studies to try and identify prognostic factors associated with survival among spinal metastasis patients and have developed various decisionmaking systems in order to estimate survival based on these prognostic factors []In a recently published metaanalysis Luksanapruksa [] have identified independent prognostic factors associated with the survival of patients with spinal metastasis Among these factors nine factors can be classified as relating to the preoperative performance or neurological status of a patient”for example the Karnofsky Performance Score or Eastern Cooperative Oncology Group grade [] Meanwhile four factors involve the presence or the number of metastases spine bone or visceral and two factors were found to be related to primary tumors in the Tomita classification scheme [] Finally male sex and the time interval from cancer diagnosis to the start of radiotherapy are the two remaining prognostic factors independently associated with survival in spinal metastasis patients Among these primary tumor histology the presence and number of metastases and performance status are proposed as the three most important prognostic factors associated with survival in spinal metastasis patients not only in this study but also in most previous investigations []In other studies the patient™s age and comorbidities assessed using the American Society of Anesthesiologists physical status [] and Charlson Comorbidity Index [] have also been identified to be prognostic factors in spinal metastasis [] Other authors have identified laboratory abnormalities such as leukocytosis and low hemoglobulin and albumin levels as prognostic factors and included these into their decisionmaking systems [] In addition previous systemic treatment or chemotherapy has also been suggested as an independent prognostic factor by multiple authors [] Further not only preoperative chemotherapy but also the presence of available systemic treatments in the postoperative period has been hypothetically regarded as a potential prognostic factor in the literature [] In a recently published study by Chang [] the authors verified the presence of the remaining systemic treatment options to be independently associated with improved postoperative performance status and survivalClassificationbased decisionmaking systemsBased on these prognostic factors numerous decisionmaking systems or œscoring systems have been developed and introduced to estimate the life expectancy among spinal metastasis patients [] In these œclassificationbased decisionmaking systems scores for each prognostic factor identified by multivariate logistic or proportional hazards regression analyses are integrated in order to obtain a total prognostic score that reflects the estimated survival of the patient Surgeons can adopt these prognostic scores to identify patients with an estimated survival profile that is sufficient to warrant surgical treatment Although the prognostic factors included in each system vary primary tumor histology and visceral metastases are included in most systems Table In the New England Spinal Metastasis Score NESMS was introduced by Ghori [] The NESMS was developed using multicenter data and retrospectively validated in the following investigations [] The NESMS consists of modified Bauer score components and score serum albumin level and ambulatory status of the patient More recently the NESMS was validated prospectively in the Prospective Observational Study of Spinal Metastasis Treatment trial which aimed to verify the 0cTreatment of Spinal Metastasis Table Prognostic factors in decisionmaking systemsReferencesBauer [] modifiedTomita [] To kuhashi [] revisedKa tagiri [] revisedGhori [] Paulino [] Primary tumorPerformance statusNo of vertebral metastasesBone metastasisVisceral metastasisPrevious systemic treatmentOther factorsOOOOOOOOOOOOOOOOOOOOOOOOOOBr ain metastasis WBC Hb platelet albumin bilirubin Creactive protein lactate dehydrogenaseSerum albuminAge WBC Hb brain metastasisWBC white blood cell Hb hemoglobin NESMS as a reliable predictive tool in spinal metastasis patients []There have been efforts to develop novel decisionmaking systems using evolving methodologies In the Skeletal Oncology Research Group S compared multiple prognostic survival algorithms including classic nomogram and boosting algorithms using the same retrospective dataset obtained from patients [] In their study the nomogram was found intuitive and demonstrated a comparable level of performance Then in the S used machinelearning algorithms to develop a novel prognostic model for metastatic spinal disease [] which was externally validated in subsequent studies []Although these various œclassificationbased decisionmaking systems are helpful and widely used for predicting the survival of spinal metastasis patients recent studies have reported that the degree of accuracy of these classic systems eg Tomita Tokuhashi decreases over time especially in cancers with poor prognoses such as lung cancer [] This pitfall of œclassificationbased decisionmaking systems reportedly stems from the inability of these systems to reflect survival improvement due to recent evolutions in systemic treatment for primary cancers [] Another existing limitation is that these systems cannot directly guide the selection of specific treatments appropriate for patients with spinal metastasisPrinciplebased decisionmaking systemsAs an alternative to these œclassificationbased decisionmaking systems that are incapable of reflecting recent advances in oncology and guiding specific treatments several authors have proposed œprinciplebased decisionmaking systems These œprinciplebased systems do not œscore the patient and estimate survival but instead provide advice regarding which treatment is more appropriate in individual cases based on the integration of rapidly evolving treatment modalities including target therapies radiosurgery and minimally invasive surgical techniquesThe neurologic oncologic mechanical and systemic NOMS decision framework was first introduced in [] The NOMS decision framework consists of neurologic N oncologic O mechanical M and systemic S considerations integrating novel multimodal therapies including SRS and minimally invasive surgical techniques [] As a neurological N assessment approach the grading system developed by Bilsky and Smith [] was used while surgical decompression is recommended for highgrade spinal cord compressions During oncological O assessment the responsiveness of spinal metastasis to currently available treatments especially the level of tumor sensitivity to radiotherapy is evaluated Mechanically M instability of the spinal column as determined by the Spinal Instability Neoplastic Score SINS indicates the need for surgical stabilization regardless of the neurologic or oncologic status [] Finally systemic S assessment focuses on the patient™s ability in tolerating the suggested treatment Meanwhile if the general condition performance status and medical comorbidities of a patient do not allow surgery to be performed radiotherapy is instead recommendedA modification of the NOMS decision framework has also been presented in the literature Paton introAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J duced the œLMNOP system as an improvement to the NOMS approach [] With this development these authors added two additional key considerations to the NOMS as follows the location and levels of metastasis L and the patient™s response to previous therapy P The P in œLMNOP stands for not only the response to prior therapy but also includes patient fitness and prognosis which was considered previously as part of the systemic S assessment in the NOMS decision framework The authors emphasized that the response of primary cancer to previous treatments including chemotherapy and radiotherapy is considered a significant factor when determining the appropriate treatment for spinal metastasis patients For instance it is anticipated that a patient diagnosed with symptomatic spinal metastasis at the initial presentation of primary cancer synchronous metastasis who would have multiple potential treatment options is more likely to experience a better prognosis than a patient diagnosed with spinal metastasis despite previous treatment metachronous metastasis Differences in the survival rates between the patients with synchronous and metachronous spinal metastasis have been confirmed in a previous research [] Therefore a more aggressive approach including surgical treatment can be considered for patients with a synchronous spinal metastatic lesion In summary œprinciplebased decisionmaking systems relative to œclassificationbased systems are able to better incorporate evolving treatment modalities and guide the selection of appropriate treatments for patients in a timely fashion Table Current trends and future directions in the development of decisionmaking systemsAdvances in cancer biology and treatment modalities are necessitating the evolution of decisionmaking systems for spinal metastasis Possible future directions to take to improve decisionmaking systems include the following the use of multicenter or multinational databases the integration of histologyspecific data the application of computational methodologies such as machinelearning algorithms and the combination of classificationbased and principlebased systems Some recent studies are already covering these trendsThe size of the study sample under assessment determines the performance and accuracy of prognostic models Although spinal metastasis is found to be relatively common data from a larger sample population beyond that of just a single institution is usually required to develop a powerful enough prognostic model For this reason recently introduced prognostic models or decisionmaking systems are generated from multicenter databases [] Future prognostic models should also have the freedom to rely on even larger databases such as multinational tumor registriesBiologic therapy including molecular target therapy and immunotherapy is believed to be an emerging gamechanger in modern cancer treatment Genetic subtype analysis of the primary cancer histology which guides selection among t hese therapies has become more essential [] In a revised prognostic system proposed by Katagiri Table The NOMS decision frameworkNeurologic NOncologic OMechanical MSystemic SDecisionLowgrade ESCCno myelopathyHighgrade ESCC±myelopathyRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadioresistantRadioresistantStableUnstableStableUnstableStableUnstableStableStableUnstableUnstablecEBRTStabilization followed by cEBRTSRSStabilization followed by SRScEBRTStabilization followed by cEBRTAble to tolerate surgeryD ecompressionstabilization followed by SRSUnable to tolerate surgerycEBRTAble to tolerate surgeryDecompressionstabilization followed by SRSUnable to tolerate surgeryStabilization followed by cEBRTNOMS neurologic oncologic mechanical and systemic ESCC epidural spinal cord compression cEBRT conventional external beam radiation SRS stereotactic radiosurgery 0c [] the authors considered the availability of molecular target therapy when classifying the primary tumor For example lung cancer treated with targeted drugs was designated as an example of a moderategrowth tumor while lung cancer without targeted drugs is regarded as an example of a rapidgrowth tumor [] This application of genetic profiles to decisionmaking systems is likely to grow more specific and tailored corresponding to the evolution of molecular genetics in the futureAs previously described machinelearning algorithms have been applied in the development of prognostic models Classically prognostic models for spinal metastasis have been developed using logistic or proportional hazards regression analyses As part of its research efforts the S was able to develop prognostic models using machinelearning algorithms such as gradient boosting decision trees random forests and neural networks [] and these algorithms were externally validated elsewhere [] Like in other fields of medicine evolving computational methodologies including machinelearning algorithms should be assessed extensively in terms of their potential in the management of spinal metastasisFinally the combination of classificationbased and principlebased decisionmaking systems should be considered Classificationbased systems or prognostic models seek to estimate the patient™s remaining survival Based on this survival estimation principlebased systems then may suggest the most appropriate treatment option Until now surgeons and physicians have been employing these two separate systems in the same decisionmaking process A novel decisionmaking system could integrate these two systems together and provide survival estimations and appropriate treatment options simultaneouslyRadiotherapy for Metastatic Spinal TumorsTreatment of Spinal Metastasis single session of SRS achieved durable longterm control of spinal metastasis regardless of histology and tumor size Of note the only significant factor associated with tumor control was the radiation dose These results suggest that SRS can be effective even in cases of metastasis previously considered to be radioresistantSRS can also be a definitive treatment for the management of solitary metastasis without spinal cord compression [] Excellent local control rates of “ have allowed SRS to replace curative surgeries with high morbidities such as total en bloc spondylectomy for addressing these solitary metastases [] In patients with highgrade spinal cord compression SRS can be applied after separation surgery which will be discussed further in the following section Overall the effectiveness of SRS has been changing the role and extent of surgical treatment and in turn shifting the focus of the treatment of spinal metastasisVertebral compression fracture following stereotactic spine radiosurgeryA pitfall of SRS is an increase in vertebral compression fracture VCF following radiotherapy Risk estimates for VCF after SRS are reported to be up to as compared with just in relation to conventional radiotherapy [] The occurrence of VCF is dosedependent and caution is required if the radiation dosage exceeds Gy per fraction in highrisk patients [] Highrisk patients of older ages with lytic lesions andor with spinal malalignment can reportedly benefit from undergoing preventive stabilization surgery before SRS When determining the necessity of stabilization surgery before SRS SINS can be used to identify potentially unstable lesions [] SINS will be further covered in the following sectionStereotactic spine radiosurgery is triggering a paradigm shiftTiming of radiotherapy after surgeryEvidence in the literature supports that radiosurgery is a safe and effective modality for local tumor control with low associated complication rates in patients with spinal metastasis [] Technical improvements including intensitymodulated and imageguided radiation delivery and processing software have allowed SRS to be a gamechanger in the treatment of spinal metastasis [] A recent study by Yamada [] reported that a highdose Adequate timing of radiotherapy following surgery whose determination is related to the risk of wound complications continues to be debated among spine surgeons and radiation oncologists It is also controversial whether the interval can be shortened in patients receiving SRS Lee [] sent questionnaires to radiation oncologists and spine surgeons to gather opinions on the optimal timing of surgery and radiotherapy in spinal metastasis Based on the procured comments the auAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J thors recommended that the interval be at minimum weeks regardless of radiation modalities Interestingly as compared with radiation oncologists surgeons tended to favor a shorter interval of time between surgery and radiotherapy when SRS is performed although there was no statistically significant difference in this regard []A recently published systemic review also advocated for weeks with a minimum of days between surgery and radiotherapy [] When the rates of wound complications were compared between SRS and conventional radiotherapy many studies reported reduced wound complications in SRS patients [] However due to limited highlevel evidence no definite conclusion was made regarding whether the interval could be reduced in patients undergoing SRSSurgery for Metastatic Spinal TumorsSurgical indicationsThe objective of surgical treatment in spinal metastasis was to provide pain relief support neurological improvement and in turn enhance the quality of life during the remaining survival period Clinical benefits of direct surgical decompression in patients with metastatic spinal cord compression MSCC have been well described in the literature [] The most important prerequisite for surgical treatment in spinal metastasis has been identified as the sufficient enough estimated survival time to make surgery a reasonable approach Researchers have largely recommended that a minimum of to months of remaining survival should exist when considering whether to perform surgery [] At this point a number of prognostic scoring systems previously described are being used to estimate a patient™s survival The patient should also have a good enough general condition or performance status to in order to endure surgery If these conditions are satisfied then surgery can be performed in patients with symptomatic MSCC or mechanical instabilityIn spinal metastasis the instability is assessed by the SINS [] Table The SINS is an independent and unique tool that integrates clinical and radiological components to help surgeons decide whether to conduct surgery for stabilization A score of to points suggests impending instability while that of points or more indicates existing spinal instability which requires stabilization As previously mentioned the SINS has also been incorporated into principlebased decisionmaking systems such as the NOMS and LMNOP systems [] Recent studies have reported the reliability and accuracy of the SINS system in predicting spinal adverse events including VCF especially in those patients who received radiotherapy [] although some components may still require revision []Separation surgery and minimally invasive surgeryWhen considering the surgical techniques used for spinal metastasis the literature shows a trend toward the adoption of less invasive techniques which is thought to have Table Spinal Instability Neoplastic Score systemComponentLocationJunctional occiput“C2 C7“T2 T11“L1 L5“S1Mobile spine C3“C6 L2“L4Semi rigid T3“T10Rigid S2“S5PainaYesOccasional pain but not mechanicalPainfree lesionBone lesionLyticMixed lyticblasticBlasticRadiographic spinal alignmentSubluxationtranslation presentDe novo deformity kyphosisscoliosisNormal alignmentVertebral body collapse collapse collapseNo collapse with body involvedNone of the abovePosterolateral involvement of spinal elementsbBilateralUnilateralNone of the aboveScoreaPain improvement with recumbency andor pain with movementloading of spine bFacet pedicle or costovertebral joint fracture or replacement with tumor 0cTreatment of Spinal Metastasis primarily resulted from recent advances in radiotherapy as previously described [] With the use of advanced radiation techniques surgeons can minimize surgical morbidities by avoiding extensive debulking surgery [] Fig During the separation surgery circumferential spinal cord decompression is performed only to the extent required to facilitate safe radiosurgery In a study by Laufer [] separation surgery followed by postoperative SRS resulted in a low local progression rate Other studies have also reported that this hybrid surgery“radiosurgery approach is a safe and effective treatment option for MSCC []BCADFig A 68yearold male with spinal metastasis of renal cell carcinoma at T9 The patient also had lung metastasis A Preoperative MRI shows spinal cord compression and involvement of posterior elements and left rib head at the T9 level B Following a separation surgery without spinal instrumentation MRI at postoperative 2weeks shows a decompressed spinal canal but residual metastatic tumor around the left 9th rib head C In the postoperative 1year MRI the patient showed a nearcomplete response following a single session stereotactic radiosurgery 18Gy1 fraction which was performed weeks after the separation surgery MRI magnetic resonance imaging D Planning images for the postoperative stereotatic radiosurgery following a separation surgery Asian Spine Journal 0cSam Yeol Chang et alAsian Spine J œMinimalaccess surgery is also used in treating spinal metastasis while reducing surgical morbidities In the anterior approach retractor systems and thoracoscopic assistance have been described [] Minimally invasive posterior approaches for decompression and corpectomy have also been introduced and reviewed in the literature [] Other minimally invasive techniques for spine surgery such as the intraoperative stereotactic navigation system and percutaneous pedicle screw instrumentation are being incorporated into spinal metastasis surgery as well []Studies comparing minimally invasive surgery and surgery showed that minimally invasive surgery provided equivalent or superior outcomes with reduced surgical morbidity and complications in spinal metastasis patients [] However because the quality of evidence is deemed low in the current literature no definite conclusion regarding the superiority of minimally invasive surgery over surgery can be derived and no strong recommendations have been made at this point []Role of curative surgery en bloc resectionMost surgeries for spinal metastasis are found palliative and the role of en bloc resection in spinal metastasis is decreasing even further due to improvements in radiotherapy Generally curative surgical resection of spinal metastasis has been considered in the context of a single metastasis of a slowgrowing tumor such as in renal cell thyroid and breast cancers Fig Favorable outcomes in this regard have been reported in the literature [] However some authors recently reported that curative ABECDFig A 63yearold male with spinal metastasis of thyroid carcinoma at T8 A Preoperative MRI shows pathologic fracture and spinal cord compression at the T8 level B Postoperative Xray at month shows removal T8 vertebra and reconstruction with an expandable cage following total en bloc spondylectomy C MRI at postoperative 3years shows a widely decompressed spinal canal with no tumor recurrence D Postoperative Xray at postoperative 5years shows wellmaintained instrumentation E Bone scan at postoperative 5years shows no evidence of bone metastasis MRI magnetic resonance imaging 0csurgical resection en bloc spondylectomy did not impact the oncologic outcomes of spinal metastasis patients [] Therefore a more careful and thorough decisionmaking process is required before performing curative resection surgery for spinal metastasis especially when the extended role of radiosurgery is consideredPostoperative complications and preventive measuresThe overall complication rate following surgery for spinal metastasis ranges from to in the literature [] Because surgery for spinal metastasis is performed to improve the quality of life of a patient surgeons should try to minimize all possible surgical and medical complications by implementing multidisciplinary interventions Among diverse complications those that require additional attention when found in spinal metastasis patients eg wound infection instrumentation failure and intraoperative bleeding are briefly discussed hereThe incidence of surgical site infection SSI is determined to be higher in spinal metastasis surgery reaching up to as compared with during other spine surgeries [] SSI has also been identified as the most common cause for reoperation in of reoperations following surgery for spinal metastasis [] Poor nutrition and exposure to adjuvant therapies chemotherapy and radiotherapy put spinal metastasis patients at risk for SSI [] Surgeons should provide adequate nutritional support perioperatively and secure a sufficient time interval between radiation and surgery as previously discussed to minimize SSIs Additional risk factors such as smoking obesity and medical comorbidities should also be considered []The second cause for reoperation in spinal metastasis surgery is the failure of instrumentation [] Risk factors associated with instrumentation failure include the number of operated levels prior chest wall resection and history of radiotherapy [] The necessity of additional fusion procedures while performing surgery for spinal metastasis is debated but highlevel evidence remains to be lacking at this time [] In future studies we suggest that instances of early and late failure be distinguished when assessing instrumentation failure because the mechanisms of failure between the two types seem to differ ie insufficient stability of the construct in the context of early failure versus the progression of deformity or lack of fusion in the context of late failureTreatment of Spinal Metastasis Intraoperative bleeding during spinal metastasis surgeries can be massive and can further lead to serious complications such as cardiovascular or cerebral events [] For the spinal metastasis of hypervascular tumors such as renal cell hepatocellular and thyroid cancers preoperative embolization is recommended Previous studies have verified the effectiveness of preoperative embolization in reducing intraoperative bleeding in spinal metastasis surgeries [] Surgery should be performed within hours following embolization to avoid the diminished effect of preoperative embolization []ConclusionsThe determination of appropriate treatment for a patient with spinal metastasis is a challenging task that requires multidisciplinary considerations Recent advances in radiotherapy and surgery for spinal metastasis have brought about improvements in the management of these patients Evolving decisionmaking systems are also crucial contributors to stateoftheart care of patients with metastatic spinal tumorsConflict of InterestNo potential conflict of interest relevant to this was reportedReferences White AP Kwon BK Lindskog DM Friedlaender GE Grauer JN Metastatic disease of the spine J Am Acad Orthop Surg Laufer I Sciubba DM Madera M Surgical management of metastatic spinal tumors Cancer Control Schiff D O™Neill BP Suman VJ Spinal epidural metastasis as the initial manifestation of malignancy clinical features and diagnostic approach Neurology Klimo P Jr Schmidt MH Surgical management of spinal metastases Oncologist Nathan SS Healey JH Mellano D Survival in patients operated on for pathologic fracture implications for endoflife orthopedic care J Clin Oncol Curtin M Piggott RP Murphy EP Spinal metaAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J static disease a review of the role of the multidisciplinary team Orthop Surg Ahmed AK Goodwin CR Heravi A Predicting survival for metastatic spine disease a comparison of nine scoring systems Spine J Barzilai O Fisher CG Bilsky MH State of the art treatment of spinal metastatic disease Neurosurgery Tomita K Kawahara N Kobayashi T Yoshida A Murakami H Akamaru T Surgical strategy for spinal metastases Spine Phila Pa Tokuhashi Y Matsuzaki H Oda H Oshima M Ryu J A revised scoring system for preoperative evaluation of metastatic spine tumor prognosis Spine Phila Pa Bauer HC Wedin R Survival after surgery for spinal and extremity metastases prognostication in patients Acta Orthop Scand Van der Linden YM Dijkstra SP Vonk EJ Marijnen CA Leer JW Dutch Bone Metastasis Study Group Prediction of survival in patients with metastases in the spinal column results based

Thyroid_Cancer

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

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"Optimizing Telemedicine Encounters for Oral and Maxillofacial Surgeons During the COVID19 Pandemic Hwi Sean Moon DDS MD1 Tim T Wang BA23 Karthik Rajasekaran MD4 Ryan Brewster BA5 Rabie M Shanti DMD MD46 Neeraj Panchal DDS MD MA7 1Resident Department of Oral Maxillofacial Surgery University of Pennsylvania Philadelphia PA 2DMD Candidate School of Dental Medicine University of Pennsylvania Philadelphia PA 3MPH Candidate Perelman School of Medicine University of Pennsylvania Philadelphia PA 4Assistant Professor of Otorhinolaryngology University of Pennsylvania Philadelphia PA 5MD Candidate Stanford University School of Medicine Stanford University Stanford CA 6Assistant Professor of Oral and Maxillofacial Surgery University of Pennsylvania Philadelphia PA 7Assistant Professor and Section Chief of Oral and Maxillofacial Surgery Philadelphia Veterans Affairs Medical Center Penn Presbyterian Medical Center University of Pennsylvania School of Dental Medicine Philadelphia PA Corresponding Author Neeraj Panchal DDS MD MA Tel Mailing Address N 39th St Philadelphia PA Email Address npanchalupennedu Disclosures None to report Abstract Word Count Manuscript Word Count Number of References Number of Figurestables Number of Supplements 0c The COVID19 pandemic has changed conventional medical practice patterns across all health disciplines including oral and maxillofacial surgery practices The use of telemedicine has rapidly expanded to uphold safety strategies of physical distancing and disease transmission reduction while maintaining uninterrupted care of patients To date there are no specific guidelines to optimize telemedicine encounters in the oral and maxillofacial surgery practice The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond Statement of Clinical Relevance The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond INTRODUCTION The COVID19 pandemic has disrupted society in a multitude of ways Healthcare is no exception the SARSCoV virus™ rapid transmission and high hospitalization rate have strained the availability of medical resources including personal protective equipment PPE respiratory ventilators and hospital beds[“] The virus also poses a major threat to healthcare personnel whose risk of exposure are compounded by the aforementioned PPE shortages[“] In response the American Association of Oral and Maxillofacial Surgeons AAOMS recommended delaying elective surgeries in accordance with the Centers for Disease Control and Prevention™s calls to 0c postpone elective medical and dental procedures[“] Similarly four out of five dental offices have closed for all except emergency procedures[] In the face of these challenges the medical and dental communities have remained steadfast in caring for patients with nonelective health needs and innovating alternate ways to deliver care One of the most important and popular alterations in the delivery of care is the increased utilization of telemedicine which allows surgeons and patients to connect virtually[ ] This has enabled patients to access muchneeded medical care while preserving PPE and minimizing exposure to pathogens Though studies have found telemedicine to decrease costs and save time without compromising patient satisfaction it was not widely used in healthcare before the COVID19 pandemic[ ] Similarly teledentistry was deemed to be œin its infancy by the founder of the American Teledentistry Association in [“] Nevertheless telemedicine has shown promise and has been incorporated into the workflow of various oral and maxillofacial surgery institutions and practices across the country Virtual visits are particularly useful in triaging patients For example patients with dentoalveolar infections can meet virtually with surgeons and receive prescriptions for appropriate analgesics and antibiotics without going to the emergency department Also patients with oral lesions can take images and show their surgeon before their inperson visit to expedite the diagnosis and treatment planning workflow This enables patients to access timely attention of providers while lightening the load on the healthcare system by reducing the number of inperson visits 0c Associated with the recent rise in telemedicine™s popularity is a learning curve for both surgeons and patients The incorporation of technology and the shift to virtual visits can be jarring for the patientsurgeon relationship and must be navigated thoughtfully While there have been helpful telehealth guides for surgeons and patients in other surgical specialties we do not know of any such guidelines for oral and maxillofacial surgery[ ] As such we detail best practices for both oral and maxillofacial surgeons OMS and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond GENERAL CONSIDERATIONS FOR TELEMEDICINE In accordance with the AAOMS White Paper on ˜Telehealth and Remote Treatment™ virtual management of any oral and maxillofacial surgical condition should only be provided by appropriately licensed oral and maxillofacial surgeons as regulated by the state law[] The delivery of patient care through telemedicine must continue to follow evidencebased guidelines to ensure quality and safety for all patients All providers must comply with the latest telehealth requirements outlined by the United State Health and Human Services to protect patient privacy and comply with the Health Insurance Portability and Accountability Act HIPAA[] Furthermore providers are ethically obligated to inform all patients about the potential benefits limitations and risks of telemedicine[] Patients requiring emergency or urgent services must be directed to the nearest hospital 0c SETTING UP FOR TELEMEDICINE While there are several modalities to conduct a telemedicine encounter we strongly recommend a live synchronous twoway interaction between the patient and the OMS incorporating both audio and visual telecommunications tools This can be achieved with a desktop computer laptop or smartphone The United States Census Bureau reports that approximately percent of households have computers or smartphones and percent have broadband internet subscriptions[] Of these options though we recommend using a desktop or a large screen laptop with a highresolution camera over smartphones even if the latter meets the minimum technical requirements Ideally telemedicine visits can offer a clinic experience that closely simulates inperson encounters Trained administrative staff members should call patients beforehand to discuss the virtual setup and basic expectations for the visit Prasad et al created Figure which exemplifies a patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the encounter [] In the following we will detail key aspects and considerations for both OMS and patients to maximize their telehealth visits 0c Insurance Coverage and Billing In an effort to reduce the burden posted on healthcare entities and facilitate mitigation efforts the Centers for Medicare Medicaid Services CMS broadened access for Telemedicine coverage with private payers following suit CMS expansion included voiceonly visits which is critical for patients without access to a smartphone or computer video capabilities Furthermore CMS allowed for parity of payment for Telemedicine visits and inperson visits so providers can bill Medicaid and Medicare at the same rate as they would for an inperson visit[] This new policy is especially relevant for older patients covered by Medicare for they are generally at higher risk for COVID19 complications[] Therefore before telehealth encounters providers should confirm if the patient™s insurance plan has Telemedicine coverage and also whether the insurance plan waives all copays for nonCOVID19 related visits to avoid a scenario where a patient receives a bill unknowingly The American Association of Oral and Maxillofacial Surgeons provide additional detailed information about telehealth billing relevant for OMSs including updated links to AMA and ADA billing codes on its website at httpswwwaaomsorgpracticeresourcestelehealthfaqs 0c Professionalism and provider attire The visit should replicate the same level of professionalism as that of inperson appointments Providers should dress professionally as they would in their office or hospitalbased practice Before the patient comes in providers should review the patient™s relevant medical records and chief complaints to save time and maximize the efficiency of the visit Also in the interest of both time and professionalism OMS and patients should both be mindful to start the visit on time During the visit OMS should communicate with patients to maintain transparency For instance if an OMS needs to document something during the visit they should respectfully inform the patient of the task to prevent any misunderstanding At the end of the visit it is important for OMS to summarize what they accomplished during the visit and provide a clear plan for appropriate next steps Physical background When possible OMS and patients should conduct virtual encounters in welllit spaces Lighting specifically can have a profound effect on video quality As such overhead lights can be helpful while lights behind the person should be avoided Care should also be taken to prevent other sources of potential disruptions such as background noise or visual distractions It may be helpful for OMS to evaluate their surroundings from the perspective of their patients 0c Technological background It is important for OMS to test their video and audio quality before visits to anticipate any potential technical difficulties that may interrupt the encounter A strong WiFi internet connection is preferred over cellular data to ensure a stable signal With the exception of electronic health records OMS should close any unnecessary programs or internet browser tabs to preserve internet bandwidth In addition OMS should be mindful that their patients may have varying internet speeds As such OMS should give approximately seconds of lag time after patients stop speaking to allow all of their words to come through completely Patient and camera position If possible patients should sit upright on a chair in front of a computer placed on top of a desk They should sit close enough to the camera so that their entire head and neck area are within the video frame The camera should be at approximately eyelevel for both OMS and patients to maintain eye contact and remain engaged during the virtual encounter For patients using a smartphone the device can be propped up at a to 90degree angle from the table surface to allow patients to free both of their hands for physical exam tasks Patient clothing Patients must be notified of the appropriate clothing well in advance prior to the appointment Ideally clothing should allow patients™ entire head and neck regions to be visualized while maintaining patient comfort and professionalism Any hat or scarves should be removed if at all possible maintaining appropriate cultural and religious norms Patient items Prior to the appointment patients should prepare the following items which can help aid OMS in visualization and retraction during the virtual physical exam Most of these items are commonplace inexpensive and available at the patient™s home 0c Flashlight A flashlight or a penlight can enhance visualization of certain obscure head and neck structures particularly those in the oral cavity The builtin flashlight of a smartphone can also be used Ruler A ruler or a measuring tape can be used to measure the patient™s maximal mouth opening and mandibular range of motion Napkins A napkin can be used to touch any intraoral landmarks and also to clean up after any inadvertent salivation from the virtual physical exam Spoon A spoon can be used to retract the cheeks or depress the tongue to evaluate structures of the oropharynx such as the soft palate and tonsillar pillars Cheek retractors A fun way for patients to achieve cheek retraction can be to use the plastic props from the board game œSpeak Out which was created in [] This game provides horseshoeshaped plastic retractors shown in Figure that can be placed along the patient™s upper and lower lips to lateralize the cheeks thus allowing handsfree visualization of the dentition and oral cavity soft tissue Patient assistant If available patients should ask a family member or friend to accompany them to the telehealth visit The assistant can help patients perform the physical tasks required during the virtual physical exam Assistants can also help position the web camera to improve the provider™s view In fact these assistants are essentially mandatory for pediatric patients or patients with disabilities Feedback It is a good practice for providers to seek feedback from patients after a visit This will help OMS hone their telehealth skills and ensure that their patients are receiving an appropriate quality of care 0c THE VIRTUAL HISTORY AND PHYSICAL Thorough patient assessment proper medical documentation and appropriate diagnostic testing are critical components of OMS practice that enable proper diagnosis and treatment planning OMS should obtain patients™ medical histories in a similar manner as they would in their offices New patients must be asked for comprehensive histories that include chief complaint history of present illness past medical history past surgical history dental history medications allergies pertinent family history social history and complete review of systems For patients of record their medical histories should be updated to reflect their current chief complaint All patients should also be screened with an up to date COVID19 questionnaire If an infection is suspected OMS should refer patients to their primary care physicians or local emergency department depending on the severity of symptoms for appropriate workup Vitals should be obtained if the patient has access to a thermometer blood pressure cuff pulse oximeter or weighing scale Even without any of these devices patients can still measure their pulse by applying two fingers on the patient™s carotid counting the number of beats per minute Also patients can calculate their respiratory rate by observing the number of chest rises in one minute Finally oxygen saturation can be measured with certain mobile health applications though OMS should not solely rely on their results for major medical decisions Finally patients with fever body temperature F warrants further work up in an emergency setting for a differential diagnosis that includes COVID19 infection The virtual physical exam will be limited to a head and neck exam and a cranial nerve exam While inspection and palpation are the basis of a focused physical examination in oral and maxillofacial surgery OMS must learn to work together with patients to achieve the same goals 0c virtually Patients must perform maneuvers on themselves with the OMS™s guidance To this end a printed stepbystep schematic as illustrated in Figure can be helpful for patients to receive before the visit During the visit the OMS can reinforce the diagram with clear verbal instructions that avoids medical jargon The exam itself must be conducted systematically with a topdown outsidein approach as is typical in an oral and maxillofacial surgery practice The exam can be further divided into head and neck subsites The OMS should ask for specific symptoms related to each subsite and carefully inspect for any abnormalities while guiding the patient or the patient™s assistant through the exam The following section offers additional details and considerations for each subsite Head The OMS should ask about any history of head trauma The head is assessed to ensure that it is normocephalic and atraumatic Face The OMS should ask the patient about any facial pain swelling weakness numbness or history of trauma to the region Then OMS can start the facial exam by asking the patient to lean close to the camera First the face is examined for any skin lesions along the forehead eyelids external ears nose malar region vermilion of the lips and the chin Patients™ left and right sides of the face should be compared for any gross asymmetry or deformities OMS can then guide patients through palpating their own face for any bony discontinuity or soft tissue swelling Patients can also tap their own face with two fingers to reveal any tenderness in the sinuses Regarding the eyes OMS can assess if the pupils are equal The extraocular muscles along with the oculomotor supratrochlear and the abducens nerves can be tested by having the 0c patient look up down left and right without moving the head The sensory portion of the trigeminal nerve can be tested by asking patients to close their eyes and slide both of their index fingers horizontally along the ipsilateral forehead ophthalmic branch cheek maxillary branch lip and chin mandibular branch The branches of the facial nerves can be tested by asking patients to raise their eyebrows close their eyes tightly puff out their cheeks smile widely and show their bottom teeth Temporomandibular joint TMJ The OMS should ask the patient about any facial jaw or ear pain trismus difficulty mastication clicking or locking of the joint Then the TMJ exam begins by asking patients to palpate their mandibular condyles and muscles of mastication to look for any tender spots Providers can ask patients to open and close their mouth while palpating the condyles to feel for any clicks or crepitus Also maximal interincisal opening can be roughly estimated by the number of fingerbreadth or precisely measured using a ruler The mandibular range of motion can be assessed or measured in protrusive and lateral excursive positions Neck The OMS should ask for any difficulty breathing dysphagia sore throat odynophagia hoarseness or new neck swelling The neck exam begins with inspection looking for any asymmetry or tracheal deviation Patients can be asked to turn the head from side to side look upwards and shrug the shoulders to assess the spinal accessory nerves OMS should ask the patient™s assistant if possible to stand right behind the patient and palpate the patient™s neck Using their fingertips on both hands the assistant can palpate the neck in a unidirectional manner from superior to inferior and then from lateral to medial Ask them to note any palpable bumps or tender spots It is particularly important to palpate the lateral neck for enlarged lymph nodes 0c Lastly OMS can identify the thyroid by asking patients to swallow while palpating the appropriate area on the neck to rule out thyromegaly Oral cavity and oropharynx The OMS should ask for any oral pain oral swelling or sores tongue numbness difficulty with tongue movement or dry mouth Examination of the oral cavity exam can be challenging because intraoral structures can be difficult to retract and illuminate The aforementioned cheek retractors whether from a board game or makeshift spoons can be helpful for retraction of soft tissue and visualization In addition patients™ friends and family can help a great deal by adjusting the camera while also properly angling an additional light source For each intraoral structure the OMS must carefully inspect for ulcers raised lesions abnormal white leukoplakic or bright red erythroplakic lesions In general OMS can best visualize structures near or at the level of the maxilla when patients lift their heads up to degrees Likewise structures near or at level of the mandible are best observed with the patient dropping the chin approximately degrees OMS may find it useful to practice these examination techniques on their own cameras before the visit Patients should be recommended to wash their hands or to use gloves before touching any intraoral landmarks The exam begins by sliding the patient™s index finger along the maxillary and mandibular vestibule to look for any swelling or fluctuance With the cheeks retracted the patient can palpate their buccal and labial mucosa using the thumb and index finger with one 0c finger compressing along the face extraorally When possible palpation should be bidigital Next the patient can use their index figures to palpate the tuberosity retromolar trigone and the hard palate for tenderness or irregularities The tongue is the most common site for oral cancer and must be thoroughly examined The dorsal surface of the tongue should be examined by asking the patient to fully protrude their tongue Providers should also ask patients to move their protruded tongues to the left and right to inspect the lateral tongue and ensure the function of the hypoglossal nerves The ventral tongue and the floor of the mouth can be observed by asking patients to touch the tip of their tongue to their hard palate The tongue can then be palpated for lumps or masses Next the sublingual and submandibular glands can be palpated for symmetry and lack of elevation by the patient with their extended index fingers on the floor of the mouth The examination of the oropharynx is mostly limited in virtual encounters Nevertheless the soft palate tonsils and uvula can be partially visualized with the patient™s mouth wide open and using a spoon to depress the tongue Although unpleasant the glossopharyngeal and vagus nerves can be tested by gently touching the soft palate using a spoon to induce a gag reflex Dentition If the patient is dentate the provider should ask about dental pain sensitivity loosening of teeth bleeding or sore gums or malocclusion The patient™s dentition can be evaluated after retracting soft tissue as described previously Dental caries missing teeth periodontal disease gingival lesions or swelling can be readily identified Mobility of teeth can be assessed by using the patient™s thumb and index finger In edentulous patients the alveolar 0c ridge should be examined for any abnormalities as part of the aforementioned oral soft tissue exam CONCLUSION The COVID19 pandemic has catalyzed an exponential increase in telemedicine usage Telemedicine helps patients maintain access to care conserves limited medical resources and protects both OMS and patients from pathogen exposure Nevertheless there is an expected learning curve that accompanies such a paradigm shift in the delivery of care As such this paper provides a guide of best practices to aid both OMS and patients to navigate this promising electronic tool In addition we provide an accessible schematic handout that can be given to patients before a telehealth appointment to help them prepare for the visit for both setting up and performing physical exam procedures Because telemedicine may have a role in oral and maxillofacial surgical care even after this pandemic we are optimistic that these best practices can be helpful and relevant for the present situation and beyond 0c FIGURE LEGEND Figure Patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the telemedicine encounter Reprinted from [] 0c Figure Horseshoeshaped plastic lipcheek retractors REFERENCES Li Q Guan X Wu P et al Early Transmission Dynamics in Wuhan China of Novel CoronavirusInfected Pneumonia N Engl J Med “ httpsdoiorg101056NEJMoa2001316 Feng S Shen C Xia N et al Rational use of face masks in the COVID19 pandemic Lancet Respir Med httpsdoiorg101016S221326002030134X Li R Rivers C Tan Q et al Estimated Demand for US Hospital Inpatient and Intensive Care Unit Beds for Patients With COVID19 Based on Comparisons With Wuhan and Guangzhou China JAMA Netw Open 3e208297“e208297 httpsdoiorg101001jamanetworkopen20208297 White DB Lo B A Framework for Rationing Ventilators and Critical Care Beds During the COVID19 Pandemic JAMA httpsdoiorg101001jama20205046 Heinzerling A Stuckey MJ Scheuer T et al Transmission of COVID19 to Health Care Personnel During Exposures to a Hospitalized Patient Solano County California 0c February MMWR Morb Mortal Wkly Rep “ httpsdoiorg1015585mmwrmm6915e5 Ng K Poon BH Kiat Puar TH et al COVID19 and the Risk to Health Care Workers A Case Report Ann Intern Med httpsdoiorg107326L200175 Halepas S Ferneini EM A Pinch of Prevention is Worth a Pound of Cure Proactive Dentistry in the Wake of COVID19 Journal of Oral and Maxillofacial Surgery httpsdoiorg101016jjoms202003036 AAOMS Member Alert COVID19 Update Healthcare Facilities Preparing for Community Transmission In Centers for Disease Control and Prevention httpswwwcdcgovcoronavirus2019ncovhcpguidancehcfhtml Accessed May CDC Guidance for Providing Dental Care During COVID19 In Centers for Disease Control and Prevention httpswwwcdcgovoralhealthinfectioncontrolstatementCOVIDhtml Accessed May Carey M Second week of HPI polling shows dentists™ response to COVID19 American Dental Association Hollander JE Carr BG Virtually Perfect Telemedicine for Covid19 New England Journal of Medicine “ httpsdoiorg101056NEJMp2003539 Prasad A Brewster R Newman JG Rajasekaran K Optimizing your telemedicine visit during the COVID19 pandemic Practice guidelines for patients with head and neck cancer Head Neck na httpsdoiorg101002hed26197 Russo JE McCool RR Davies L VA Telemedicine An Analysis of Cost and Time Savings Telemedicine and eHealth “ httpsdoiorg101089tmj20150055 Cain SM Moore R Sturm L et al Clinical assessment and management of general surgery patients via synchronous telehealth Journal of Telemedicine and Telecare httpsdoiorg1011771357633X16636245 Teledental Practice and Teledental Encounters An American Association of Teledentistry Position Paper Jampani ND Nutalapati R Dontula BSK Boyapati R Applications of teledentistry A literature review and update J Int Soc Prev Community Dent “ httpsdoiorg1041032231076297695 Wicklund E Dentists Use Telehealth to Improve Access to Care And Fight a Phobia In mHealthIntelligence httpsmhealthintelligencecomnewsdentistsusetelehealthtoimproveaccesstocareandfightaphobia Accessed May 0c Smith WR Atala AJ Terlecki RP et al Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program during the COVID19 Pandemic Journal of the American College of Surgeons httpsdoiorg101016jjamcollsurg202004030 AAOMS White Paper on Telehealth and Remote Treatment Notification of Enforcement Discretion for Telehealth Remote Communications During the COVID19 Nationwide Public Health Emergency In HHSgov httpswwwhhsgovhipaaforprofessionalsspecialtopicsemergencypreparednessnotificationenforcementdiscretiontelehealthindexhtml Accessed May Ethical Practice in Telemedicine In American Medical Association httpswwwamaassnorgdeliveringcareethicsethicalpracticetelemedicine Accessed May Ryan C Computer and Internet Use in the United States United States Census Bureau Additional BackgroundSweeping Regulatory Changes to Help US Healthcare System Address COVID19 Patient Surge CMS In CMSgov httpswwwcmsgovnewsroomfactsheetsadditionalbackgroundsweepingregulatorychangeshelpushealthcaresystemaddresscovid19patient Accessed Jun Medicaid Learning Network Telehealth Services Hasbro Brings Mouth Piece Challenge to the Masses with New SPEAK OUT Game In Business Wire httpswwwbusinesswirecomnewshome20160624005633enHasbroBringsMouthPieceChallengeMassesNew Accessed May 0c"

Thyroid_Cancer

Patients with resected oral cavity squamous cell carcinoma OCSCC are often treated with adjuvantradiation RT ± concomitant chemotherapy based on pathological findings Standard RT volumes include all surgicallydissected areas including the tumour bed and dissected neck RT has significant acute and longterm toxicitiesincluding odynophagia dysphagia dermatitis and fibrosisThe goal of this study is to assess the rate of regional failure with omission of radiation to the surgically dissectedpathologically node negative pN0 heminecks compared to historical control and to compare oncologic outcomestoxicity and quality of life QoL profiles between standard RT volumes and omission of RT to the pN0 neckMethods This is a multicentre phase II study randomizing patients with T1“ N0“ OCSCC with at least one pN0hemineck in a ratio between standard RT volumes and omission of RT to the pN0 heminecks Patients will bestratified based on overall nodal status nodal involvement vs no nodal involvement and use of concurrentchemotherapy The primary endpoint is regional failure in the pN0 heminecks we hypothesize that a 2year regionalrecurrence of or less will be achieved Secondary endpoints include overall and progressionfree survival localrecurrence rate of salvage therapy toxicity and QoLDiscussion This study will provide an assessment of omission of RT to the dissected pN0 heminecks on oncologicoutcomes QoL and toxicity Results will inform the design of future definitive phase III trialsTrial registration Clinicaltrialsgov identifier NCT03997643 Date of registration June Current version onJuly Keywords Head and neck cancer Oral cavity Radiotherapy Recurrence Survival Quality of life Randomized controlledtrial Deescalation Correspondence pencillalanglhsconca1Division of Radiation Oncology London Health Sciences Centre Commissioners Rd E London ON N6A 5W9 CanadaFull 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 0cLang Radiation Oncology Page of BackgroundPatients with resected squamous cell carcinoma of the oralcavity OCSCC are at risk of locoregional failure recurrence LRR at either the tumour surgical bed or in theneck Postoperative radiotherapy PORT is often addedafter surgical resection to reduce the risk of local and regional recurrence in patients with highrisk featuresGuidelines generally recommend PORT in patients withmore than one lymph node involved pT3 or pT4 primarydisease lymphovascular invasion LVI perineural invasion PNI close or positive margins extranodal extensionENE and sometimes for patients with lymph node involvement of neck levels IV or V [“]ENE and positive surgical margins are considered thehighestrisk pathological features whereas the other adverse features are considered ˜intermediate risk™ Patientswith highrisk features are generally offered concurrentchemotherapy with PORT based on a posthoc analysistwo of randomized trials [“] In patients with otheradverse features there is limited randomized evidence ofthe benefits of PORT alone [“] Retrospective seriesand comparisons with historical controls have shown reductions in LRR and improvement in overall survivalOS with PORT [“]RT treatment volumes after surgery generally includethe entire surgical bedincluding the dissected heminecks and may include the contralateral undissectedneck at the discretion of the treating physician []However PORT is associated with significant acute andincluding dysphagia mucositis xerostolate toxicitiesmiavoicechanges ototoxicity and hypothyroidism [ ] LargerRT treatment volumes are associated with increasingtoxicity These large treatment volumes are based onhistorical practice without guidance from randomizedevidence The benefit of treating the nodal regions in thepathologically node negative pN0 neck is unknownosteoradionecrosisdermatitisfibrosisRetrospective studies looking at series of oral cavitycancer patients in whom PORT was omitted altogetherhave also demonstrated low rates of isolated nodal recurrence ranging from to [“] In their seriesof patients with mostly T1T2 oral tongue tumourswith a pN0 neck treated with surgery alone PORTto the primary site or PORT to the primary siteand neck So reported a rate of isolatednodal recurrence of “ in all groups [] Mizrachi described a series of patients with T1T4 disease and pN0 neck with not receiving PORT with aneck recurrence rate of [] In Ganly patients with pT1T2 pN0 oral tongue cancer withoutPORT had an isolated regional recurrence rate of with an additional having a simultaneous locoregional recurrence In this study depth of invasion was predictive of neck recurrence []In a series of patients with a pN1 neck the neck recurrencefree survival was in those receiving PORT vs in thosewithout PORT [] It is difficult to draw comparisonsbetween groups receiving or not receiving PORT inthese retrospective studies since those receiving PORThad more risk featuresRetrospective data from a small number of PORT volume studies also demonstrate good oncologic outcomesin patients where radiation was omitted to the contralateral clinically or pathologically N0 neck [ ] In Vegeer patients with welllateralized oral cavity ororopharynx squamousSCC weretreated with unilateral PORT with of patients N0and N1 or N2 Contralateral metastases developedin only of patients with most successfully receivingsalvage therapy []carcinomacellA recent nonrandomized prospective phase II trialeliminated PORT to the pN0 neck in patients withhead and neck squamous cell carcinoma HNSCCdemonstrating excellent results with no isolated failuresand control in the unirradiated pN0 neck Twentypercent of patients included in the study had oral cavitytumours []Taken together the existing retrospective and prospective data suggests that omitting PORT in patientswith a pN0 neck likely has a recurrence rate less than“ However no randomized studies have directlyexamined the omission of PORT in the pN0 neck Thereis also a paucity of data examining the effects of omission of PORT on QoL We hypothesize that omittingPORT to the heminecks that have been dissected andshown to be pN0 will be associated with acceptable ratesof regional recurrence and will improve quality of lifeQoL The goal of this randomized phase II study is toassess oncologic outcomesfunctional outcomes andQoL in patients treated with PORT to the historicallystandard volumes usually including the primary site andall dissected neck areas vs PORT only to the primarysite and pathologically involved hemineck omitting radiation to the pN0 heminecksMethods designObjectivesThe objectives of this trial are to Compare regional recurrence rate to historicalcontrols with omission of PORT to the pN0 neck Compare oncologic outcomes toxicity and QoL forPORT ± chemotherapy based on standardtreatment volumes including the primary sitetumour bed dissected neck ± elective nodalregions vs PORT [± chemotherapy] that avoidstreating the dissected pN0 neck 0cLang Radiation Oncology Page of anisation for Research and Treatment of CancerEORTC Quality of Life Cancer Patients generalQLQC30 [] and head neck HN35 scales[] the EuroQOL 5Dimension 5Level EQ5D5L [] and the Neck Dissection Impairment IndexNDII [] 0f QoL measured at treatment completion and and monthsmeasured with MDADI EORTC QLQC30HN35 EQ5D5L and NDII 0f Rate of feeding tube insertion after start of radiationeither gastric gastrojejeunal or nasogastric andrate of feeding tube use at 1year postrandomization 0f Swallowing function at 1year assessed by ModifiedBarium Swallow MBS study and measured by theModified Barium Swallow Impairment ProfileMBSImP„¢ score [] the Dynamic ImagingGrade of Swallowing Toxicity DIGEST„¢ score []and the Functional Oral Intake Scale FOIS [] 0f Toxicity assessed using the National CancerInstitute Common Toxicity Criteria NCICTCversion 0f Rate of failure in the clinically node negative neck ifapplicable ie the undissected nodenegative neckfor welllateralized tumoursInclusion criteria 0f Age years or older 0f Willing to provide informed consent 0f Eastern Cooperative Oncology Group ECOG 0f Resected OCSCC with at least an ipsilateral selectiveneck dissection The oral cavity includes lips buccalperformance status “It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin Our hypothesis is that for patients with SCC ofthe oral cavity T1“ N0“ as per AJCC 8th editionwith at least one surgically dissected pN0 hemineck theregional failure rate will be or less at years whentreated with PORT omitting the pN0 neckStudy designThis is an label phase II multicentre randomizedtrial Patients will be randomized between current standard of care treatment Arm vs omission of radiationto the pN0 dissected heminecks Arm in a ratioFig The required sample size is patients Stratification factors include neck nodal status pN0 vs pNand use of chemotherapy Patients will be recruited fromtertiary care centres full list of participating sites available on clinicaltrialsgov NCT03997643Primary endpoint 0f Regional failure in the pN0 heminecksSecondary endpoints 0f OS 0f Diseasefree survival DFS 0f Local recurrence 0f Regional recurrence 0f Locoregional recurrence 0f Rate of salvage treatment surgery ± radiotherapy inthe pN0 neck and freedom from unsalvageable neckrecurrence 0f QoL at year assessed with the MD AndersonDysphagia Inventory MDADI [] the EuropeanFig Study schema 0cLang Radiation Oncology Page of mucosa oral tongue floor of mouth gingivaretromolar trigone and hard palate 0f Patient has at least one pathological feature that isan indication for PORT positive or close ‰¤ mmmargin presence of LVI or PNI pT3 or pT4 diseasepositive lymph nodes or ENE 0f PORT is recommended by the treating physician 0f Pathologically lymph node negative in at least onedissected hemineck with at least nodes recovered in each pN0 hemineck after a dissection thatat minimum includes nodal levels “ in the pN0heminecks 0f Radiation contours have been peerreviewed andapprovedExclusion criteria 0f Patients with an ipsilateral neck dissection only withpositive lymph nodes unless they undergo acontralateral neck dissection that is pN0 0f Patients with bilaterally involved neck nodes 0f Patients with pT3T4 tumours involving midlinewho undergo an ipsilateral neck dissection unless acontralateral neck dissection is performedcontraindications to radiotherapy 0f Serious medical comorbidities or other 0f Prior history of head and neck cancer within years 0f Any other active invasive malignancy except noncavity or neckmelanotic skin cancers lowrisk prostate cancer andStage IIVA papillary or follicular thyroid cancer 0f Prior head and neck radiation at any time 0f Prior oncologic head and neck surgery in the oral 0f Known metastatic disease 0f Locoregional disease recurrence identified followingsurgical resection but prior to start of radiotherapy 0f Inability to attend full course of radiotherapy or 0f Unable or unwilling to complete QoL questionnaires 0f Pregnant or lactating womenfollowup visitsPretreatment evaluation 0f History and physical examination by a radiationoncologist within weeks prior to randomization 0f Staging prior to randomizationCT or MRI of the neck with contrast unlesscontraindicated within weeks ofrandomization This can include the preoperativeCT or the radiation therapy CT or MRI simulation if reviewed by a radiologist–ª In some instances suspicious lymph nodes arevisible on the scan after surgery In suchinstances recurrence must be ruled outpathologically before enrollment either with aneedle biopsy or resection of these nodesCT of the chest or whole body PETCT usually prior to surgery must be within weeks ofrandomizationedentulous patientswithin weeks of randomization 0f Histological confirmation of SCC 0f Pregnancy test for women of childbearing age 0f Dental evaluation prior to starting treatment except 0f Assessment of dysphagia using NCICTC version 0f Completion of QoL scoring prior to initiation of 0f Prior to randomization radiation contours are to be 0f MBS at baseline prior to initiation of treatment with within weeks of treatment initiationpeerreviewed and approvedtreatmentdocumentation of the MBSIimP„¢ DIGEST„¢ andthe FOIS scoresresectionTreatment planSurgicalandchemotherapy will be delivered in accordance with National Comprehensive Cancer Network NCCN ClinicalGuidelines []and adjuvantradiotherapyPrimary tumours should be resected en bloc wheneverpossible with the goal of achieving clear margins Patients with midline involvement of the primary tumourshould receive a bilateral neck dissectionAdjuvant cisplatinbased chemotherapy concurrentwith radiotherapy is at the discretion of the treatingmedical oncologist and is recommended for patientswith positive margins or ENE for patients who can tolerate chemotherapy For patients who are deemed unfitor too elderly years of agefor cisplatinbasedchemotherapy the standard dose andor schedule canbe modified alternative systemic therapy regimensmaybe used eg weekly carboplatin Calais regimen orsystemic therapy can be omitted at the discretion of thetreating physicians It is strongly recommended that radiation starts within weeks of the date of surgery andit is mandatory to start no more than weeks after thedate of surgeryDuring treatment supportive care should be in accordance with local standard of care which often includes speech language pathology SLP assessment AnySLP interventions eg providing swallowing exercisesshould be the same in both arms and conform to localstandard of careDosefractionation In both arms a dose of Gy in fractions will be delivered to the operative bed targetvolumes Centres that normally treat dissected nodenegative levels to Gy in fractions will be permitted 0cLang Radiation Oncology Page of to do so if used consistently for all patients on trialAreas of positive margins or ENE should receive Gyin fractions if those areas can be localized Undissected areas that require coverage in the opinion of thetreating radiation oncologist eg low neck retrostyloidspace should receive Gy in fractions Table Immobilization and localization All patients will beimmobilized in a custom thermoplastic shell and willundergo a planning CT simulation with or withoutIV contrast encompassing the head and neck tobelow the clavicles The planning CT will be fusedwith other diagnostic imaging eg MRI scans or preoperative CT where necessary Bite blocks tonguedepressors jaw separators may be used as per institutional protocolthetime of planning CT prior to contour generation andpatient randomizationthese must be determined atRadiotherapy volume definitions A randomization volume will be defined as the nodal volume in the dissectedpN0 heminecks The randomization volume will depend on the laterality of the neck dissection performedipsilateral vs bilateral and whether pathologically involved nodes are present in the neck dissection definedin Fig Patients with bilaterally involved neck nodesare ineligible Patients with a unilateral neck dissectionwith positive lymph nodes are ineligible unless theyundergo a staged neck dissection of the opposite sidethat is pN0Standard treatment volumes Arm If randomizedincluding thecontoured volumesto Arm randomization volume will be treatedallOmission of pN0 neck Arm If randomized toArm the randomization volume will be omitted fromtreatment planningClinical Target Volumes CTVThe following radiation volumes will be contoured forall patients prior to randomization Table The suffixœpos denotes CTVs in the nodepositive hemineck andthe suffix œneg denotes the CTVs in the nodenegativehemineck Highrisk volume CTV64 regions correspondingto positive margins or ENE if present and if thoseareas can be localized Primary tumour operative bed CTVp60surgically dissected areas corresponding to theresected primary tumour typically including thepreoperative tumour area and any flap reconstructions and clips with a margin Involved necka Surgically dissected involved neck nodalvolume CTVn60pos surgically dissected necklevels in the nodepositive hemineck if applicable In the dissected neck some centres routinely treat only involved nodal areas to Gyand the remainder of the dissected neck to Gy For centres where this is standard this approach is acceptable but all patients enrolledfrom these centres must be treated with this approach The areas treated to Gy would be included in a CTVn54posb Optional uninvolved lowrisk neck on the involved side CTVn54pos lowrisk undissectedneck nodal volume on the involved side if applicable at the discretion of the radiation oncologist This may include the standardlymphatic drainage sites not dissected at thetime of surgery in the node positive heminecksuch as the nodal levels below or above the dissected areas eg level IVb retrostyloid space œRandomization Volume corresponding to theUninvolved necka Surgically dissected involved neck nodalvolume CTVn60neg surgically dissectedneck levels in the node negative hemineck Incentres that routinely treat the pN0 neck to Gy that dose is acceptable but all patients mustbe treated with that approach In such centresthese areas treated to Gy would be includedin a CTVn54negb Optional uninvolved lowrisk neck on theuninvolved side CTVn54neg lowrisk undissected neck nodal volume on the uninvolvedside if applicable at the discretion of theTable Radiation treatment volumes and dosesPTV VolumePTV64Areas of positive margin or ENEPTV60Dissected neckPTV54optionalNot surgically dissected elective nodal regionsCTV Volumes IncludedArm CTV64CTVp60 CTVn60posCTVn60negCTVn54posCTVn54negArm CTV64CTVp60 CTVn60posCTVn54posDose infractions Gy Gy Gy 0cLang Radiation Oncology Page of Fig The œRandomization Volume corresponds to the pN0 heminecks The neck volumes included in the œRandomization Volume dependon whether the patient had an ipsilateral vs bilateral neck dissection and the pathological findings in each heminecks Patients with bilaterallyinvolved neck nodes are ineligible Patients with an ipsilateral neck dissection with positive lymph nodes are ineligible unless they undergo acontralateral neck dissection that is pN0 If randomized to standard treatment volumes Arm all contoured volumes including theœRandomization Volume will be treated If randomized to omission of the pN0 neck Arm the œRandomization Volume will be omitted fromtreatment planningradiation oncologist In a patient who has had aunilateral neck dissection that was pN0 thismay include the contralateral clinically nodenegative cN0 neck if that is standard institutional practiceIf randomized to omission of PORT to pN0 neckArm the CTVn60neg and CTV54neg contours aredeleted after randomization prior to treatment planningTo prevent bias contours cannot be changed afterrandomization Local peer review of contours must takeplace before randomization occurs An overview of theprotocol timeline is shown in Fig Planning Target Volumes PTVA “ mm expansion is used around the combinedCTVs Table to create the PTV as per institutionalsetup and protocolRadiotherapy planning Intensity modulated radiotherapy IMRT photon therapy or proton therapy will beused for all patients in this study IMRT can be deliveredusing staticbeam techniques or rotational techniqueseg Tomotherapy or Volumetric Modulated Arc Therapy [VMAT] If protons are used the dose will be reported in Gy relative biological effectiveness [RBE]where the proton dose is multiplied by an RBE of All reported doses in Gy are considered equivalent Centres with proton therapy will use their institutionalstandard planning and delivery techniquesAll plans will be normalized to ensure that ofeach PTV is covered by of the prescription dosefor that volume An exception will be allowed forcentres that normally prescribe in such a manner that of the PTV be covered by of the prescription dose and such centres must prespecify this before enrolling the first patient and all subsequentpatients must be planned in the same manner Amodified PTV cropped mm from the externalcontour for dose evaluation may be used as per institutional guidelines 0cLang Radiation Oncology Page of 0f All dose delivery for intensitymodulated plans including arcbased treatments will be confirmed before treatment by physics staffConebeam CT andor orthogonal xrays will be usedon a daily basis to verify treatment positioning as per institutional standard practiceCredentialling Prior to enrolling patients each centrewill be given a sample CT dataset through secure filetransfer protocol FTP for contouring planning andphysics QA Enrollment can begin once the plan andQA have been approved at the London Regional CancerProgram Centres who have been accredited for ORATOR [] or ORATOR2 [] are exempt from thisrequirementParticipant discontinuation withdrawalParticipants may voluntarily discontinue participation inthe study at any time If a participant is removed fromthe study the clinical and laboratory evaluations thatwould have been performed at the end of the studyshould be obtained If a participant is removed becauseof an adverse event they should remain under medicalobservation as long as deemed appropriate by the treating physicianFollowup evaluationDay of followup will be the first day of radiotherapyFollowup will consist of history and physical examination with laryngopharyngoscopy CT imaging of theneck ± thorax QoL assessments and MBS The followup schedule is shown in Additional file Additional imaging or laboratory investigations should be carried outat the discretion of the oncologist based on findings inthe history or physical examination Additional treatment eg salvage treatment with surgery or furtherradiotherapy is at the discretion of the treating physicians but will be recorded in the case report formMeasurement of outcomes 0f pN0 neck failure measured as time fromrandomization until disease recurrence in theinitially pN0 heminecks Patients with prior orsimultaneous recurrence at the primary site or inthe initially pN hemineck will be censored for thisoutcome as of that timepoint The primary endpointis a comparison of pN0 neck failure in Arm vshistorical controls this endpoint will also be compared between the two arms as a secondaryendpoint 0f OS measured as time from randomization untildeath from any causeFig Flowchart showing timing of randomization with respect topeer review and treatment planning Contours must be finalizedbefore randomization and may not be changed after randomizationThe maximum dose to PTV64 or PTV60 if no PTV64present should not exceed of the prescribed doseand no volume cc outside of these PTVs should receive of the prescription doseans at Risk OAR definitions dose constraints andplanning priorities are adapted from the following protocols RTOG protocols [] Arm and RTOG [] Arm NCICCTG HN6 ORATOR [ ] andORATOR2 [] and are described in Additional file Quality assurance QAIn order to ensure patientsafety and effective treatment delivery a robust QAprotocolis incorporated The following requirementsmust be completed for each patient 0f Prior to randomization each set of contours will bepeerreviewed either by another individual radiationoncologist or at a team head and neck QA rounds 0cLang Radiation Oncology Page of 0f DFS measured as time from randomization toeither recurrence at any location or deathwhichever occurs first New primary tumours willnot count as DFS events 0f Local recurrence measured as time fromrandomization until disease recurrence at theprimary site 0f Regional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck 0f Locoregional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck or at the primary site whichever occursfirst 0f Recurrence in the pN0 neck without otherlocoregional recurrence measured as time fromrandomization until disease recurrence in the pN0neck alone without recurrence at the primary site orpN neck 0f Rate of salvage surgery andor radiation in pN0neck measured as time from randomization tosalvage intervention surgery ± radiation in the pN0neck Freedom from unsalvageable neck recurrencewill be reported as the time from randomization todevelopment of a neck recurrence in the pN0 neckthat could not be salvaged 0f Feeding Tube Insertion Rate of feeding tubeinsertion after start of radiation either gastricgastrojejeunal or nasogastric and rate of feedingtube use at 1year postrandomization Patients withfeeding tubes inserted prior to randomization will becensored for this endpoint 0f Rate of failure in the clinically node negativeneck for patients who have unilateral neckdissections the cN0 neck may be treated withradiation or observed at the discretion of thetreating oncologist see section This is measuredas the time from randomization to failure in the cN0neck and will be reported for the whole group ofpatients who had unilateral dissections and alsostratified by whether radiation was delivered to thatareaEnrollment randomization and allocationPatients will be enrolled by dedicated clinical trials staffandor the investigator at each participating institutionPatients will randomized in a ratio to standard radiation volumes Arm vs omission to the pN0 neckArm A permuted block design with two stratification factors will used with the size of the blocks knownonly to the statistician stratified based upon overall necknodal status pN0 vs pN and use of concurrentchemotherapy yes vs no Randomization sequences aregenerated for each strata separately with a randomnumber generator based on permuted block design Thisgets formatted as a CVS file which gets uploaded intoREDCap []Statistical considerationsSample size The primary endpoint is the rate of relapsein the pN0 neck ie the regional relapse rate in the pN0neck in Arm compared to historical controls Allother endpoints are a comparison between Arm andArm It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin and therefore we wish to exclude a risk of regionalrecurrence in the pN0 neck of at 2years Using aonesided onesample binomial test allowing for dropout a sample size in Arm of patients provides power at a significance level to detect a regional control rate in the pN0 neck of compared toan unacceptable level of This sample size in Arm and in Arm alsoallows for power to detect a 10point difference inthe total MDADI score at year a secondary endpointassuming the scores are normally distributed with astandard deviation of in each arm It is generally believed that a 10point difference in standardized QoLscores represents a clinically significant difference inQoL [] It is assumed that the QoL scores will be normally distributed with a standard deviation of in eacharm The MDADI will be calculated as the compositescore but will also be reported for each of the subscalesAnalysis planPatients will be analyzed in the groups to which they areassigned intentiontotreat Oncologic outcomes andOS will be calculated from date of randomization usingthe KaplanMeier method A onesided onesample binomial test will be used for the primary endpoint as described above For actuarial comparisons between armsthe stratified logrank test will be used stratified bystratification factors An twosample Ttest will be usedto compare QoL scores at 1year The percentage of patients in each arm who experience a clinically significantQoL decline on the MDADI points will also bereportedMBSImP„¢ scores will be compared at 1year including the total score and the scores on the oral andpharyngeal subscales using a twosample Ttest or aWilcoxon rank sum test as appropriate DIGEST„¢ andFOIS scores will be compared using the Chisquare testPreplanned subgroup analysis will occur based on thestratification variables nodal status [pN0 vs pN] anduse of concurrent chemotherapy as well as based onthe neck dissection performed unilateral vs bilateral 0cLang Radiation Oncology Page of and Tstage T1“ vs T3“ There will also be a preplanned analysis based on the extent of nodes harvestedin the pN0 neck vs or more depth of invasionof the primary tumour mm vs mm or more andphoton versus proton treatmentMultivariable Cox proportional hazards or logistic regression analysis as appropriate will be used to determine baseline and pathologic factors predictive of pN0neck failure DFS OS locoregional recurrence and salvage therapy For the secondary endpoints involvingQoL scales linear mixed effects models will be used forthe MDADI the total scores will be compared betweenthe two arms whereas for the EORTC QLQC30 andHN35 scales each of the subscales eg pain swallowing etc will be compared between the two armsRates of grade ‰¥ toxicity and use of feeding tubes willbe compared between arms using the Chisquare orFisher™s exact test as appropriateUtilities will be calculated from the EQ5D5L whichwill be administered at baseline and at month intervalsQuality adjusted life years QALYs will be assessed asthe area under the preferenceweighted survival curveOverall costs of each treatment strategy will be abstracted from the available literature The incrementalcost effectiveness ratios ICERs between treatment armswill be compared through the standard method of ratiobetween differences in costs and QALYs Point estimatesfor these differences can be derived from multivariablegeneralized estimating equations GEE or generalizedlinear model GLM analysesData safety monitoring committeeThe Data Safety Monitoring Committee DSMC consisting of at least one radiation oncologist and one medical oncologist not involved in the study will meet every months after study initiation Toxicity outcomes willbe monitored but since the experimental arm involvessmaller radiation volumes it is extremely unlikely thattoxicity would be higher in the experimental arm andtherefore no stopping rules for toxicity are includedInterim analysis The DSMC will conduct one interimanalysis once patients have been accrued and completed the 6month QoL questionnaires For this analysis the DSMC will be blinded to the identity of eachtreatment arm but QoL recurrence in the pN0 neckOS and DFS estimates at 2years will be presented foreach armThe DSMC will recommend stopping the trial if thereis an OS difference that is statistically significant with athreshold of p using the stratified logrank testbased on the HaybittlePeto stopping rule this retainsan overall alpha of Ethical considerationsThe Principal Investigator will obtain ethical approvaland clinical trial authorization by competent authoritiesaccording to locallaws and regulations The WorldHealth anization WHO Trial Registration Data Setis shown in Additional file Institutional review board IRB research ethicsboard REB The protocol and any

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

thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples œedgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients™ prognosis was verified using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identification of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore it™s very important to find eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression profiles of specific cancers are easy to obtainwhich helps us better find more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to define and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenverified However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identification of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionprofiles of THCA patients were accessed from TCGA databaseand modules associated with THCA were identified by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression profiles of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentification and Confirmation ofTHCAAssociated GenesœedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionprofiles In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients™ prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset œglmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentification of THCAAssociatedModulesAs shown in A a total of DEGs were identifiedlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of ‰ ‰ log k and log Pk were calculated for linearmodels™ construction respectively is the squared value of thecoefficient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the first time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and significance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were verified to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identification of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology fitting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene significance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore — GHR —GPR125 — Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was verified Figure 2E From the heatmaps of the expression profiles of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3A“C THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3D“FFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patients™prognosis F Heatmap of the genes expression profilesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model A“C Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset D“F ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identificationof the important genes during tumorigenesis and the definitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq profiles andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been verifiedto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is firstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction It™sreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereverified as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further verified by ROC and KaplanMeier curvesAs the expression profiles of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and it™s necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the finalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci “ Carling T Udelsman R Thyroid cancer Annu Rev Med “ 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma “pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope “ Zhao QJ Zhang J Xu L Liu FF Identification of a fivelong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol “ 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentification ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival“ 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA ™ endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res “ Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit “ 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is specifically expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model “ 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identification of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics “ 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep“ 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117“ 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConflict 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 Zhao Zhang Shao and Fang 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

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

generate a map of the copy number variations CNV segregating in a population of Murciano‘Granadina goats the most important dairy breed in Spain and to ascertain the main biologi‘cal functions of the genes that map to copy number variable regionsResults Using a dataset that comprised Murciano‘Granadina goats genotyped with the Goat SNP50 Bead‘Chip we were able to detect and autosomal CNV with the PennCNV and QuantiSNP software respec‘tively By applying the EnsembleCNV algorithm these CNV were assembled into CNV regions CNVR of which of the total CNVR count were consistently called by PennCNV and QuantiSNP and used in subsequent analyses In this set of CNVR we identified gain loss and gainloss events The total length of all the CNVR Mb represented of the goat autosomal genome Mb whereas their size ranged from kb to Mb with an average size of kb Functional annotation of the genes that overlapped with the CNVR revealed an enrichment of pathways related with olfactory transduction fold‘enrichment q‘value — ˆ’ ABC transporters fold‘enrichment q‘value — ˆ’ and bile secretion fold‘enrichment q‘value — ˆ’Conclusions A previous study reported that the average number of CNVR per goat breed was CNVR50 breeds which is much smaller than the number we found here CNVR We attribute this difference to the fact that the previous study included multiple caprine breeds that were represented by small to moderate numbers of individuals Given the low frequencies of CNV in our study the average frequency of CNV is such a design would probably underestimate the levels of the diversity of CNV at the within‘breed level We also observed that functions related with sensory perception metabolism and embryo development are overrepresented in the set of genes that overlapped with CNV and that these loci often belong to large multigene families with tens hundreds or thousands of paralogous members a feature that could favor the occurrence of duplications or deletions by non‘allelic homologous recombinationCorrespondence marcelamillsuabcat Centre for Research in Agricultural Genomics CRAG CSIC‘IRTA‘UAB‘UB Universitat Aut²noma de Barcelona Bellaterra SpainFull list of author information is available at the end of the BackgroundCopy number variations CNV encompass genomic deletions or duplications with sizes ranging from base pairs bp to several megabases Mb and which display polymorphisms in terms of copy number among individuals of a particular species [“] In livestock a broad array of phenotypes related with among others morphology [ ] pigmentation [“] sexual development 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 0cGuan a0et a0al Genet Sel Evol Page of [] and susceptibility to disease [] is caused by the segregation of CNV Genome scans to detect structural variations in cattle have revealed that CNV regions CNVR are often enriched in genes that are involved in immunity [“] metabolism [ ] embryo development [ ] and sensory perception [ ] There is evidence that the dNdS ratios of genes that map to taurine CNV are generally higher than those of genes that do not overlap with CNV which indicates that CNV genes probably evolve under reduced selective constraint [] The analysis of gene networks has also shown that genes that colocalize with duplications tend to have fewer interactions with other genes than loci that do not overlap with CNV reinforcing the idea that genes mapping to duplicated regions have fewer essential housekeeping functions than nonCNV genes and also have reduced pleiotropy []Although structural chromosomal variations can have strong effects on gene expression and phenotypic variability technical limitations and the moderate quality of genome assemblies have hampered CNV mapping in livestock [] Until recently this has been particularly true for goats In Fontanesi et a0al [] published the first caprine CNV map by identifying with the Bovine a0k aCGH array CNVR including and copy loss and gain variants respectively Later on resequencing the genome of individuals from several caprine breeds made it possible to identify CNV that overlap with pigmentation genes and to detect an association between increased ASIP copy number and light pigmentation [] The first worldwide survey of copy number variation in goats was performed within the Goat ADAPTmap Project httpwwwgoata daptm ap and involved the genomewide genotyping of goats from breeds [] This study resulted in the identification of CNVR among which several overlapped with genes that are functionally related with local adaptation such as coat color muscle development metabolic processes and embryonic development [] Moreover the patterns of the diversity of CNV differed according to geographic origin which indicates that they have been influenced by population history [] In another study on individuals from East African goat breeds Nandolo et a0al [] detected CNVR More recently Henkel et a0al [] demonstrated the existence of complex patterns of structural variation in the regions containing the caprine ASIP and KIT genes with potential causal effects on pigmentation In spite of these efforts the description of structural chromosomal variation in goats is still lagging behind that of other domestic species Most of the CNV surveys in goats have analyzed large populations that represent a mixture of different breeds each with a limited number of individuals [ ] thus making it difficult to assess the magnitude of the CNV diversity at the withinbreed level Our goal was to fill this gap by analyzing a population of individuals from a single Spanish breed MurcianoGranadina and to investigate the functional roles of genes that map to CNVR and compare these results with data obtained in composite goat populationsMethodsGenomic DNA extraction and a0high‘throughput genotypingBlood samples from MurcianoGranadina female goats from farms that are connected through the use of artificial insemination were collected in EDTA K3 coated vacuum tubes and stored at ˆ’ a0 °C before processing Genomic DNA was isolated by a modified saltingout procedure [] Four volumes of red cell lysis solution Tris“HCl a0 mmolL pH EDTA a0mmolL Tween were added to a0mL of whole blood and this mixture was centrifuged at —g Pelleted cells were resuspended in a0mL lysis buffer Tris“HCl a0 mmolL pH EDTA a0 mmolL SDS NaCl a0 mmolL plus µL proteinase K a0 mgmL The resulting mixture was incubated at a0°C for h followed by centrifugation at —g in the presence of a0mL of ammonium acetate a0molL The supernatant a0mL was mixed with a0mL of isopropanol which was subsequently centrifuged at —g for a0min The supernatant was removed and the DNA pellet was washed with a0 mL of ethanol After centrifuging at —g for a0min the DNA precipitate was dried at room temperature and resuspended in a0mL of TE buffer a0mmolL Tris pH a0mmolL EDTA pH Highthroughput genotyping of the MurcianoGranadina DNA samples was carried out with the Goat SNP50 BeadChip [] according to the manufacturer™s instructions Illumina Signal intensity ratios ie log R Ratio or LRR the total probe intensity of a SNP referred to a canonical set of normal controls [] and B allele frequencies or BAF relative quantity of one allele compared to the other one [] were exported for each single nucleotide polymorphism SNP with the GenomeStudio software Illumina https emeaillum inacom Then SNP coordinates were converted to the latest version of the goat reference genome ARS1 [] After filtering out unmapped and nonautosomal SNPs and those with a call rate lower than a set of SNPs remained for CNV mappingCopy number variant calling with a0PennCNV and a0QuantiSNPBased on their excellent performance in comparative studies we selected two software packages PennCNV v105 [] and QuantiSNP v2 [] to call CNV in the MurcianoGranadina population [ ] The PennCNV software [] detects CNV by applying the default 0cGuan a0et a0al Genet Sel Evol Page of parameters of the HiddenMarkov model Population frequencies of B alleles were compiled based on the BAF of each SNP in the population We used the œ“gcmodelfile option to adjust œgenomic waves [] The number of goat chromosomes was set with the œ“lastchr  instruction The QuantiSNP analysis [] assumes an objective Bayes hiddenMarkov model to improve the accuracy of segmental aneuploidy identification and mapping This CNV calling software was run under default parameters by modifying the œ“chr  option The CNV that were supported by less than three SNPs were removed from the filtered set used hereDefinition and a0functional annotation of a0copy number variant regionsWe used the EnsembleCNV algorithm beta version [] to assemble CNVR All CNV called by PennCNV andor QuantiSNP were combined to generate a set of initial CNVR by using the heuristic algorithm threshold of minimum overlap described in [] Subsequently CNVR boundaries were refined by considering the local correlation structure of the LRR values of the SNPs mapping to CNVR [] Then we reassigned the CNV calls that were initially obtained with PennCNV and QuantiSNP to each refined CNVR so that the final set of CNVR comprised only those that were simultaneously detected by both callers The resulting CNVR were matched to gene features that are annotated in the National Center for Biotechnology Information NCBI https wwwncbinlmnihgov by using BEDTools v2250 [] In addition we performed gene ontology GO enrichment and pathway analyses using the DAVID Bioinformatics Resources [ ] based on human and goat background gene sets The statistical significance was set to a qvalue ‰¤ Confirmation of a0copy number variant regions by a0quantitative real‘time PCRIn order to evaluate the rate of false positives in our experiment we conducted quantitative realtime PCR qPCR experiments to obtain an independent estimate of the copy number of four putative CNVR CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 Primers were designed with the Primer Express software Applied Biosystems to amplify specific regions of the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes see Additional file a0 Table a0 S1 As reference genes we used the melanocortin receptor MC1R and glucagon GCG genes see Additional file a0 Table a0S1 loci [ “] Quantitative PCR reactions contained a0ng genomic DNA µL — SybrSelect Master mix Applied Biosystems a0pmol of each forward and reverse primer and ultrapure water to a maximum final volume of µL Each sample was analyzed in triplicate in order to obtain averaged copy number estimates Reactions were loaded onto 384well plates and run in a QuantStudio a0K Flex RealTime PCR System instrument Applied Biosystems The specificity of the PCR reactions was evaluated with a melting curve analysis procedure and the efficiency was assessed with standard curves Thus relative copy number was inferred with the qbase software Biogazelle Ghent Belgium by using the ˆ’ΔΔCt approach [] Copy number values were calibrated by taking as a reference four samples which according to Goat SNP50 BeadChip data had two copies of the investigated genomic lociResultsDetection of a0copy number variation in a0Murciano‘Granadina goatsThe initial calling with PennCNV and QuantiSNP yielded and autosomal CNV respectively By using the EnsembleCNV tool [] we assigned these CNV into CNVR with refined boundaries of which of the total CNVR count were detected simultaneously by PennCNV and QuantiSNP The resulting CNVR included copy gain copy loss and copy gainloss variants Fig a0 and Table a0 and see Additional file a0 Table a0 S2 The total length of the CNVR covered a0Mb of the goat autosomal genome a0Mb whereas their individual size ranged from a0kb to a0Mb with an average of a0kb Fig a02a and Table a0 Moreover we found that of the CNVR showed minimum allele frequencies lower than with an average frequency of Fig a02b In addition CNVR with frequencies higher than were distributed over seven caprine chromosomes With a frequency of CNVR_1229_chr21 was the CNVR with the highest frequency in the whole dataset see Additional file a0 Table a0S2 By using the BEDTools v2250 program [] of the CNVR that we detected overlapped with unique CNVR published by Liu et a0al [] Fig a0 and see Additional file a0 Table a0S2 The CNVR that were detected in both studies are referred to as œshared CNVR whereas those that were identified in our study only are referred to as œnonshared CNVR Fig a0 Six of the ten œshared CNVR with frequencies higher than show positional concordance with six CNVR detected by Liu et a0al [] see Additional file a0 Table a0S2Functional annotation of a0the a0genes that a0are located in a0copy number variable regionsWithin the CNVR defined in our study we detected proteincoding genes according to the goat reference genome annotation ARS1 [] from the NCBI database see Additional file a0 Table a0S2 and Additional file a0 0cGuan a0et a0al Genet Sel Evol Page of Fig Genomic distribution of CNVR detected with the PennCNV and QuantiSNP software on the caprine autosomes Squares triangles and circles represent copy number gain loss and gainloss events respectively Red and black colors represent shared and non‘shared CNVR respectively Shared CNVR are those detected both in our study and in Liu et al [] while non‘shared CNVR are those identified only in our studyTable a0S3 In a survey of the diversity of CNV in goats with a worldwide distribution Liu et a0 al [] detected copy number variable genes of which were also identified in our study and are referred to as œshared copy number variable genes see Additional file a0 Table a0S3 Among the œshared copy number variable genes the ASIP and ADAMTS20 genes are particularly relevant they are involved in pigmentation [ “] and colocalize with selection signals detected in a worldwide sample of goats [] In addition we found that about of the annotated genes that colocalize with CNVR are olfactory receptors or olfactory receptorlike genes see Additional file a0 Table a0S3 Consistently the most significantly enriched pathway was œOlfactory transduction qvalue — ˆ’ Table a0 followed by œABC transporter qvalue — ˆ’ Table a0 A significant pathway related with immunity ie Fc epsilon RI signaling qvalue was also identified based on a human background gene set Table a0 Several overrepresented GO terms were related with embryonic skeletal system morphogenesis qvalue — ˆ’ and Gprotein coupled purinergic nucleotide receptor activity 0cGuan a0et a0al Genet Sel Evol Page of Table Main features of a0 copy number variation regions CNVR detected in a0 MurcianoGranadina goatsSummary statisticsTotalGainLossGainlossTotal length MbTotal number of CNVRNumber of CNVR kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR “ kbNumber of CNVR kb“ MbNumber of CNVR ‰¥ MbAverage number of SNPs per CNVRMinimum size of CNVR kbMaximum size of CNVR kbAverage CNVR size kbStandard deviation of CNVR size kb qvalue — ˆ’ Table a0 Interestingly the copy number variable genes were also enriched in pathways with metabolic significance such as prolactin signaling and insulin signaling as well as GO terms related with feeding behavior but none of these pathways reached the significance threshold qvalue ‰¤ after correction for multiple testing see Additional file a0 Table a0S4 Several of the pathways outlined in Additional file a0 Table a0S4 play important roles in immunity eg chemokine signaling B cell receptor signaling and T cell receptor signaling cancer eg endometrial cancer proteoglycans in cancer thyroid cancer as well as in oncogenic signaling eg Ras and ErbB signaling see Additional file a0 Table a0S4 but most of them are not significant after correction for multiple testingFig Histograms displaying the distribution of CNVR according to their size a and frequency b CNVR that were longer than kb were included in the ‘kb bin whereas those with frequencies above were grouped in the bin The histograms were drawn by using the ggplot2 package httpggplo t2tidyv erse implemented in R https wwwr‘proje ct 0cGuan a0et a0al Genet Sel Evol Page of Table Functional enrichment of a0genes colocalizing with a0CNVR detected in a0 MurcianoGranadina goatsBackground gene setCategory IDTermNumber of a0genesFold enrichment P valueq‘valueGoatGoatGoatHumanHumanHumanHumanHumanHumanHumanKEGGKEGGKEGGKEGGGOBPGOBPGOBPGOCCGOMFGOMFOlfactory transductionABC transportersBile secretionFc epsilon RI signaling pathwaychx04740chx02010chx04976hsa04664GO0009952 Anteriorposterior pattern specificationGO0048704 Embryonic skeletal system morphogenesisGO0035589 G‘protein coupled purinergic nucleotide receptor signaling pathwayGO0016020 MembraneGO0003677 DNA bindingGO0045028 G‘protein coupled purinergic nucleotide receptor activity126Eˆ’ 161Eˆ’333Eˆ’ 427Eˆ’446Eˆ’ 570Eˆ’140Eˆ’ 176Eˆ’936Eˆ’ 161Eˆ’713Eˆ’ 122Eˆ’418Eˆ’ 716Eˆ’145Eˆ’ 198Eˆ’110Eˆ’ 160Eˆ’424Eˆ’ 622Eˆ’KEGG Kyoto Encyclopedia of Genes and Genomes pathway GOMF gene ontology GO term related with molecular function GOBP GO term related with biological process GOCC GO term related with cellular componentValidation of a0four copy number variants by a0real‘time quantitative polymerase chain reactionIn order to confirm our results we selected four CNVR ie CNVR_371_chr5 CNVR_506_chr6 CNVR_160_chr2 and CNVR_1229_chr21 that colocalized with the ADAMTS20 BST1 NCKAP5 and TNFAIP2 genes respectively the primers used to amplify these CNVR are listed in Additional file a0 Table a0 S1 As shown in Fig a0 the estimated copy numbers obtained by qPCR analysis of MurcianoGranadina goat samples were to copies relative to the calibrator ADAMTS20 to copies BST1 to copies NCKAP5 and to copies TNFAIP2 According to D™haene et a0 al [] copy number estimates between and most likely correspond to a normal copy number of whereas any number below or above these thresholds could represent a deletion or a duplication respectively Thus based on these values evidence of copy number variation was inferred for three of the four genes analyzed by qPCRFig Relative quantification of four copy number variation regions by real‘time quantitative polymerase chain reaction analysis a CNVR_371_chr5 ADAMTS20 b CNVR_506_chr6 BST1 c CNVR_160_chr2 NCKAP5 d CNVR_1229_chr21 TNFAIP2 The x and y axes represent sample ID and relative quantification of CNVR mean ± standard error with each sample analyzed in triplicate respectively As calibrator we used the average of four samples estimated to have two copies diploid status based on the Goat SNP50 BeadChip analysis 0cGuan a0et a0al Genet Sel Evol Page of DiscussionIn this work our aim was to characterize copy number variation in MurcianoGranadina goats a native Spanish breed used for milk production By genotyping MurcianoGranadina goats with a SNP array we were able to identify CNVR covering of the goat genome whereas Liu et a0 al [] identified CNVR that covered of the goat genome The latter higher percentage reported by Liu et a0al [] can be explained by the fact that they analyzed breeds with different geographical origins ie a composite population that is probably much more diverse than that used in our work Besides the pipeline that we used to identify CNVR is more stringent than that employed by Liu et a0al [] removing CNVR that were not consistently detected by PennCNV and QuantiSNP In the literature estimates of to for CNVR coverage in the human genome are reported [] Our results and those obtained by Liu et a0al [] are consistent with these valuesIndeed when Liu et a0 al [] calculated the CNVR length for each breed normalized by the goat genome size their results agreed well with our estimate of For instance this parameter reached values of in goats from Southeastern Africa and in goats from Northwestern Africa and Eastern Mediterranean whereas it was lowest for individuals from West Asia [] The number of CNV detected at the withinbreed level by Liu et a0 al [] was on average CNV per breed and ranged from to whereas the average number of CNVR was only per breed [] Since the number of detected CNVR is proportional to population size for most of the breeds investigated in [] the level of withinbreed CNV variation is probably underestimated In summary one important conclusion from our study is that the magnitude of CNV diversity at the withinbreed level is likely to be much larger than that previously reported in studies that analyzed multiple populations each represented by a small or moderate number of individualsMost of the CNVR that we report here ranged in size from to a0kb with a mean size of a0kb Similarly the average CNVR size reported by Liu et a0al [] was a0kb Both estimates are quite large and reflect that mediumdensity SNP arrays are not well suited to detect small CNVR in spite of their high abundance In cattle the average sizes of CNVR detected with the Illumina BovineHD Genotyping BeadChip a0K SNPs [] Illumina wholegenome sequencing and PacBio sequencing [] were and a0 kb respectively Another consistent feature of CNVR is that in general their frequencies are low or very low In our study approximately of the CNVR had frequencies lower than and the average frequency was Liu et a0al [] reported lower CNVR frequencies ranging from Alpine and Northern European goats to Northwestern African goats This decreased average CNVR frequency is not very significant and probably reflects differences in sampling size and the use of composite populations with multiple breeds each one with its specific CNVR frequenciesThe CNVR detected in our study covered proteincoding genes Pathway analyses reflected a substantial enrichment of genes that are involved in olfactory perception which is consistent with previous reports in cattle [ ] In this regard there is an important difference between our results and those by Liu et a0al [] Whereas in the study of Liu et a0al [] the term œsensory perception was underrepresented among the CNV genes fold enrichment in our work the terms œolfactory transduction fold enrichment and œGprotein coupled purinergic nucleotide receptor activity fold enrichment were overrepresented and many CNV genes were olfactory receptors The two terms mentioned before are closely related because a broad array of purinergic receptors are differentially expressed in the olfactory receptor neurons that modulate odor responsiveness [] Moreover purinergic nucleotides are important neuromodulators of peripheral auditory and visual sensory systems [] In cattle Keel et a0al [] reported that œsensory perception of smell and œGprotein coupled receptor signaling pathway were significantly overrepresented in the proteincoding genes that overlapped with CNVR Similarly Upadhyay et a0 al [] showed that œsensory perceptions of smell and œchemical stimuli are enriched in their set of CNV genes A potential explanation for the underrepresentation of the œsensory perception functional category among the genes overlapping CNV reported by Liu et a0al [] could be that in goats these genes are not well annotated yet so the majority of them are identified with a LOC prefix and a number and as a consequence of this they are not correctly detected by PANTHER [] thus biasing the results obtained in the gene ontology enrichment analysisLoci belonging to large multigene families might be more prone to colocalize with CNV because paralogous genes can act as templates in nonallelic homologous recombination events which promote increases or reductions in copy number [] It should be noted that olfactory receptor genes constitute the largest gene superfamily and in humans more than genes and pseudogenes have been identified [] In cattle olfactory receptor genes and pseudogenes are distributed in clusters across bovine chromosomes [] and similar numbers have been reported for pigs [] Moreover purifying selection against CNV is probably less 0cGuan a0et a0al Genet Sel Evol Page of intense in regions that contain olfactoryreceptor genes than in genomic regions that contain genes with essential functions [] Interestingly copy number changes in the olfactory receptor genes of wild and domestic mammals might have consequences on food foraging as well as on mate and predator recognition [ ]In the set of genes that colocalize with CNVR we also detected an enrichment of loci related with the multigene family of ATP binding cassette ABC transporters a result that agrees well with previous findings in humans [“] and cattle [ ] In mammals ABC transporters fulfill the mission of carrying a broad array of endogenous substrates such as amino acids peptides sugars anions and hydrophobic compounds and metabolites across lipid membranes At least ABC genes that belong to eight subfamilies have been identified in the human genome [] Copy number variation in the human ABCC4 and ABCC6 genes is associated with susceptibility to esophageal squamous cell carcinoma [] and to the rare autosomal recessive disease pseudoxanthoma elasticum [] respectively Moreover largescale deletions of the human ABCA1 gene are a causative factor for hypoalphalipoproteinemia [] a disease that is characterized by the complete absence of the apolipoprotein AI and extremely low levels of plasma highdensity lipoprotein HDL cholesterol We also found a highly significant enrichment of pathways related with embryo development anteriorposterior pattern specification embryonic skeletal system morphogenesis as previously reported [] These pathways are featured by genes that belong to the Hox multigene family of transcription factors possibly reflecting the genomic instability of certain homeobox gene clusters as evidenced by the existence of many syntenyparalogy breakpoints and assembly gaps as outlined in comparative studies []Although not significant after correction for multiple testing we detected an enrichment of pathways with metabolic significance such as prolactin and insulin signaling which could have an impact on milk production and growth [“] Interestingly the comparison of our work with that of Liu et a0al [] revealed proteincoding genes that colocalize with the set of shared CNVR One of the most relevant shared genes encodes ASIP a protein that increases the ratio of pheomelanin to eumelanin by binding to the melanocortin receptor and delivering an antagonist signal that blocks the downstream expression of eumelanogenic enzymes [] Mutations in the ASIP gene play critical roles in animal pigmentation [] For instance the causal factor of the white color typical of many sheep breeds is the ubiquitous expression of a duplicated copy of the ASIP coding sequence which is regulated by a duplicated promoter corresponding to the itchy E3 ubiquitin protein ligase gene [ ] Although some studies proposed that the ASIP CNV might be associated with different pigmentation patterns in goats [ ] no functional assay has verified an association of ASIP copy number with ASIP mRNA levels Another interesting shared copy number variable gene is ADAMTS20 which was also identified in two previous CNV surveys [ ] This gene encodes a metalloproteinase with an important role in melanoblast survival by mediating Kit signaling [] and in palatogenesis [] Bertolini et a0al [] performed a selection scan in white vs colored black and red goats and detected a selective sweep in the ADAMTS20 gene In the light of these results the potential involvement of a structural variation in ADAMTS20 in goat pigmentation should be explored further Moreover it is worthwhile to mention that several CNVR genes have functions related with production and reproduction traits For instance the NCKAP5 gene which colocalizes with CNVR_160_chr2 frequency is associated with milk fat percentage in cattle [] Taking the above evidence into account the implication of structural chromosomal variations in the genetic determinism of traits of economic interest with a complex inheritance deserves further exploration by designing tools that allow inferring CNVR genotypes with high confidenceConclusionsWith the PennCNV and QuantiSNP software we detected CNVR in the genome of the MurcianoGranadina breed In a a0 previous study [] that used a less stringent pipeline only PennCNV was used and included multiple populations with small to moderate sample sizes the average number of CNVR events per breed was One conclusion of our study is that CNV surveys which are based on a broad array of breeds represented by only a few individuals underestimate the true levels of the CNV diversity at the withinbreed level The main reason for this outcome is that since the majority of CNV have very low frequencies they cannot be detected efficiently when sample size is small and in consequence much of the existing variation is missed We have also found that genes that overlap with CNV are functionally related with olfactory transduction embryo development ABC transporters and Gprotein coupled purinergic nucleotide receptor activity Most of these genes belong to large multigene families encompassing tens hundreds or thousands of paralogous genes that could act as substrates in nonallelic homologous recombination events which is one of the main mechanisms generating duplications and deletions in humans and other species Finally we detected CNV that colocalize with the ASIP and ADAMTS20 pigmentation genes 0cGuan a0et a0al Genet Sel Evol Page of which according to previous studies have been subjected to positive selection for coat color in goatsSupplementary informationSupplementary information accompanies this paper at https doi101186s1271 ‘‘ ‘ Additional file a0 Table a0S1 List of primers used in the real‘time quanti

Thyroid_Cancer

"Laryngology COVID19 Coronavirus Head and Neck Cancer Otolaryngology Key Points Journal Preproof 0c The landscape of ever evolving information about COVID19 during the pandemic has hindered the transition to normal clinical volume and efficiency COVID19 should not be a reason for delay in diagnosis or treatment with patients who have upper aerodigestive tract pathology or malignancy The approach to resection reconstruction and surveillance for patients with head and neck cancer may need to be altered to consider severity of disease patient comorbidity and prevalence of regional COVID19 infections amongst other factors In light of the significant number of prolonged intubations there may be an increase in the number of patients who develop early and late sequelae of treatment for COVID19 Tracheostomy should be performed in a safe and efficient manner when specific indications are met Synopsis This review explores the changes to practice associated with COVID19 for providers treating patients with head and neck cancer and laryngeal pathology The aim of the review is to highlight some of the challenges and considerations associated with treating this patient population during the pandemic Additionally it seeks to discuss some of the areas of concern related to ramping up clinical volume IntroductionHistoryDefinitionsBackground The downstream effects of COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 have now pervaded most aspects of society and have made an indelible mark on the way that medicine and specifically otolaryngology is being practiced Of note the Journal Preproof 0cdisease represents a threat to an aging population throughout the world but also has dangerous implications for providers Among the most œatrisk group of medical providers may be those within the fields of otolaryngology and ophthalmology An otolaryngologist was among one of the first providers to succumb to the illness in its early days as it spread through Wuhan China In light of the risk to both patients healthcare workers and society atlarge a push has been made to mitigate the risk of transmission within the field of Otolaryngology Head and Neck Surgery As of June 22nd there are a total of COVID19 cases reported worldwide with a total of deaths The United States has the highest number of cases at with the total number dead at Given the high mortality associated with the novel virus much of the world has enacted significant social distancing restrictions and facial covering mandates to curb the spread of the disease The origin of the virus is not well understood but it is thought that a bat or pangolin vector might have served as the primary reservoir The disease tends to be marked by fever of patients and cough of patients however a litany of other symptoms have also been described including gastrointestinal upset diarrhea shortness of breath headache loss of smelltaste among others Severe disease is characterized by an acute respiratory distress syndrome ARDS with a mortality for patients that require mechanical ventilation The disease has a slight male predominance at Severity of disease seems to correlate to age as patients who are aged have a mortality while those over present with a mortality approaching in early studies Journal Preproof 0cThe nasal cavity and nasopharynx seem to harbor the highest viral load concentration and thus the nasopharynx is the preferred location for acquisition of samples for diagnostic testing Nasal swabs oropharyngeal swabs bronchial alveolar lavage saliva and tracheal aspirates have also been suggested as possible testing sites The current preferred diagnostic assay is RTPCR which has a variable sensitivity of depending on the institution and type of test During the months of May and June many cities states and countries have focused on a return to normal activity and a ramp up of commercial activities During this time many otolaryngology practices have aimed to ramp up activity as well while employing telehealth social distancing and utilization of personal protective equipment PPE The American Academy of Otolaryngology recently published return to practice guidelines which are detailed below As the world continues to move forward during the COVID19 era considerations such as testing including preoperativepreprocedure COVID testing surgical triage clinic workflow and practice management continue to evolve as more information becomes available This review is intended to highlight some of the current recommendations for patient care within the Laryngology and Head and Neck Surgical Oncology scope of practice Discussion Laryngology Journal Preproof 0cAs cases continue to rise increased emphasis has been placed on protection for the provider in the clinical setting Over the last decade officebased management of many common laryngeal disorders has significantly expanded This includes but is not limited to officebased laser ablation of papilloma or dysplasia transoral or transcervical injection laryngoplasty for vocal fold paralysis and EMGguided injection of Botox for spasmodic dysphonia Given the high number of clinicbased aerosolgenerating procedures practiced by today™s laryngologists many providers have seen a marked reduction in their ability to treat patients and their clinical productivity Within the category of aerosolgenerating procedures is flexible fiberoptic laryngoscopy one of the most widely used diagnostic tools for all otolaryngologists and speech pathologists A consensus statement reported by Rameau et al from a virtual webinar attended by approximately participants in the American laryngology community recommended flexible laryngoscopy should be reserved for critical cases in which the findings may have an immediate impact on diagnosis or treatment œIndications include hemoptysis odynophagia limiting hydration and nutrition or airway compromise”notably secondary to infectious and malignant conditions Some have advocated for preclinic COVID testing prior to any aerosol generating procedure however given the high false negative rate of many available tests the use of universal personal protective precautions is recommended According to Givi and colleagues examinations should take place in negative pressure rooms if possible with avoidance of topical lidocaine spray although other groups recommend administration of a topical anesthetic to theoretically decrease the coughsneeze reflex unpublished online chats A substitute to standard aerosolized anesthesia may be pledgets soaked in lidocaine and Journal Preproof 0c oxymetazoline The group also suggests using videolaryngoscopy whenever possible to keep the practitioner and the patient farther apart Disposable laryngoscopes should be used whenever possible Most studies universally recommend the following personal protective equipment PPE N95 mask or powered airpurifying respirators PAPRs gloves gown eye shield or goggles and cap It has also been suggested that the patients wear a mask covering the mouth during flexible laryngoscopy to reduce aerosolization from phonatory maneuvers and in case of coughing or sneezing At this time transoral rigid and mirror laryngoscopy are discouraged unless flexible laryngoscopy cannot be performed due to the increased risk of gagging and coughing as well as the need for the patient to phonate with the mouth uncovered to allow visualization of the larynx Additionally universal masking is encouraged in all clinical spaces in accordance with many state policies Patients in the waiting rooms are encouraged to physically distance or wait in their car for a phone call prior to presenting for their appointment Crosby also suggests offering personal protective equipment for the friends and family accompanying the patient during laryngoscopy and other sites prevent friends and family from accompanying patients inside for the visit Some alternatives to flexible laryngoscopy have been raised including transcervical laryngeal ultrasound which has a reported concordance of in identifying vocal fold motion abnormalities Another key consideration for the laryngologist in the COVID19 era is the approach to sanitization and room turnover after aerosol generating procedures AGPs Laryngoscope turnover should include highlevel disinfection including the use of such chemical disinfectants as glutaraldehyde chlorine dioxide or orthophthaladehyde OPA Some authors recommend immediate placement of the scope after use into a covered receptacle for transport from the Journal Preproof 0cexamination room to the sterile processing areas After completion of laryngoscopy room sanitization with an EPAregistered hospitalgrade disinfectant is recommended with a hydrogen peroxide solution gL chlorine disinfectant or alcohol According to the CDC website it is unknown how long the air inside a particular examination room remains infectious and likely relates to the room size rapidity of air exchange patient factors like viral shedding amount of coughingsneezing and length of time patient was in the room The CDC suggests that rooms with air changeshour ACH take about and minutes to purify the air with and efficiency respectively As the number of air changeshour decreases the time between patients should be increased to allow for appropriate dissipation of theoretical infectious agents As such many hospitals have recommended a turnover time of 4x the time it takes to purify the air with efficiency which may be either minutes or minutes depending on the level of air turnover or could be no additional time if any additional HEPA filtration system and negative pressure has been added Limited data exists to support this approach for SARSCoV2 Laryngology patients are quite diverse with respect to their level of acuity Some patients require more urgent intervention while others may have their care deferred1221 Most guidelines advocate for a tiered approach to ramping up both clinicbased and surgical activity The American Academy of Otolaryngology published guidelines for ramping up clinical activity on May The AAO recommends limiting patient care to individuals with œtimesensitiveurgent and emergent medical conditions This approach is also echoed in the care of head and neck cancer patients see below According to the guidelines emergent conditions include œimpending airway obstruction due to infection neoplasm stenosis foreign body which may Journal Preproof 0cwarrant the following intervention œflexible and rigid laryngoscopy with intervention direct laryngoscopysuspension laryngoscopy bronchoscopy and tracheostomy Urgent conditions include œmoderate or impending airway obstruction progressive dysphonia progressive dysphagia glottic incompetence causing aspiration or impaired pulmonary toilet which warrant the previously described procedures in addition to œstroboscopy functional endoscopic evaluation of swallow esophagoscopy with or without intervention open airway procedures for cancer Time sensitive conditions include œT1 glottic carcinoma or carcinoma in situ stablemild dysphonia stable dysphagia which adds œtranscervical Botox injection to the above list of procedures Routine conditions which may be deferred for days or more include œmildmoderate dysplasia nonobstructive benignphonotraumatic lesions of the vocal folds glottic incompetence glottic incompetence with mild to moderate dysphonia gender affirmation globuscough without alarm signs Comparing acuity of patients also raises an important point about the subset of patients who are typically seen for benign phonotraumatic voice disorders Many live vocal performance venues have shut down concerts have been cancelled or postponed and some studies point to live singing as being a potential source of massive spread For this reason one might assume that the percentage of patients being seen for acute phonotraumatic voice disorders diminishes somewhat Conversely as patients continue to recover from hospitalizations related to COVID19 it is anticipated that there may be a number of patients with sequelae of prolonged intubation including posterior glottic stenosis vocal fold granulomas and trachealsubglottic stenosis Laryngeal surgery in the era of COVID has had to undergo some significant changes in the approach to patient triage surgical technique and management of the airway Preoperative Journal Preproof 0cevaluation of patients must weigh the risk of delaying surgery with the risk of complications related to COVID19 infection Lei et al studied a group of operative patients in whom all were COVID19 positive within the incubation period Mortality was for this group and required ICU admission25 Of note all patients in this study underwent surgery about days prior to demonstrating signs or symptoms of COVID19 pneumonia This suggests there is significant risk associated with elective surgery in seemingly asymptomatic patients who are infected with COVID19 For this reason many authors have suggested preoperative COVID testing although it is a subject of some debate Some advocate for a negative test within hours followed by selfquarantine until the time of surgery while others favor a negative test 48hrs from the time of surgery and a point of care negative test on the day of surgery17 This is not always possible given the limitations of the institution where the patient is undergoing surgery Just as discussed earlier with regard to personal protective equipment in clinic universal precautions should be taken including full PPE and all patients should be presumed positive Airway management in the COVID19 era has become a point of focus for quality improvement and safety groups Endotracheal intubation is cited as one of the procedures which seems to have the highest aerosol generating burden It is recommended that intubation be performed by the most experienced practitioner Additionally some recommend early intubation for patients that are high risk for decompensation while others have advocated delaying intubation in favor of noninvasive means of ventilation This may include high flow nasal cannula which actually has minimal dispersion of exhaled air if appropriately fitted according to Cheung It is recommended that flexible fiberoptic intubation be avoided whenever possible Journal Preproof 0cAdditionally excessive bagmask ventilation should be avoided due to the risk of dispersion of exhaled air Furthermore jet ventilation is considered particularly high risk and should be avoided if possible Management of the surgical airway and the topic of tracheostomy has been well represented in the recent literature During the SARS outbreak in open tracheostomy was the most common surgical procedure performed on infected patients Most studies seem to favor open tracheostomy over percutaneous tracheostomy however consideration may be given for percutaneous dilatation tracheostomy in some patients if the anatomy is favorable and the practitioner has sufficient expertise with the procedure Tay and colleagues advocate for use of PAPR during tracheostomy based on the experience of countries during the SARS crisis Other authors have suggested the use of an N95 mask appropriate eye protection gown double gloves and cap To decrease the risk of autocontamination some have recommended an infection control nurse be available to monitor donning and doffing procedures during tracheostomy Additional proposals include trach teams which may consist of a surgeon anesthetist and scrub nurse to increase efficiency and create an environment of consistent verbal and nonverbal communication especially important given the burdens of communicating through a mask or PAPR Portugal et al discuss a surgical safety checklist for performing tracheostomy in patients with COVID1932 This includes performing tracheostomy in the ICU whenever possible decreasing the number of personnel in the room and having a specific tracheostomy bundle in the ICU room to decrease the number of times providers and nurses need to break scrub to leave the room They also recommend donning inner gloves prior to gown and outer gloves after donning gown to maintain clean inner gloves for the removal and Journal Preproof 0cdisposal of the rest of the PPE Two universally agreed upon maneuvers include stopping ventilation prior to entrance into the airway and holding ventilation until after the tracheostomy tube cuff has been inflated Givi and colleagues suggest that a smaller tracheotomy cuffed may be preferred to decrease the spread of aerosolized particles Miles discusses the New York experience suggesting that for intubated patients the cuff pressure should be checked every hours with a goal of cm H2O greater pressure predisposes tracheal pressure necrosis33 The group also suggests delaying the timing of tracheostomy until days after onset of symptoms when feasible Finally some have advocated for the use of specific air containment setups including plastic draping smoke evacuator tubing or specifically designed negative pressure box The field of laryngology has had to undergo significant change in the setting of the COVID pandemic As the numbers of COVID19 patients have continued to increase during the month of June it is clear that practice of laryngology in the postCOVID era will need to be carefully ramped up to protect patients and providers alike Additionally one would expect a continued increase in the number of recovered patients being seen for sequelae of prolonged intubation Decisions to relax restrictions on flexible laryngoscopy and other AGPs will depend on the local incidence of COVID19 infection availability and accuracy of preprocedure testing sustainable supply of PPE the ability to properly sanitize rooms and ultimately development of an effective vaccine Head and Neck Surgical Oncology Journal Preproof 0cSimilar to laryngology the approach to head and neck surgical oncology continues to evolve as more information becomes available during the COVID era During the early weeks of the pandemic the aspect of cancer care most concerning to patients and providers involved potential delays in therapy Finley suggests that delaying cancer surgery should be done with extreme caution despite COVID1937 Additionally delays beyond weeks could significantly affect longterm outcomes and morbidity of treatment Among patients diagnosed with severe COVID19 requiring ICU admission patients with cancer deteriorated faster than noncancer patients Desai and colleagues discovered a higher risk of severe events in patients recently treated with chemotherapy or surgery in the past days compared to noncancer COVID16 patients38 Additionally patients with advanced stage cancer tended to have a higher rate of severe events when compared to early stage cancer Cancer patients undergoing active treatment are predisposed to COVID19 related complications and critically ill patients with cancer have a higher predisposition to death Head and neck cancer patients especially are considered a highrisk population for complications associated with COVID19 infection8 making safe coordination of care difficult but imperative Head and neck cancer patients are an atrisk group for a number of reasons Silverman points out that head and neck cancer patients tend to present with advanced age history of tobacco and alcohol abuse and cardiac and pulmonary comorbidities which are similarly found in COVID19 Risk of respiratory sequelae in patients who have received chemotherapy andor radiation therapy are high with increased rates of dysphagia aspiration and pneumonia Additionally head and neck cancer patients have an increased risk of respiratory infections and aspiration pneumonitis These factors may expedite deterioration to Journal Preproof 0csevere adverse events in patients with COVID19 Additionally head and neck patients who are actively receiving chemotherapy or immunotherapy may have depressed immune function malnutrition and older age For this reason the patients need to be carefully selected and comorbidities strongly considered when constructing a treatment plan for patients with head and neck cancer Within the United States mortality for patients of color African American and Latinx with COVID19 is significantly higher than for Caucasian patients40 Unfortunately this is a consequence of inequality within society and the healthcare system rather than a biological or pathological difference41 Correspondingly these communities also tend to present with more advanced disease and have significantly worse mortality compared to their Caucasian fellow citizens This pandemic has laid to bear some of the gross inequities within the American health care system and highlighted the need for equitable decision making for all patients with a diagnosis of head and neck cancer during the COVID19 era Another consideration for the head and neck cancer patient during the COVID19 era may include the financial burden and cost of survivorship associated with undergoing cancer treatment and financial hardship related to COVID19™s effect on the world economy and increasing levels of unemployment Given the significant job losses across the United States there is preliminary data to suggest that there will be at least million newly unemployed people who will also lose their insurance coverage in the wake of the pandemic Increased financial strain has been associated with decreased quality of life scores and subsequently mortality in head and neck cancer patients Journal Preproof 0c Recommendations for head and neck clinic are similar to what was previously discussed for laryngology Providers are expected to take universal precautions regardless of the patient™s COVID status Flexible fiberoptic laryngoscopy is considered a highrisk aerosol generating procedure Due to this laryngoscopy should be reserved for instances in which it is likely to change management One of the beneficial consequences of the COVID19 era is the increased access of care through the widespread adoption of telehealth clinics among most hospitals49 Providers may use telemedicine as an initial preoperative assessment or prescreen for patients that will later be seen in clinic or prior to surgery While telehealth is wonderful for obtaining a detailed history reviewing dataimaginglabs and discussing surgical optionsrisksbenefits a big drawback is the inability to perform a comprehensive head and neck physical exam Physical examination with or without fiberoptic laryngoscopy is important to define the extent of tumor and formulating an ablative and reconstructive plan Fortunately some workarounds include anatomic and physiologic imaging for ablative planning and CT angiography and virtual planning sessions for microvascular reconstruction Telemedicine also serves a vital role in triage of posttreatment head and neck cancer patients who may not be able to be seen as frequently due to the pandemic Telemedicine also serves a vital role in the coordination of care between multiple oncologic disciplines Dharmarajan and colleagues highlighted the University of Pittsburgh approach to a virtual multidisciplinary tumor board clinic MDC This strategy has been adopted by multiple institutions and works quite well to coordinate care between specialties In their study they found that of virtual tumor board participants preferred virtual multidisciplinary clinic to Journal Preproof 0cthe inperson format Additionally about of participants indicated that they would prefer to continue the virtual multidisciplinary format once in person meeting restrictions had been lifted Through multiple virtual meeting applications practitioners can share imaging and laryngoscopy which may assist with decision making for patients Similar to the guidelines published for laryngeal surgery the American Academy of Otolaryngology has published recommendations for ramping up clinical volume as it relates to triage for head and neck surgical oncology Setzen et al note that most head and neck cases fall within the urgent category The guidelines list emergent procedures as being tumor obstructing airway significant bleeding acute or impending neurological change and salivary gland or deep neck abscesses Urgent procedures within days include all head and neck squamous cell carcinomas of the upper aerodigestive tract benign tumors with impending complications or morbidity anaplastic thyroid cancer medullary thyroid cancer bulky differentiated thyroid cancer with regionaldistant metastasis locally aggressive or large nodules 4cm Bethesda high grade salivary malignancies skin cancers and parathyroid carcinomas with significant systemic effects Time sensitive procedures include lowrisk differentiated thyroid cancer lowgrade salivary neoplasms and slower growing basal cell carcinomas in favorable locations Routine procedures include benign thyroid pathology parathyroid disease without significant systemic effects benign salivary lesions and low risk skin cancers and posttreatment disorders Ranasinghe and colleagues recommend a tiered approach to surgical triage with more aggressive pathology being prioritized in a similar fashion to the AAO guidelines Similarly the review recommends considering alternatives to longduration microvascular reconstructive cases It is recommended that the focus shift to simplifying reconstruction and Journal Preproof 0creducing surgical duration when it™s feasible and appropriate However it is also acknowledged that this approach may lead to an increase in downstream complications Endocrine surgery is similarly tiered in a memo by Ashok Shaha which outlines a strategic approach to thyroid surgery during the pandemic Similar to other strategies anaplastic thyroid cancer medullary thyroid cancer and locally aggressive differentiated thyroid cancer specifically with impending concern for airway obstruction take precedent However some alternatives are also discussed for instance in patients with BRAF V600E mutations who may have surgery delayed while being treated with appropriate targeted therapies Additionally deescalation of surgical care is advocated for benign conditions like thyroid goiters that are nonobstructive and even papillary thyroid microcarcinoma which may be followed with serial ultrasonography until resource allocation has returned to preCOVID levels As institutions attempt to weigh the pros and cons of elective and essential surgery in the midst of the pandemic some authors have advocated for creating rating systems to allow for appropriate surgical triage during periods of limited clinical output and resource reallocation The medically necessary timesensitive MeNTS procedures scoring system aims to œethically and efficiently manage resource reallocation and risk to healthcare providers during the COVID19 pandemic51 The scoring system which uses procedural disease and patient factors within a 5point Likert scale to determine the potential risk of proceeding with surgery The cumulative MeNTS score may range between and with score above being considered within the high risk or resource heavy procedures either due to patient factors AgeComorbidities or procedure factors head and neck surgical site high anticipated blood loss ICU admission requirement Using scoring systems like MeNTS should help hospitals more Journal Preproof 0cappropriately and objectively triage elective and essential surgeries in the setting of a resurgence of caseslimiting of resources Given the significant lack of available knowledge regarding SARSCoV2 and its associated complications it is difficult to characterize risk for patients undergoing ablative and reconstructive head and neck surgery As mentioned earlier in asymptomatic COVID19 positive patients undergoing elective surgery mortality approached COVID19 has demonstrated myriad manifestations which might interfere with the success and management of patients undergoing head and neck surgery Tang demonstrated that coagulopathy was more common in patients with severe disease and nonsurviving COVID patients52 In this study Ddimer fibrin

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

"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

Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the ‚uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these findings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insufficient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social financialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreflect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Specifically procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and specificity[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical efficacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the ‚uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBGž level ‰¥ mmolling mgdl or h blood glucose ‰¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ‰¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushing™s syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may ‚uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coefficients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Student™s ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe Mann“Whitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ””””””” ± ± ± ± ” ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r ˆ’P There was a significant negative correlationbetween OC or CTX and TG rs ˆ’ P rs ˆ’ P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs ˆ’ P OC rs ˆ’ P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Thesefindings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH ˆ’ P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC ˆ’ P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars “P lmgp HTPlmgn XTC𝛽r “P HbA1c brs “P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearson™s correlation coefficient rs Spearman™s correlation coefficient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coefficients Standardized coefficients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the ‚uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [“] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ‰¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These findings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ‚ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ‚ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ‚ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ‚ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen fibres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and confirmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliflozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliflozin dapagliflozin and empagliflozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the ‚uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an ‚uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third the‚uence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespecific alkalinephosphatase BAP vitamin D or steroids all of which ‚uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its specificity for bone metabolism is lacking [] BAPis a more bonespecific marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high specificity in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreflects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These findings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DM‚uencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneficial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientific Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang œIDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and  Diabetes Research and Clinical Practicevol pp “ [] A V Schwartz D E Sellmeyer K E Ensrud œOlderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp “ [] L Forsen H E Meyer K Midthjell and T H Edna œDiabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp “ [] Y Fan F Wei Y Lang and Y Liu œDiabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp “ [] M Janghorbani R M Van Dam W C Willett and F B HuœSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp “ [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides œVitamin D and glycemic control in diabetes mellitus type  Therapeutic Advances in Endocrinologyand Metabolism vol no pp “ [] H Miyake I Kanazawa and T Sugimoto œAssociation ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalcified Tissue International vol no pp “ [] E S Siris R Adler J Bilezikian œThe clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp “ [] M B Greenblatt J N Tsai and M N Wein œBone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp “ [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell œMarkers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp “ [] H W S Cabral B F G Andolphi B V C Ferreira œTheuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp “ [] P Iglesias F Arrieta M Pi±era œSerum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp “ [] J M Wishart A O Need M Horowitz H A Morris  andB E C Nordin œEï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp “ [] P Szulc P Garnero F Munoz F Marchand and P D Delmas œCrosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp “ [] K G M M Alberti P Z Zimmet and WHO ConsultationœDefinition diagnosis and classification of diabetes mellitusand its complications Part diagnosis and classification ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp “ [] J StarupLinde and P Vestergaard œBiochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp “ [] L Achemlal S Tellal F Rkiouak œBone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp “ [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin œAssociation of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp “ [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira œMetabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp “ [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco œBone turnover markers in men andwomen with impaired fasting glucose and diabetes CalcifiedTissue International vol no pp “ [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann œLower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp “ in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu œSignificant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp “ [] A Tan Y Gao X Yang œLow serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp “ [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang œThe relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp “ [] Y Chen Q Zhao G Du and Y Xu œAssociation betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a

Thyroid_Cancer

Rapid repair of human disease‘specific single‘nucleotide variants by One‘SHOT genome editingYuji Yokouchi12 Shinichi Suzuki2 Noriko Ohtsuki12 Kei Yamamoto12 Satomi Noguchi12 Yumi Soejima3 Mizuki Goto34 Ken Ishioka5 Izumi Nakamura12 Satoru Suzuki6 Seiichi Takenoshita7 takumi era123Many human diseases ranging from cancer to hereditary disorders are caused by single‘nucleotide mutations in critical genes Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases However current procedures for repairing deleterious single‘nucleotide mutations are not straightforward requiring multiple steps and taking several months to complete In the current study we aimed to repair pathogenic allele‘specific single‘nucleotide mutations using a single round of genome editing Using high‘fidelity site‘specific nuclease AsCas12aCpf1 we attempted to repair pathogenic single‘nucleotide variants SNVs in disease‘specific induced pluripotent stem cells As a result we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single‘nucleotide marker followed by absolute markerless scarless repair of the RET SNV with no detected off‘target effects The markerless method was then confirmed in human type VII collagen‘encoding gene COL7A1 Thus using this One‘SHOT method we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one resulting in allele‘specific repair that can be completed within weeks with or without a single‘nucleotide marker Our findings suggest that One‘SHOT can be used to repair other types of mutations with potential beyond human medicineThe human genome contains extensive variation including an estimated — singlenucleotide variants SNVs that determine how we look and function as well as our specific disease tendencies1“ Some SNVs are pathogenic and either directly or indirectly cause hereditary disorders4“ such as multiple endocrine neoplasia type 2B MEN2B7 and dystrophic epidermolysis bullosa DEB8 MEN2B is an autosomal dominant syndrome characterised by thyroid adrenal gland and neuronal tumours and skeletal abnormalities The majority of MEN2B cases result from a single aminoacid substitution Met918Thr in the RET protooncogene which is caused by a pathogenic SNV RET c2753TC at the second base of the codon7 DEB is an inherited disease characterised by severe recurrent skin ulcers and blistering It is caused by genetic mutations in the human type VII collagenencoding gene COL7A1 the product of which is an anchoring fibril connecting the epidermis to the dermis8 To model diseases such as these in a0vitro diseasespecific induced pluripotent stem cells iPSCs carrying pathogenic SNVs or other genetic mutations can be obtained from patients9“ Repairing these iPSCs to generate isogenic revertant cells is a promising strategy for genome repair and s up new avenues for drug 1Pluripotent Stem Cell Research Unit in Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Hikarigaoka Fukushima Japan 2Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Fukushima Japan 3Department of Cell Modulation Institute of Molecular Embryology and Genetics IMEG Kumamoto University Kumamoto Japan 4Department of Dermatology Faculty of Medicine Oita University Yufu Japan 5Department of Microbiology School of Medicine Fukushima Medical University Fukushima Japan 6Office of Thyroid Ultrasound Examination Promotion Radiation Medical Science Centre for the Fukushima Health Management Survey Fukushima Medical University Fukushima Japan 7Fukushima Medical University Fukushima Japan email yokouchyfmuacjpScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c discovery1314 However the repair process remains problematic and a precise and convenient genome editing procedure has not yet been developedArtificial genome repair andor modification generally starts from a targetspecific doublestrand break generated by sitespecific nucleases1516 Doublestrand breaks are then repaired by a cell™s own genome repair machineries1516 However most of the break sites are incorrectly repaired by nonhomologous end joining and can result in gene knockout through the generation of nonspecific insertions or deletions indels1718 If a repair template carrying a repair base is coadministrated however the cleavage sites can be accurately repaired via homologydirected repair HDR1516 Sitespecific nucleases such as transcription activatorlike effector nucleases or the Streptococcus pyogenes Sp Cas9 nuclease are typically used as genome editing tools for human iPSCs19“SpCas9 is a type IIA endonuclease in the class clustered regularly interspaced short palindromic repeats CRISPRCas system that has been repurposed as a programmable sitespecific nuclease for genome engineering22“ Indeed SpCas9 has become a popular genome editing tool for genetically modifying human pluripotent stem cells19 Despite its efficient cleavage activity wildtype SpCas9 has a low DNA repair rate using HDR following plasmidbased administration It recuts the repaired site because the guide RNA has a “2base mismatch tolerance during sequence recognition leading to incorrect repair by nonhomologous end joining1718 To prevent recutting a blocking mutation must be introduced into the seed sequence of the guide RNA or into the protospaceradjacent motif PAM26“However for œscarless genome editing repairs with wildtype SpCas92749 each method has its obvious strengths and weaknesses For example CORRECT which includes excellent tricks to prevent recutting of the edited target by the editing tool can be performed even if the target recognition ability of the genome editing tool is insufficient however the need for two consecutive editing steps27 In comparison MhAX has high genome editing efficiency but cannot achieve completely scarless editing because singlebase markers are required Further as with the CORRECT method MhAX editing requires two consecutive edits49 increasing cost and time requirementsAnother recentlyidentified bacterial programmable sitespecific nuclease CRISPRCas12aCpf1 is a type VA endonuclease belonging to the class CRISPRCas system32 Among identified Cas12a enzymes those from Acidaminococcus sp BV3L6 As and Lachnospiraceae bacterium ND2006 show strong cleavage activity in mammalian cells32 These Cas12a endonucleases have unique features that complement Cas9 and expand the genome editing range First Cas12a recognises a Trich PAM upstream of the protospacer whereas Cas9 recognises a Grich PAM downstream of the protospacer32 Second two PAMinteracting variants have been generated that expand the Cas12a target range3334 Third Cas12amediated cleavage generates a staggered cut on the PAMdistal region of the target sequence as opposed to the PAMproximal blunt ends generated by Cas932 Finally and perhaps most importantly the guide or CRISPR RNA crRNA exhibits highfidelity target recognition meaning that Cas12a can precisely distinguish the target sequence at a singlebase resolution3536 Consequently the resulting offtarget effects are kept to a background level These features suggest that Cas12a might be suitable for precise diseasespecific iPSC repair because its recut activity is lowCas12a has already been used to knock out pathogenic genes in cancer cells37 generate insertions or twonucleotide substitutions in iPSCs38 and to induce exonskipping in diseasespecific iPSCs39 Thus we investigated whether Cas12a could be used to carry out allelespecific singlenucleotide repair of iPSCs carrying the pathogenic SNVs found in MEN2B and DEB patients in a single round of genome editing To accomplish this we used an AsCas12a PAM variant and singlenucleotide mismatch detection polymerase chain reaction SNMDPCR analysis in diseasespecific iPSCs to develop a precise convenient genomeediting procedure we have called OneSHOT One allelespecific single HDR and singlestranded oligodeoxynucleotide ssODN transient drug selection with SNMDPCR screening OneSHOT provides scarless singlenucleotide substitution of a pathogenic SNV in diseasespecific iPSCs within a0weeks The final modification rate is within a practical range for handpicking cloning Our findings suggest that this simple low cost procedure could be used for genome editing in a single step drastically reducing the time currently needed for scarless SNV repairResultsPrinciples of One‘SHOT repair of single‘nucleotide mutations AsCas12a is a highfidelity RNAguided sitespecific nuclease that binds to the target genomic DNA site via a 20nt guide sequence in the crRNA allowing it to discriminate the target sequence at the single nucleotide level Fig a0 Following the addition of a crRNA designed for a specific target sequence containing a singlenucleotide mutation AsCas12a selectively binds to the target sequence on the mutant allele and induces a doublestrand break leaving the wildtype sequence on the alternative allele unaffected Fig a0 In the presence of a ssODN wildtype sequence template the mutant nucleotide in the target sequence can be œrepaired to the wildtype sequence via the cellular HDR machinery To mark the repaired allele we labelled the ssODN with a singlenucleotide marker in the vicinity of the mutant nucleotide This label allowed us to easily identify the generepaired clones by allelespecific amplification40“42SNMDPCR detection of the singlenucleotide marker Fig a0 A complete outline of the OneSHOT workflow for SNV repair is provided in the Supplementary Information and in Supplementary Fig a0S1Allele‘specific single‘nucleotide substitution in MEN2B‘specific iPSCs Before carrying out allelespecific singlenucleotide repair of the pathogenic RET mutation we assessed whether the OneSHOT approach could be used to accomplish allelespecific singlenucleotide substitution of the wildtype alleleWe established FB414 human iPSCs from a patient with MEN2B using a Sendai viral vector protocol43 We then confirmed that the FB414 cells exhibited an embryonic stem celllike morphology and expressed pluripotent gene markers indicating that they were authentic iPSCs Supplementary Fig a0S2 and X7 To identify Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cguide seqcrRNAHigh Fidelity SiteSpecific Nuclease SSN AsCas12aCpf1target alleleDSBTYCValleleTYCVHDRTYCVTYCVby SNMDPCR ntssODN w markerFigure a0 OneSHOT principles AsCas12a pale yellow and crRNA orange and grey lines selectively bind to a target sequence containing a pathogenic SNV red triangle on the target allele Binding leads to a doublestrand break in the target sequence on the target allele left but not in the corresponding wildtype sequence containing the wildtype nucleotide blue triangle on the alternative nontarget wildtype allele right When the ssODN repair template bluegreen line with the wildtype nucleotide blue triangle and a singlenucleotide marker a silent mutation for SNMDPCR screening green triangle is cotransfected with AsCas12a into the cells the target site on the pathogenic allele is repaired using the template by the endogenous HDR machinery In this case the intended geneedited clones are easily identified by positive screening for the singlenucleotide marker because the repaired expathogenic allele now carries the singlenucleotide markerpossible target sites for AsCas12a around the SNV of interest we first searched for PAMs recognised by wildtype AsCas12a or the RR and RVR variants which recognise TYCV and TATV PAMs respectively3334 We identified two PAM sites for the RR variant TYCV Y CT V ACG TTCC located 12bp upstream of the target nucleotide on the sense strand and TTCA located 7bp upstream of the target nucleotide on the antisense strand Fig a02a magenta lines Based on this information we designed two pairs of crRNAs crRNA_RET1 and crRNA_RET1 a0m and crRNA_RET2 and crRNA_RET2 a0m which contain guide sequences that specifically recognise wildtype and mutant target sequences respectively Fig a02aTo test the cleavage activity and targetrecognition specificity of AsCas12a_RR using these crRNAs we performed a T7E1 assay using 409B2 human iPSCs carrying the wildtype RET sequence in the target site Fig a02a middle The crRNAs for the wildtype sequence crRNA_RET1 and crRNA_RET2 each exhibited significant cleavage activity towards the wildtype target sequence Fig a02b P and P respectively By contrast the crRNAs for the mutant sequence crRNA_RET1 a0m and crRNA_RET2 a0m showed extremely weak activity Fig a02b P for both A more accurate ICE analysis showed no significant activity of the crRNAs on the WT allele Supplementary Fig a0X2a These results indicate that the crRNAs for the mutant sequence do not have significant if any activity on the WT alleleHowever the observed cleavage activity of AsCas12a_RR in conjunction with crRNA_RET1 was significantly higher than that with crRNA_RET2 Fig a02b P Supplementary Fig a0X2a Puromycin treatment further promoted the cleavage activity of AsCas12a_RR with crRNA_RET1 Fig a02b P To test the applicability of the method to carry out allelespecific singlenucleotide substitution in human iPSCs we attempted to replace the wildtype nucleotide RET c2753T at the Met918 site in the wildtype allele in FB414 MEN2BiPSCs Fig a02c Following electroporation of the pY211puro vector which expresses AsCas12a_RR and crRNA_RET1 Fig a02c blue line and a ssODN modification template ssODN_RET_M918T_I913silentC carrying both a variant nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a02c red C and light green C respectively into FB414 cells we conducted SNMDPCR screening Overall clones were positive for the substitution Fig a02c d and GE1 in Table a0 Direct sequencing of the target sequence revealed that clones contained the wildtype allelespecific introduction of the mutant nucleotide at the target site T C substitution resulting in the Met918Thr substitution Fig a02e red arrow along with the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a02e blue arrow The HDR efficiency was Table a0We then searched for offtarget sequences corresponding to the target sequence using the web tool CHOPCHOP v244 and detected no indels in either of the predicted two offtarget sites by Sanger sequencing Table a0 GE1 and by AmpliSeq Supplementary Table a0X4 These results indicated that the OneSHOT method could be used to replace a single nucleotide in an allelespecific manner while minimising offtarget effects As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE1Scientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c–¸Figure a0 Singlenucleotide substitution of the RET wildtype sequence in MEN2B iPSCs a Human RET locus containing the MEN2B mutation and crRNA of AsCas12a_RR for the mutation Top exon of the RET locus Middle the wildtype WT allele sequence Blue letters indicate the wildtype nucleotide at Met918 underlined Bottom the mutant allele sequence Red letters indicate the single missense mutation caused by a TC substitution producing a Met918Thr substitution underlined Coloured lines indicate the guide sequence template for the crRNA The pink line indicates the AsCas12a_RR PAM Coloured dashed lines indicate the sites cleaved by AsCas12a_RR with the corresponding crRNA b T7E1 assay using human wildtype iPSCs 409B2 electroporated with AsCas12a_RR and the different crRNAs crRNA_RET1 crRNA_RET1 a0m crRNA_RET2 or crRNA_RET2 a0m targeting exon Left the cropped gel images Arrowheads indicate cleaved bands The fulllength gels are presented in Supplementary Figure a0S7 Right statistical analysis of the cleavage activity and specificity of AsCas12a_RR with the crRNAs following selection with different concentrations of puromycin c HDRmediated editing for generating artificial homozygous MEN2B using AsCas12a_RR with crRNA_RET1 selectively targeting RET_Met918 in the wildtype allele d SNMDPCR analysis of the first round of screening The cropped gel image is shown here The arrowhead indicates positive PCR amplicon a0bp The fulllength gel is presented in Supplementary Figure a0S8 e Sequencing of the original and modified MEN2B iPSCs FB414 Top original sequence of RET exon with a T C substitution in the MEN2B mutant allele Bottom the modified RET sequence The T C substitution resulting in a homozygous Met Thr substitution Red and blue arrows indicate the positions of the pathogenic SNV and the singlenucleotide marker respectively Underlining indicates the codons affected by the editing A more detailed explanation is provided in the œExtended Figure Legends in the Supplementary InformationAllele‘specific single‘nucleotide repair of a pathogenic RET variant To repair the pathogenic SNV RET c2753TC in the mutant allele in FB414 cells we first tested the cleavage activity and target recognition specificity of AsCas12a_RR using crRNA_RET1 a0m and crRNA_RET2 a0m Fig a02a in a homozygous MEN2B iPSC line with mutations in RET exon in both alleles GE19 genotype RETMet918ThrMet918Thr RETIle913 silentC Fig a02e bottom The T7E1 assay confirmed that the MEN2B target sequence was selectively cleaved by AsCas12a_RR with either crRNA_RET1 a0m or crRNA_RET2 a0m but not with crRNA_RET1 or crRNA_RET2 Fig a03a The ICE analysis revealed that only the AsCas12a_RR with crRNA_RET1 a0m exhibited strong cleavage activity on the target sequence Supplementary Fig a0X2b therefore we selected the crRNA_RET1 a0m for use in subsequent experimentsWe then carried out OneSHOT repair in FB414 cells using AsCas12a_RR with crRNA1m and a ssODN repair template containing a repair nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a03b T in blue and C in light green respectively Subsequent SNMDPCR screening showed that clones were positive Fig a03c GE2 in Table a0 while direct sequencing confirmed that of the positive clones contained the introduced wildtype nucleotide at the target site C T substitution leading to a Thr918Met substitution repair Fig a03d red arrow These clones also contained the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a03d blue arrow The overall HDR efficiency was Table a0 GE2 and we detected no offtarget effects by Sanger sequencing Table a0 GE2 and by AmpliSeq Supplementary Table a0X4 As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE2Allele‘specific single nucleotide repair of a pathogenic variants in RET and COL7A1 without a single‘nucleotide marker We next investigated whether the OneSHOT method could be used to repair the pathogenic SNV in RET without including the singlenucleotide marker which would achieve true scarless repair We therefore performed OneSHOT repair in the FB414 cells using AsCas12a_RR crRNA_RET1 a0m and the ssODN repair template with only a wildtype nucleotide at Met918 In the subsequent SNMDPCR screening for the pathogenic SNV no amplicons were obtained from repaired clones because the pathogenic SNV was lost from the mutant allele Fig a04a Overall we identified negative clones by SNMDPCR screening for the pathogenic SNV and direct sequencing revealed that carried only the wildtype nucleotide at Met918 Fig a04cd and GE4 in Table a0 In this experiment the overall HDR efficiency was Table a0 GE4 and no indels were detected in the two predicted offtarget sites by Sanger sequencing Table a0 GE4 and by AmpliSeq Supplementary Table a0X4We next attempted to perform scarless repair of a pathogenic SNV in iPSCs derived from a patient with DEB to confirm the applicability of the approach for other hereditary diseases We generated iPSCs from a patient with DEB autosomal recessive compound mutation COL7A1pGly2138Ter COL7A1c3591del13insGG and aimed to substitute the pathogenic SNV c6412G T pGly2138Ter in exon Supplementary Fig a0S4ab Scarless OneSHOT using AsCas12a_RR with crRNA_COL7A11 a0m plus the repair template scarlessly repaired the pathogenic SNV in the mutant allele Supplementary Fig a0S4cde and GE5 in Table a0 with a substitution rate of No indels were detected in the seven predicted offtarget sites Supplementary Table a0S2 Supplementary Table a0X4 Unlike the scarless OneSHOT for RET_Met918Thr in FB414 cells Fig a0 GE4 in Table a0 identical sequences within the target site were observed among the unintended geneedited clones suggesting that these clones were likely duplicated Supplementary Fig a0S3 GE5Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cabcRET locus on Chr 10q11exon M918PAM crRNA_ RET1 bpWT allelecrRNA_RET2 PAMPAM M918TcrRNA_RET1mMutant allelecrRNA_RET2m PAM M NCpuro crRNA_RET 1m 2m T7E1 assay in WT iPSC 409B2 nsns xedni ledniNCcr2mpuro cr2mpuro cr2mpuro cr2puro cr2puro cr1puro cr1puro cr1mpuro cr1mpuro cr2puro cr1puro cr1mpuro crRNApuro treatmentRETI913 C T PAMRET M918 C TcrRNA_RET1WT allele sequencessODN_RET_M918T_I913silentCModified WT allele sequence Mutant allele sequencede1KM M1KIle913 A T T A T TCMet918 A TC G A C GIleIleMetThrThrScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0cGene editing CellGE1GE2GE3GE4GE5FB414FB414FB414FB414B1173Genotype phenotypeOriginal †’ DestinationRETM918T †’ RETM918TM918T I913_silentCMEN2Ba †’ MEN2B homo with SN MarkerRETM918T †’ RET I913_silentCMEN2Ba †’ MEN2B revertant with SN MarkerRETM918T †’ RET I920_silentCMEN2Ba †’ MEN2B revertant with SN markerRETM918T †’ RETMEN2Ba †’ MEN2B scarless revertantCOL7A1G2138X del13 ins GG †’ COL7A1 del13 ins GGDEBb †’ DEB scarless revertantNo of total picked clones TCNo of 1st screening passed clones SNMD PCRNo of 2nd screening passed clonessequencing1stTC 2nd1st 2ndTC Table OneSHOT and scarless OneSHOT gene editing GE experiments After electroporation of the AsCas12a_RR expression vector and the ssODN template into the cells the crude DNA samples from the singlecell derived colonies that expanded on the master plates were subjected to SNMDPCR in the first screening round For positive screening colonies with amplifiable “200bp fragments from the SNMDPCR primer pair were the intendedclone candidates GE13 For negative screening colonies lacking PCR amplification were the intendedclone candidates GE4 and In the second screening round we directly read the sequences around the target site of the DNA fragments amplified by Tks Gflex DNA polymerase in each sample silentC a silent mutation generated by replacement with a cytidine for SN marker a Multiple endocrine neoplasia type 2B B Dystrophic epidermolysis bullosa Positive screening results Negative screening resultsSampleSiteGenomic location No of mis matchesOriginalRET exon target1chr10 GE1GE1GE2GE2GE3GE3GE4GE4Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget chr15 chr4 chr15 chr4 chr15 chr4 chr15 chr4 Sequencea including mismatchesTTCC AGT TAA ATG GAT GGC AAT TGTTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG Indel ratio b Table Offtarget effects of AsCas12a_RR in gene editing experiments “ GE1GE4 After amplifying the offtarget candidates predicted by CHOPCHOP v2 from the intended geneedited iPSC clones we directly read the sequences around the candidate sites after Sanger sequencing with specific primers a Underline indicates the PAM of the AsCas12a_RR variant Lower letters indicate mismatched bases in the offtarget candidates as compared with the original target sequence b Number of indel clones relative to the number of analysed clonesDiscussionMany hereditary human diseases are caused by singlenucleotide mutations These singlebase alterations have the potential to drastically alter protein structure and function Although most singlenucleotide mutations are completely harmless silent repair of pathogenic SNVs would significantly improve the quality of life and life expectancy of patients with hereditary diseases Thus in the present study we investigated whether we could achieve scarless repair of pathogenic SNVs in pluripotent stem cells from patients with two different types of hereditary disease MEN2B and DEB More importantly we aimed to carry out the repairs in a single stepUsing the OneSHOT approach developed in this study we successfully repaired a RET gene SNV in MEN2B iPSCs with the addition of a singlenucleotide selective marker in a single step We then confirmed that the same technique could be used to carry out scarless repair in MEN2B and DEBspecific iPSCs without the need for the singlenucleotide marker Scarless repair where no trace of gene editing is left around the target sequence is the goal of any gene editing technique because it safely repairs mutations in noncoding genomic regions without any secondary effects In contrast the inclusion of marker sequences during gene editing can have downstream effects Such secondary effects include the introduction of noncoding SNVs to cryptic splice sites Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0ccausing abnormal RNA splicing4546 and mutations that introduce a premature termination codon resulting in unstable mRNA46 Noncoding mutations affecting regulatory elements can also interfere with gene regulation through loss of function resulting in reduced gene expression or gain of function resulting in gene mis or overexpression4748 Therefore scarless repair is crucial for maintaining genome integrity and preventing unknown secondary effects in the target geneSeveral other methods of pathogenic SNV repair have been developed including CORRECT2627 and MhAX49 However all currently available methods have inherent obstacles to achieving scarless SNV repair in a fast and errorfree manner To overcome some of these obstacles we used the AsCas12a nuclease which has highfidelity targetrecognition3536 circumventing the need for a blocking base to inhibit recutting as is required in other methods2627 We also performed SNMDPCRbased negative screening for the pathogenic SNV which easily detects candidate clones containing the intended alteration As a result of these modifications we achieved absolute scarless editing of the RET and COL7A1 SNVs see Fig a0 Supplementary Fig a0S4 and GE4 and GE5 in Table a0 Another advantage of the AsCas12a nuclease was the ability to carry out SNV repair in a single step because only one round of HDR is required for gene editing Fig a0 The OneSHOT method was used to repair the SNVs in RET and COL7A1 within a 3week period with sufficient efficiency for handpicking In contrast other methods can take up to “ a0months to generate the intended geneedited clone because two rounds of HDRMMEJ may be required262749 However similar to our approach the CORRECT method can achieve scarless singlenucleotide substitution thus ensuring high sequence fidelity around the target site in geneedited cells Fig a0 and Supplementary Fig a0S4 Conversely MhAX leaves a silent single nucleotide mutation around the target site for use in screening49 Another difference is that the dsDNA template in MhAX can be randomly integrated into the genome outside of the target site by nonhomologous end joining50 whereas the ssODN templates used for OneSHOTscarlessOneSHOT and CORRECT approaches are not randomly integrated51 Thus the OneSHOT method developed for SNV repair in the current study appears to have several advantages over currently available methods see Supplementary Table a0S3In the CORRECT procedure the cuttomutation distance the distance between the CRISPRSpCas9 cleavage site and the blocking mutation is a crucial factor for HDR efficiency and zygosity determination2627 We therefore searched for more appropriate sites for the singlenucleotide markers by first comparing the efficacies of three singlenucleotide markers set in different positions around the target site using a PCRrestriction fragment length polymorphism RFLP assay52 We found that two of the markers showed similar HDRspecific cleavage activity while no cleavage activity was detected for the third marker Supplementary Information and Supplementary Fig a0S5 suggesting that Ile920 could be used as an alternative singlenucleotide marker Testing of HDR efficiency in FB414 cells following OneSHOT repair using the alternative marker again confirmed that the singlenucleotide substitutions in the geneedited clones were effectively detected by positive screening using SNMDPCR for a singlenucleotide marker Supplementary Information and Supplementary Fig a0S5 We do note however that the efficiency of identification might depend on the position of the singlenucleotide marker and the primers used for SNMDPCRDespite our success in repairing the pathogenic SNVs in a single step the study has several limitations The OneSHOT method only requires one PCR run thereby reducing the time and cost compared with standard PCR“RFLP screeningbased methods which require up to three steps5253 However we found that falsepositive clones are included in the population after the first SNMDPCR screen Supplementary Fig a0X8 Therefore we are currently designing a simple way to discriminate false clones from authentic clones using a PCRbased procedure We also noted that the geneedited cell lines generated by OneSHOT are not always clonal This situation arises because high cell densities occur in the culture during puromycin selection a0days and in the recovery culture “ a0days prior to clonal expansion However assessment of our data suggests that a 1day recovery culture and sufficient singlecell suspension at the reseeding stage can prevent duplication and ensure clonal establishment of the geneedited cells Using the current protocol we estimate that the HDR substitution rate is “ While this is sufficient to permit a handpicking cloning protocol it is lower than that achieved by Cas12a in fertilised eggs from model animals3954 We hoped to improve this rate by combining OneSHOT with other procedures based on alternative principles such as introducing a blocking base into the repair template2627 andor using HDRNHEJ modification compounds3863“ We have examined whether the modification compounds can promote HDR however the compounds examined in this study had no HDRpromoting effects in our experimental system Supplementary Fig a0X4It is important to emphasise though that the procedure depends on highfidelity target recognition by the sitespecific nuclease Thus the only enzymes appropriate for the OneSHOT procedure include highfidelity variants of engineered SpCas955“ or naturally highfidelity Cas9 orthologues59“ Finally while we confirmed the expression of pluripotency markers in the geneedited clones data not shown we next aim to carry out functional analyses to confirm the differentiation potential of the repaired cells Therefore further work is needed to finetune the protocol and to confirm differentiation potential and functionality of the proteins in the corrected cell populationsTo increase the reliability of the OneSHOT method it is important to show the robustness of OneSHOT and the fidelity of the repair In order to demonstrate these issues we performed targeted NGSbased deep AmpliSeq analysis of the target sequence With regard to repair fidelity the AmpliSeq analysis showed that accurate singlenucleotide substitutions were achieved by HDR that were faithful to the ssODN template and occurred at sufficient frequency Supplementary Fig a0X3ac “ These results suggest that the method has good repair fidelity With regard to the robustne

Thyroid_Cancer

Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earth™s species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleep™s quotidian œnormalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brain™s clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patient™s desired sleep time or augmenting factors that entrainthe body™s clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require “ h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon œsiesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period œCatchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances œmorning larks latephase delays œnight owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of “ weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ‰¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway™s soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisorders”specifically sleep walking”also occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women “ in males and “ in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [“] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economo™s encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while œasleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of “ OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in œthe loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleep”or its encephalopathic analog”may affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i

Thyroid_Cancer

"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine œLa Sapienza University Viadi Grottarossa Rome ItalyFull 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 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a “7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders [“]its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[“] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [“] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures “ °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no “ revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITO“Luc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kgˆ’ STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of “ g·kgˆ’ ·dayˆ’ [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at ˆ’ °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect„¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect„¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of Sant™Andrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withœAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of field™s area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect„¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe œResults sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturer™s protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fisher™s exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and “ animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr    P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see œResults section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fisher™s exact test P vsCtr  P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see œResults section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ±     ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr   P or  P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fisher™s exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKras“driven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [“] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they 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 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [“] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative real‘time polymerase chain reaction qRT‘PCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne„¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ˆ’ΔΔCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of × cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit‘ CCK8 assayMM cells were plated in 96well plates at the density of × cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur Becton“Dickinson San Jose CA USA and analyzed using Cell Quest software Becton“DickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur Becton“Dickinson and analyzed using Cell Quest software Becton“DickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dual‘luciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturer™s instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNA‘pull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturer™s instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Student™s t test or oneway analysis of variance ANOVA followed by Tukey™s test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearman™s correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells What™s more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR‘‘3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if œUGC UGG  in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its œUGC UGG  sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR e“k MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR‘‘3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearman™s coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR‘‘3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearman™s correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR‘‘3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells a“i MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5b“h The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR‘‘3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearman™s coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells a“i MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCs‘generated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07e“k sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis a“c Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins d“f The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 What™s more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings

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 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Thyroid_Cancer

Drug repurposing identifying novel indications for drugs bypasses common drug development pitfalls to ultimately deliver therapies to patients faster However most repurposingdiscoveries have been led by anecdotal observations eg Viagra or experimentalbasedrepurposing screens which are costly timeconsuming and imprecise Recently more systematic computational approaches have been proposed however these rely on utilizing theinformation from the diseases a drug is already approved to treat This inherently limits thealgorithms making them unusable for investigational molecules Here we present a computational approach to drug repurposing CATNIP that requires only biological and chemicalinformation of a molecule CATNIP is trained with diverse small molecules and uses different drug similarity features such as structural target or pathway based similarityThis model obtains significant predictive power AUC Using our model we createda repurposing network to identify broad scale repurposing opportunities between drugtypes By exploiting this network we identified literaturesupported repurposing candidatessuch as the use of systemic hormonal preparations for the treatment of respiratory illnessesFurthermore we demonstrated that we can use our approach to identify novel uses fordefined drug classes We found that adrenergic uptake inhibitors specifically amitriptylineand trimipramine could be potential therapies for Parkinson™s disease Additionally usingCATNIP we predicted the kinase inhibitor vandetanib as a possible treatment for Type Diabetes Overall this systematic approach to drug repurposing lays the groundwork tostreamline future drug development effortsa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gilvary C Elkhader J Madhukar NHenchcliffe C Goncalves MD Elemento O Amachine learning and network framework todiscover new indications for small moleculesPLoS Comput Biol e1008098 101371journalpcbi1008098Editor Avner Schlessinger Icahn School ofMedicine at Mount Sinai UNITED STATESReceived September Accepted June Published August Copyright Gilvary 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 Data is available atthe following URL wwwgithubcomcoryandarCATNIPFunding JE is supported by NLM of the NationalInstitutes of Health under award numberF31LM013058 The content is solely theresponsibility of the authors and does notnecessarily represent the official views of theNational Institutes of Health OE and his laboratoryare supported by NIH grants 1R01CA1945471U24CA210989 P50CA211024 UL1TR002384PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingThe funders had no role in study design datacollection and analysis decision to publish orpreparation of the manuscriptCompeting interests OE is cofounder and equityholder in OneThree Biotech and Volastra twocompanies that use data science and machinelearning to develop novel therapies In addition OEis an advisor and equity holder in Freenome andOwkin NM is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies CG is cofounder and equity holder inOneThree Biotech a company that use datascience and machine learning to develop noveltherapies This does not alter our adherence to allPLOS Computational Biology policies on sharingdata and materialsAuthor summaryCurrently clinical approval of a drug is an arduous process that results in an overwhelming number of compounds failing due to safety or efficacy concerns which leaves patientswithout novel lifesaving treatments The idea of drug repurposing is to take approveddrugs or compounds that were shelved due to reasons other than safety and identify newdiseases for them to treat This would allow drugs if they are sufficiently effective toquickly go through the FDA approval process and be available to patients quicker whichalso cuts the ever growing cost of novel compound research and development Here weintroduce CATNIP a computational model that can predict novel indications for specificdrugs or entire drug classes This approach analyzes drug similarity across a wide range ofbiological chemical and clinical features giving a complete picture of each drug™s mechanism and possible indications Interestingly CATNIP can be used for drugs that not onlyare previously approved but also shelved compounds which are often overlooked in previous repurposing analyses Most importantly CATNIP successfully identified noveltreatments for both Parkinson™s disease and Type Diabetes which are currently undergoing preclinical validationIntroductionWith over million spent bringing a single drug to market over the course of yearsdrug development has remained a costly and timeconsuming affair[] In response there hasbeen an increase in interest in drug repurposing the identification of novel indications forknown safe drugs Successes in this area have been seen in the past most notably in sildenafileg Viagra which was originally intended to treat hypertension and angina pectoris but waslater repurposed to treat erectile dysfunction Other examples of compounds repurposed fornew therapeutic applications include minoxidil[] and raloxifene[] which are now used totreat androgenic alopecia and osteoporosis respectively However most of these repurposingopportunities were discovered through inefficient approaches including anecdotal observations or hypothesisdriven investigations and a more efficient approach could lead to manymore repurposing opportunitiesComputational approaches for repurposing drugs are appealing in that they can be systematically and quickly applied to many drugs at a low cost compared to their experimental counterparts One computational approach that has proven to be invaluable in other areas of thedrug development pipeline is machine learning Machine learning is the use of computationalalgorithms to learn from available data to make novel predictions and gain new insight Usingthis technique one can create unbiased algorithms to match seemingly disparate drugs bycomparing their common features[] such as clinical indication toxicity profile[] or therapeutic target[ ] Previously our lab used a ˜similarity™ approach leveraging the principlethat similar drugs tend to have similar characteristics to predict a drug™s target by investigatingthe known targets of other drugs that were predicted to be œsimilar to the investigated drugbased on shared features[] We found that DRD2 a dopamine receptor was the predicted target for the compound ONC201 After identifying and experimentally validating this targetclinical trials were shifted to focus on gliomas which are now successfully completing phasetwo trials at the time of this publication[] The approach of leveraging drug similarity couldimmensely aid drug repurposing efforts with the appropriate dataPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingOthers have successfully used this ˜similarity™ approach to repurpose drugs and demonstrated high predictive power when tested against FDA approved drugdiseases[] However these methods have primarily linked drugs together using a diseasecentric approachinstead of using features related to the drug itself ie drugcentric These repurposingopportunities are identified by predicting diseases similar to the diseases a drug is alreadyknown to treat Disease similarities can be based on semantic pathophysiological or clinicalsimilarities related to the drug™s clinical indication For example PREDICT a repurposingmethod developed by Gottlieb et al[] exploits the semantic similarity of disease terms asa form of diseasedisease similarity Such approaches while reliable limit the scope of therepositioning effort in several ways First the vast majority of small molecules never reachclinical approval and would be overlooked in this type of analysis Second the use of a diseasecentric approach biases repurposing predictions toward exclusively similar clinical diseases ie cancer drugs to other cancer types [] We postulated that using solely druginformation such as chemical and biological features would be a more effective andbroader approach to drug repurposingHere we propose a novel approach to drug repurposing which operates by a platform wecall Creating A Translational Network for Indication Prediction CATNIP CATNIP is amachinelearning algorithm that learns to predict whether two molecules share an indicationbased solely on the drug™s chemical and biological features using unique drugs The systematic application of CATNIP to molecule pairs creates a network with million nodesthat can then be used to identify potential drug repurposing opportunities By identifying feature importance through the use of chemical structure and target information to make broadscale predictions CATNIP is able to effectively bridge between different therapeutic indications to advance methods of drug repurposing In this report we have identified various candidate drug classes that are predicted to have therapeutic activity outside of their intendedindication in diseases such as Parkinson™s disease and Type DiabetesResultsVariance in drug indication nomenclature can be standardizedWe collected a wide variety of drugs N including both approved and investigationalmolecules with a diverse set of indications to ensure that our drug network covered a largeportion of the known chemical space A subset of these drugs FDA approved drugs and indications taken from DrugBank [] were used as a goldstandard of drugindicationassociations in the training set for the model Disease names are often not standardized whichcan lead to many diverse names for the same disease This problem leads to many drug pairsappearing to not have shared indications when they are associated with two different namesfor the same disease To address inconsistencies in nomenclature for drug indications such asœprostate carcinoma and œcarcinoma of the prostate the MetaMap tool [] was applied tomap disease names to UMLS concepts Methods This standardization of medical terminologies allowed us to reconcile various variations in the database allowing us to confirm thatdrugs did in fact treat the same disease Examples of these variations and their mappingsmay be seen in Table Using MetaMap we clustered the DrugBank indications into standardized indications A multitude of indication types were included in this standardization including but not limited to oncological mental health and neurological diseasesS1A Fig Our rigorous standardization of drug indications ensured an accurate training setallowing for the discovery and modeling of drugindication relationshipsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cTable Indication nomenclatures and their mappingsMetamap MappedIndicationIndicationDrugBankIndication IDDrugBankNumber of unique drugsassociated with IndicationUnique drugs associated with Indication IDMachine learning approach to drug repurposingProstateCarcinomaAcne VulgarisAdvanced ProstateCarcinomaAdvanced carcinomaof the prostateSevere AcneAcneDBCOND0070333DBCOND0020265DBCOND0077433DBCOND0019842DementiaVascularIdiopathicPulmonaryFibrosisPaget DiseaseModerate AcnevulgarisMild VascularDementiaDementia VascularDementiasIdiopathic PulmonaryFibrosis IPFMild IdiopathicPulmonary FibrosisPaget™s DiseasePaget™s Disease ofBoneDBCOND0022329DBCOND0022662DBCOND0029264DBCOND0060453DBCOND0031843DBCOND0093824DBCOND0038793DBCOND0030189101371journalpcbi1008098t001IDCyproterone acetate Esterified estrogensGoserelinCyproterone acetate Doxycycline TetracyclineAloe Vera Leaf Benzoyl peroxide Chloramphenicol ClioquinolGlycolic acid Linoleic acid Octasulfur Salicylic acid SilverSpironolactoneEthinylestradiol Minocycline Nestimate TazaroteneMemantineDonepezilGalantamine Trazodone TrifluoperazineNintedanib PrednisolonePirfenidoneAlendronic acid Pamidronic acid Risedronic acid ZoledronicacidEtidronic acidDrug pairs sharing indications have other similar characteristicsWe hypothesized that pairs of drugs that shared at least one indication would have other similar drug characteristics S1 Table To test this hypothesis we integrated the similarity of twodrugs across chemical and biological drug properties and created a computational model topredict if two drugs will share an indication Fig All of the drug similarity features S1Table collected could significantly distinguish between drug pairs known to share an indication and those not known to share an indication S2“S5 Figs For example we found thatdrug pairs with a shared clinical indication according to their listed DrugBank indicationstended to have significant overlap in targets Dstatistic pvalue S2A FigThe feature which best discriminated between drug pairs that shared a clinical indication versus drug pairs that do not was the similarity between the KEGG pathways that each drug™s targets are involved in Dstatistic p S4C Fig Pathway similarity was calculatedas the Jaccard Index between the KEGG pathways that contain each drug™s gene targets Methods The difference in effect size between the target similarity and the pathway similarity Dstatistic vs respectively indicates that the drugs do not necessarily have to targetthe same exact genes but rather the same biological pathway in order to share a clinical indication Additionally we found that drug pairs that share an indication had a more similarchemical structure than drug pairs that did not share an indication Dstatistic pvalue S5A Fig A biological network containing both physical and nonphysicalinteractions was curated containing proteincoding genes drugs and TFsThis curated network provided another feature for our model allowing us to utilize previouslyestablished interactions between proteins to aid with distinguishing drug pairs that share anindication Overall these features seem to indicate sufficient power in differentiating drugsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig Schematic of CATNIP repurposing approach A The use of drug similarity properties to predict if two drugs will share an indication using agradient boosting model the model is referred to as CATNIP B Schematic showing the use of CATNIP output scores to create a network with the scoresused as edge weights The colors of each drug represent the known disease and this demonstrates how one could identify novel indications for drugsthrough the network101371journalpcbi1008098g001PLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingFig CATNIP model accurately predicts drugs that share an indication and can be used for repurposing A Receiveroperating characteristic curve for CATNIP the performance for drug pairs with high and low structural similarity is alsoshown B A network of all drug pairs with a CATNIP score higher than Nodes drugs are colored based on ATCclassification and a specific example of repurposing between ATC classifications is highlighted C A graph of all ATCclassification and the median CATNIP score between the drugs belonging to each of them only including drug pairswith CATNIP score The edges between ATC Classifications with the highest median CATNIP scores are colored red101371journalpcbi1008098g002that share and do not share indications which we hypothesized can then be leveraged to createa predictive modelDrug pairs that share indications can be predicted by modelUsing these diverse drug properties as features we trained a Gradient Boosting model to predict if two drugs share a clinical indication A Gradient Boosting model showed superiorresults when compared with other algorithms Methods S2 Table The model output is adrug similarity score hereby referred to as a œCATNIP score which allows us to classifydrug pairs that share clinical indications We performed a 5fold crossvalidation analysis andachieved significant predictive performance with an areaunderthereceiveroperator curveAUC of Fig 2A We confirmed the statistical significance of our model with aPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingprecisionrecall curve PRC because of the class imbalance in our dataset between drug pairsthat share indications against those that do not Shared Not Shared Whencompared to random predictions our model showed significant improvement vs areaunder PRC S6 Fig We retained a low percent of false positive predictions at various cutoffs false positives and false positives at a model prediction probability oftwo drugs sharing an indication of and respectively providing extra confidencethat our predictions can lead to strong repurposing candidatesWe found that the predictive model greatly benefited from the addition of diverse datatypes While structure similarity showed the highest feature importance of any single featureS11 Fig when used as a single feature within a gradient boosting model it only achieved anAUC of S12 Fig Interestingly when only supplying the model with ontology featuresa Jaccard index for the GO terms of the known targets of each drug within a drug pairachieved an AUC of However even at the highest AUC of any single feature typeit is significantly below the performance when combining all feature typesIn certain cases a high predictive performance is expected such as when two drugs arestructurally similar or share targets It has been shown before that structurally similar drugshave a high probability of treating the same indication[] However there continue to bedrug pairs known to treat the same indication that are not structurally similar For exampletamoxifen[] and anastrozole[] are structurally dissimilar compounds Dice similarity that treat the same indication Metathesaurus term Cancer Breast We recalculated our performance metrics to evaluate how our model performed in classifying drug pairsthat shared indications when only exposed to drug pairs with low structure similarityDice High performance was retained under with an AUC Fig 2A Additionally we found that our model performed similarly well when only exposed to drug pairs thatdid not have any known shared targets AUC Fig 2A These performance metricsconfirm that our model is robust enough to predict if a drug pair will share an indication evenfor more difficult prediction tasksNetwork clusters identify drugs with similar clinical characteristicsWe constructed a repurposing network by calculating a CATNIP score for all possible drugpairs found within DrugBank and assigning the drugs as nodes and the CATNIP score as theedge weight We pruned the network using a cutoff value of for the CATNIP scoresFig 2B which included different drug pairs This cutoff is equivalent to a predictedprobability of to share an indication and allowed for a balance between confidencewithin our predictions and drug diversity and availabilityWe hypothesized that drugs sharing at least one indication would cluster together in ournetwork To confirm this theory we classified each drug per its 1st order Anatomical Therapeutic Chemical ATC classification This identification is a method of distinguishing theclinical use of a drug that is widely used in European and North American chemoinformaticsdatabases[] Using ATC we observed clearly defined clusters within the repurposing network Fig 2B Many clusters featured multiple ATC classifications suggesting potential repurposing opportunities For example one cluster included the thiazolidinediones rosiglitazoneand pioglitazone ATC classification ˜Alimentary Tract and Metabolism™ and the fibratesfenofibrate and bezafibrate ATC classification ˜Cardiovascular system™ These two clusteredATC classifications were connected by a high CATNIP score between bezafibrate andpioglitazone an antidiabetic drug a relationship driven by the shared targeting of PPARa andPPARg resulting in the improvement of lipid and glucose metabolism Bezafibrate has shownefficacy in the treatment of Type Diabetes in numerous retrospective and preclinical studiesPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingATC Code ReferenceRespiratory SystemRespiratory SystemRespiratory System[“][“][“][ ]Table Literature Support for ATC Repurposing PredictionsATC Code DermatologicalsAlimentary Tract and MetabolismSensory ansSystemic Hormonal Preparations Excluding Sex Hormones AndRespiratory SystemInsulinsSensory ans101371journalpcbi1008098t002Alimentary Tract and[“]Metabolismincluding Phase trials[“] however is still not an approved antidiabetic The identification of bezafibrate as a potential diabetes treatment is a key example of how CATNIP can beused to identify repurposing opportunitiesWe reasoned that the connections between ATC classifications across all the drug clusterscould provide additional aid for drug repurposing purposes Using the pruned network CATNIP Score we collected all the scores between drugs of differing ATC classificationsFrom this collection we were able to determine the median score associated between each pairof ATC classifications The ATC classifications with the highest median CATNIP scores hadliterature support for numerous repurposing efforts between them Table For exampledrugs with the ATC classifications of œRespiratory System and œSystemic Hormonal Preparations excluding sex hormones and insulins were strongly connected to each other median CATNIP score This connection was driven by highly scored pairs of drugs includingrimexolone to mometasone CATNIP score and prednisone to triamcinolone CATNIP score These connections are supported by the fact that hormonal agents like glucocorticoids and beta adrenergic agonists have been used for decades to relax the airway musculature in patients with reactive airways disease and chronic obstructive pulmonary disease[]Interestingly our analysis identified glucagon a peptide hormone that increases blood glucoselevels as a candidate for œRespiratory System repurposing and this use already has clinicalsupport[][] Additionally drugs classified as œRespiratory System and œDermatologicalwere also observed to be highly associated because of interactions such as the one betweenciclesonide and hydrocortisone CATNIP score Ciclesonide and hydrocortisone do infact share a clinical indication œAsthma Bronchial giving added confidence to our findingsThese types of network observations are important in laying the groundwork for suggestingnovel clinical repurposing strategies for FDAapproved drugsCATNIP identifies novel disease areas for drug classesWe investigated the ability to leverage CATNIP scores to identify repurposing opportunitiesby evaluating specific drug classes Drug classes are predefined in DrugBank In order to identify actionable repurposing possibilities we narrowed this list down to classes containinginhibitors antagonists or agonists of specific gene or protein families We focused our attention on specific disease areas that are attractive for drug repurposing opportunities due to alack of current treatments or high rates of acquired resistance The specific disease areas wereœmental disorders œneurological diseases œdiabetes and œcancer cancer was furtherdivided into specific cancer types due to the large variance in disease pathology between typesMethodsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingWe hypothesized that CATNIP scores could be used to identify specific drug classes thatwould be efficacious for a new disease area For each drug class and disease area we foundthe statistical difference in the CATNIP score distribution between two sets of drug pairsThe first set included pairs that had one drug within the drug class and the other drugapproved for the disease in question while the other set included drug pairs that had onedrug within the drug class and the other drug not approved for the disease in questionMethods We compared the effect size estimated by the Wilcoxon location shift for alldrug classdisease pairs that had a significant difference in distribution compared to drugclassnondisease pairs FDR Supplementary Data By using CATNIP scores wefound that many wellknown drug classdiseases associations could be recovered For example œmuscarinic antagonists were highly ranked for œneurological diseases and many suchagents are FDAapproved for this indication[] In addition we found that œkinase inhibitors were closely associated with the treatment of cancer and œdopamine antagonists forthe treatment of œmental disorders[ ] Wilcoxon Location Shift “ forœkinase inhibitors and select cancer types Location Shift for œdopamine antagonists and œmental disorders pvalue S7 Fig In fact almost all drug classdiseaseassociations contained at least one FDAapproved drug for the respective disease giving usadded confidence in our model Of note each drug was allowed to be categorized intonumerous drug classes leading to unexpected yet easily explained results for exampleœdopamine antagonists appearing as a top drug class for œneurological diseases This isdue to risperidone a drug traditionally used for schizophrenia and mood disorders alsohaving a secondary indication of Alzheimer™s type severe dementiaOur method reached significant levels of predictive power for predicting both drug class”disease associations and individual drugdisease association When predicting drug classdisease associations under our most lenient conditions calling cases where at least one drugwithin the class was known to treat the disease a true positives our method achieved a sensitivity of greater than However this improved to a sensitivity of when we implementedstricter cutoffs ie only calling drug classdisease associations true positives if of drugswithin the class treated that disease S10 Fig We additionally compared our method™s abilityto predict individual predictions to that of a previously highlighted method Gottlieb et al™sPREDICT[] We found our method had a slightly higher AUPRC vs andhigher sensitivity vs S4 Table S1 Methods While these results indicate modest improvements over PREDICT it is important to note that unlike in PREDICT diseaseinformation is not a required feature in CATNIP™s machine learning approach This meansthat CATNIP can be applied towards investigational molecules with no previously knownindications Additionally by not using disease information as a feature repositioning of drugswith known indications using CATNIP is not directly biased by the associated disease indication and instead uses mechanistic features chemical structure and properties targets etc aspart of the repositioning strategyNext we further interrogated the drug classes associated with œneurological diseases andœdiabetes specifically CATNIP scores could correctly identify drug classes known to treatthese diseases Table To identify possible repurposing candidates we focused our attentionon drug classes shown to have a large positive effect size with this CATNIP analysis but are notcurrently approved for treatment For œneurological diseases the use of adrenergic uptakeinhibitors traditionally used as antidepressants was the top repurposing candidate for œdiabetes alpha antagonists and kinase inhibitors were identified as possible novel treatmentsTable We believe further investigation into these drug classes and diseases could lead tosuccessful clinical applicationsPLOS Computational Biology 101371journalpcbi1008098 August PLOS COMPUTATIONAL BIOLOGY 0cMachine learning approach to drug repurposingTable Top Predictions of Drug Class Repurposing OpportunitiesClassDiabetesDiseasePrediction RankAlpha1 AntagonistsKinase InhibitorProtein Kinase InhibitorsProtein Synthesis InhibitorsCytochrome P450 CYP2E1 InhibitorsMonoamine Oxidase InhibitorsNeurologicalAdrenergic Uptake InhibitorsAdrenergic alpha AgonistsProtease Inhibitors101371journalpcbi1008098t003CATNIP interpretability reveals reasoning for repurposing candidatesFrom our list of repurposing candidates we chose two novel drug classdisease associations tofurther investigateAdrenergic uptake inhibitors applied to Parkinson™s disease First we evaluated therelationship between œneurological diseases and œadrenergic uptake inhibitors We focusedon the drug pairs with the highest CATNIP scores ie those predicted with the highest confidence to share at least one indication Fig 3A Of all the adrenergic uptake inhibitors wefound that amitriptyline and trimipramine two antidepressants had the highest CATNIPscores with the œneurological diseases drugs The drugs that shared the strongest connectionswith amitriptyline and trimipramine were drugs approved for Parkinson™s disease PD Specifically metixene atropine pergolide and benzatropine were associated with amitriptylineaccording to CATNIP and trimipramine was associated to benzatropine and rotigotine Trimipramine was also strongly connected with orphenadrine which is sometimes used off labelin PD but will not be included in the following analysesUsing the CATNIP model we evaluated which features contributed towards the predictionof amitriptyline and trimipramine to share an indication with PD drugs We found that targetgene ontology and pathway similarity all strongly contributed to the predictions for both amitriptyline and trimipramine Fig 3B S8 Fig Since target similarity and distance between targets in a proteinprotein interaction network were among the top contributing features weinvestigated which gene targets were shared amongst these drug pairs We found that amitriptyline targets three specific gene classes that are also targeted by at least one of the PD drugsmuscarinic acetylcholine receptors Gcoupled protein receptors GPCRs and alpha adrenergic receptor Trimipramine also targets muscarinic acetylcholine receptors alphaadrenergicreceptors and dopamine transporters which is similar to benzatropine a PD drug All thesereceptors have welldefined relationships with PD and other neurological diseases[ ]which adds support for repurposing amitriptyline andor trimipramineAmitriptyline may be an ideal candidate for use in PD patients We evaluated the sharedmolecular function gene ontology terms shared between amitriptyline and all four PD drugsGPCR activity was once again identified S1“S4 Files We then interrogated the biologicalpathways these drug targets are involved in and found many broad GPCR pathways overlapping between amitriptyline and the PD drugs S9 Fig including the Reactome pathway œGASTRIN_CREB_SIGNALLING PATHWAY VIA PKC AND MAPK Several recent studiessupport the link between gastrinreleasing peptide signaling to brain function[] ThroughCATNIP we have identified œadrenergic uptake inhibitor

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

significant genebiomarkers of primary colorectal cancerJing Han Xue Zhang Yan Liu Li Jing Yibing Liu andDepartment of Medical Oncology The Fourth Hospital of Hebei Medical University Jiankang Road Shijiazhuang Hebei PR ChinaLi FengCorrespondence Li Feng lifeng191gqsinacomBackground Primary colorectal cancer PCRC is a common digestive tract cancer in theelderly However the treatment effect of PCRC is still limited and the longterm survival rateis low Therefore further exploring the pathogenesis of PCRC and searching for specificmolecular targets for diagnosis are the development trends of precise medical treatmentwhich have important clinical significanceMethods The public data were downloaded from Gene Expression Omnibus GEOdatabase Verification for repeatability of intragroup data was performed by Pearson™s correlation test and principal component analysis Differentially expressed genes DEGs between normal and PCRC were identified and the protein“protein interaction PPI networkwas constructed Significant module and hub genes were found in the PPI network A total of PCRC patients were recruited between and from the Fourth Hospital of Hebei Medical University RTPCR was used to measure the relative expression ofCLCA4 and MS4A12 Furthermore the study explored the effect of expression of CLCA4and MS4A12 for overall survivalResults A total of DEGs were identified between PCRC and normal colorectal tissuesTen hub genes concerned to PCRC were screened namely CLCA4 GUCA2A GCG SSTMS4A12 PLP1 CHGA PYY VIP and GUCA2B The PCRC patients with low expressionof CLCA4 and MS4A12 has a worse overall survival than high expression of CLCA4 andMS4A12 P005Conclusion The research of DEGs in PCRC DEGs hub genes especially CLCA4and MS4A12 and related signaling pathways is conducive to the differential analysis of themolecular mechanism of PCRCIntroductionPrimary colorectal cancer PCRC is a common digestive tract cancer in the elderly The primary lesioncan be seen in the left colon the right colon the upper or lower rectum [] PCRC is the second mostcommonly diagnosed cancer in women and the third most commonly diagnosed cancer in men and theprevalence of male is higher than that of female in most areas [] With the social environment lifestyleand dietary structure changes the incidence of PCRC is on the rise and there is a trend of rejuvenationThis is a social issue worthy of attention [] At present there is controversy about the pathogenesis ofPCRC It is generally believed that smoking drinking greasy diet obesity lack of exercise colorectalinflammation and genetic factors are all involved in the onset of cancer But these factors are also the causeof many other tumors Therefore the specific etiological mechanism of PCRC has not yet been elucidated[] Some scholars believe that some genes or molecules are involved in the development of PCRC Thesefindings promote the research and treatment of PCRC [“] At present the treatment of PCRC includestraditional surgery chemotherapy radiotherapy emerging immunotherapy molecular targeted therapyetc Clinically the single or combination therapy that best suits the condition is usually selected accordingto the actual situation of the patient [] However the treatment effect of PCRC is still limited andReceived March Revised August Accepted August Accepted Manuscript online 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 Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963the longterm survival rate is low The early prognosis of patients with early diagnosis is often better [] Thereforefurther exploring the pathogenesis of PCRC searching for specific molecular targets for diagnosis and treatment realizing early diagnosis targeted treatment and individualized treatment are the development trends of precise medicaltreatment which have important clinical significancePersonalized medicine refers to the treatment of existing diseases based on the information of each person™s disease genome [] It is now widely believed that majority of individual differences in drug response are due to geneticfactors Personalized medicine is a discipline that emphasizes studying the effect of genetic factors on a drug [] Recently due to the smooth implementation of the human genome project and the rapid development of bioinformaticspersonalized medicine has been strongly promoted and the concept has been gradually developed []Bioinformatics is a method to process and analyze biological data by combining biological knowledge with information processing technology It is commonly used in highthroughput data analysis such as gene and proteomicsAs a frontier interdisciplinary subject bioinformatics analysis technology can realize the biological analysis of thestructure and function of histological data find the genes or proteins most relevant to diseases and further analysis may find the molecules most relevant to diseases and can be used as disease markers [] At present a largenumber of scholars have applied this technology to tumor research that is processing gene sequence or omics databy bioinformatics analysis technology to find genes or molecular markers most relevant to tumors [“]Therefore the present study aimed to use the bioinformatics to identify the hub genes of PCRC and to verify theirrole on the overall survival of patients with PCRC based on the clinical data And the research would provide novelinsights for the personalized medicine on the treatment of patients with PCRCMaterial and methodsLease start with dates and time location of study and the recruitmentsof patientsThe present study recruited a total of PCRC patients between and from the Fourth Hospital of HebeiMedical University Shijiazhuang of Hebei province Clinical and histopathological characteristics and followup andsurvival information were available for all patients and were collected retrospectively from medical records Patientsaged “ years old histologically confirmed as PCRC not received tumor treatment and no history of gastrointestinal surgery will be screened for inclusion criteria Exclusion criteria included age years old or yearsold combined with other malignant tumors operation time more than one month after the last examination andsevers heart diseaseEthical clearance and informed contentThe research conformed to the Declaration of Helsinki and was authorized by the Human Ethics and Research EthicsCommittees of the Fourth Hospital of Hebei Medical University The written informed consents were obtained fromall participatesDownload public dataThe Gene Expression Omnibus GEO database [] httpwwwncbinlmnihgovgeo is the largest most comprehensive and publicly available source of gene expression data It contains information about the expression levels ofmultiple genes in different groups of clinical samples such as the differences in gene expression between tumor tissues and normal tissues GSE41258 GPL96 [HGU133A] Affymetrix Human Genome U133A Array and GSE81558GPL15207 [PrimeView] Affymetrix Human Gene Expression Array were obtained from the GEO database A totalof samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues wereselected from GSE41258 A total of samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues were selected from GSE81558Verification for repeatability of intragroup dataFirst repeatability of intragroup data were verified by the Pearson™s correlation test The heatmap was drew via the Rlanguage environment and presented the correlation among intragroup data Second principal component analysisPCA was the general method for sample clustering and is commonly performed for diversity analysis resequencinggene expression and other sample clustering based on various variable information The verification for repeatabilityof intragroup data was executed by PCA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Differentially expressed genes DEGs between normal and PCRCGEO2R [] httpwwwncbinlmnihgovgeogeo2r could import data of the GEO database into the R languageand perform differential analysis essentially through the following two R packages including limma packages andGEOquery Therefore through the GEO2R tool DEGs were identified between normal and PCRC group The adjusted Pvalues adj P and the fold change FC ‰¥ or ‰¤ ˆ’ were defined as significance SangerBoxshengxinren one tool was used to draw volcano maps [] Venn diagrams were delineated using anonline Venn tool httpbioinformaticspsbugentbewebtoolsVenn which would visualize common DEGs sharedbetween GSE41258 and GSE81558Protein“protein interaction PPI networkThe common DEGs shared between GSE41258 and GSE81558 were converted into differently expressed proteinsThe STRING Search Tool for the Retrieval of Interacting Genes online database tringdb could construct PPI network which was visualized by Cytoscape version []GO and KEGG analysis via DAVID toolOne online tool DAVID davidncifcrfgovhomejsp version Maryland America was applied to carriedout the functional annotation for DEGs Gene Ontology GO [] generally perform enrichment analysis of genomesAnd there are mainly cellular components CC biological processes BP and molecular functions MF in theGO analysis Kyoto Encyclopedia of Genes and Genomes KEGG wwwkeggjp [] is a comprehensivedatabase of genomic chemical and systemic functional information Therefore DAVID was used to make analysisof GO and KEGGSignificant module and hub genesMolecular Complex Detection tool MCODE version [] an plugin of Cytoscape was performed toidentify tested most significant module from the PPI network and the criteria was that the maximum depth MCODE scores cutoff kscore and node score cutoff Then cytoHubba [] a free plugin of Cytoscape was applied to authorize the hub genes when the degree ‰¥ChiaHao Chin™s [] research introduce a novel Cytoscape plugin cytoHubba for ranking nodes in a network by theirnetwork features CytoHubba provide a userfriendly interface to explore important nodes in biological networksWhen the degree‰¥ in the cytoHubba the hub genes would be obtained And in the former publications [“]numerous researchers chose hub genes out of the DEGs Therefore the present study chose hub genes out of DEGsInteraction between the hub genesPearson™s correlation analysis was also performed to present the interaction between the hub genes The cBioPortalhttpwwwcbioportal [] one online software constructed the coexpression network of these hub genesSimultaneously the coexpression network of hub genes in the field of PCRC was also analyzed via Coexpedia a freeand online toolhttpwwwcoexpedia []Expression analysis of hub genesUCSC Xena xenaucsceduwelcometoucscxena could integrate the public genomic data sets to analyzeand visualize the expression level of hub genes Then the clustering analysis of expression level of hub genes wasperformed using heatmaps based on the GSE41258 and GSE81558 Also the expression profiles of hub genes in thehuman different ans were displayed with Gene Expression Profiling Interactive Analysis GEPIA httpgepiacancerpkucn [] In order to compare the expression of hub genes in the various tumors GEPIA was used Andthe expression profiles of hub genes in the PCRC and normal groups were analyzed using GEPIAEffect of hub gene expression for pathological stage and overall survivalEffect of hub gene expression for pathological stage and overall survival was analyzed by the GEPIA Finally the correlation and linear regression analysis between PCRC and hub gene expression were performed And the receiveroperator characteristic ROC curve analysis was performed to test the sensitivity and specificity of hub gene expression for diagnose PCRC The SPSS software version IBM New York America was used to conduct all thestatistical analysis A Pvalue was defined as statistical significance The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Summaries for the function of hub genesNoGene symbolFull nameFunctionCLCA4GUCA2AChloride channel accessory Guanylate cyclase activator 2AGCGSSTGlucagonSomatostatinMay be involved in mediating calciumactivated chloride conductanceEndogenous activator of intestinal guanylate cyclase It stimulates this enzyme through thesame receptor binding region as the heatstable enterotoxinsRegulates blood glucose by increasing gluconeogenesis and decreasing glycolysisGLP1 is a potent stimulator of glucosedependent insulin release GLP2 stimulatesintestinal growth concomitant with increased crypt cell proliferationSomatostatin inhibits the release of somatotropin This hormone is an important regulatorof the endocrine system through its interactions with pituitary growth hormone thyroidstimulating hormone and most hormones of the gastrointestinal tractMS4A12Membrane spanning 4domainsMay be involved in signal transduction as a component of a multimeric receptor complexA12Silencing of this gene in colon cancer cells inhibits the proliferation cell motility andchemotactic invasion of cellsPLP1CHGAPYYVIPProteolipid protein This is the major myelin protein from the central nervous system It plays an important rolein the formation or maintenance of the multilamellar structure of myelinChromogranin AThis gene product is a precursor to three biologically active peptides vasostatinpancreastatin and parastatinPeptide YYThis gut peptide inhibits exocrine pancreatic secretion has a vasoconstrictory action andinhibitis jejunal and colonic mobilityVasoactive intestinal peptideVIP causes vasodilation lowers arterial blood pressure stimulates myocardial contractilityincreases glycogenolysis and relaxes the smooth muscle of trachea stomach andgallbladderGUCA2BGuanylate cyclase activator 2B May be a potent physiological regulator of intestinal fluid and electrolyte transport May bean autocrineparacrine regulator of intestinal salt and water transportRTqPCR assayTotal RNA was extracted from tumor colorectum tissues from PCRC patients and adjacent normal colorectum tissuesby the RNAiso Plus Trizol kit Thermofisher Massachusetts America and reverse transcribed to cDNA RTqPCRwas performed using a Light Cycler® System with specific primers for the ten hub genes Table presents theprimer sequences used in the experiments The RQ values ˆ’ 01 01Ct where Ct is the threshold cycle of each samplewere calculated and are presented as fold change in gene expression relative to the control group GAPDH was usedas an endogenous control The expression level of CLCA4 and MS4A12 in PCRC patients was measured by RTqPCROverall survival analysis of the PCRCThe Kaplan“Meier method was performed to analyze the overall survival All statistical analyses were conductedusing SPSS software version and P005 was considered statistically significantResultsHigh repeatability of dataThere exist strong correlations among samples in the PCRC group and also strong correlations among samples in thecontrol group in the GSE41258 via the Pearson™s correlation test Supplementary Figure S1A And there also existstrong correlations among samples in the PCRC group and also strong correlations among samples in the controlgroup in the GSE81558 via the Pearson™s correlation test Supplementary Figure S1B Furthermore PCA was performed to verify the repeatability of data Through the PCA the repeatability of the data in GSE41258 was fine Thedistances between per samples in the PCRC group were close and the distances between per samples in the controlgroup were also close in the dimension of PC1 Supplementary Figure S1C Through the PCA the repeatability ofthe data in GSE81558 was fine The distances between per samples in the PCRC group were close and the distancesbetween per samples in the control group were also close in the dimension of PC1 Supplementary Figure S1DDEGs between control and PCRCThere are plenty of DEGs on the all chromosomes between PCRC and control samples Supplementary Figure S1EOne volcano plot presents the DEGs in the GSE41258 Figure 1A and another volcano plot presents the DEGs in theGSE81558 Figure 1B In the volcano plots the green nodes indicate the downregulated DEGs and the red nodesindicate the upregulated DEGs The Venn diagram manifested that a total of DEGs were exist in the two datasetsGSE41258 and GSE81558 simultaneously Figure 1C After construction of PPI network for the common DEGsthere are nodes and edges in the PPI network Figure 1D The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The differently expressed genes and PPI networkA One volcano plot presents the DEGs in the GSE41258 B Another volcano plot presents the DEGs in the GSE81558 In thevolcano plots the green nodes indicate the downregulated DEGs and the red nodes indicate the upregulated DEGs C TheVenn diagram manifested that a total of DEGs were exist in the two datasets GSE41258 and GSE81558 simultaneously DThe PPI network of the common DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963The functional enrichment analysis of DEGs via GO and KEGGGO analysis manifested that variations in DEGs related with biological processes BP were significantly enrichedin bicarbonate transport onecarbon metabolic process cell surface receptor signaling pathway collagen catabolicprocess transport xenobiotic transport body fluid secretion axon development positive regulation of guanylate cyclase activity drug transmembrane transport response to steroid hormone response to tumor necrosis factor positiveregulation of peptidyl“threonine phosphorylation cell proliferation and regulation of intracellular pH Figure 2AThe variations in DEGs related with cellular components CC were significantly enriched in extracellular space extracellular region anchored component of membrane proteinaceous extracellular matrix plasma membrane apicalplasma membrane integral component of plasma membrane apical part of cell extracellular exosome and basolateralplasma membrane Figure 2B The variations in DEGs related with molecular functions MF were significantly enriched in hormone activity carbonate dehydratase activity xenobiotictransporting ATPase activity arylesterase activity metalloendopeptidase activity neuropeptide hormone activity and ˜hydrolase activity hydrolyzing Oglycosylcompounds™ Figure 2C KEGG pathway enrichment analysis showed that the top pathways related with DEGs werenitrogen metabolism bile secretion proximal tubule bicarbonate reclamation and pancreatic secretion Figure 2DSignificant module network and identification of hub genesA significant module was screened from the PPI network and the module network consisted of nodes and edgesFigure 2E And ten hub genes were identified including CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGAPYY VIP and GUCA2B Figure 2F The function of hub genes were summarized in the Table Strong interaction among the hub genesThrough the Pearson™s correlation test heat maps manifested that there were strong correlations among hub genes inthe GSE41258 Supplementary Figure S2A and GSE81558 Supplementary Figure S2B datasets PYY SST GCG andVIP existed simultaneously in the coexpression network via cBioPortal Supplementary Figure S2C And throughthe analysis of Coexpedia there were strong interactions among PYY SST GCG CHGA CLCA4 GUCA2B andMS4A12 Supplementary Figure S2DDifference of expression of hub genes between PCRC and controlsamplesHeat map showed that the expressions of all the hub genes were lower in the PCRC samples than the control samplesSupplementary Figure S2E Hierarchical clustering allowed for simple differentiation of PCRC tissues from normalcolorectal tissues via the expression levels of hub genes in the GSE41258 Supplementary Figure S3A and GSE81558Supplementary Figure S3B datasets The expressions of all the hub genes were lower in the PCRC group than thecontrol groupThe analysis of expression level of hub genesThe hub genes in the human different ans were expressed in the Supplementary Figure S3C The pink presents thetumor individuals and the green presents the normal individuals The expression of hub genes in the colorectum washigher in the normal individuals compared with the tumor samples Supplementary Figure S3C When we comparedthe expression of hub genes in the various tumors the all hub genes were downregulated in the PCRC samples alsonamed colon adenocarcinoma COAD Supplementary Figure S3D Through GEPIA analysis the expressions ofhub genes in the PCRC patients were lower than the normal individuals Supplementary Figure S4AAssociation between hub gene expression pathological stage andoverall survivalThe results of GEPIA manifested that the expression of VIP was significantly positively related with pathological stageP0027 while the expression of CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGA PYY and GUCA2B wasnot as Supplementary Figure S4B Kaplan“Meier analysis showed that PCRC patients with low expression levelsof CLCA4 and MS4A12 had poorer overall survival times than those with high expression levels P005 Figure3AE PCRC patients with high expression levels of GCG SST PLP1 and CHGA had poorer overall survival timesthan those with low expression levels P005 Figure 3CDF Supplementary Figure S5A However there was nostatistically significant effect on OS associated with the expression of GUCA2A PYY VIP and GUCA2B P005Figure 3B Supplementary Figure S5B“D The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The enrichment analysis for DEGs and the identification of hub genesA Detailed information relating to changes in the biological processes BP of DEGs in PCRC and control colorectal samplesB Detailed information relating to changes in the cellular components CC of DEGs in PCRC and control colorectal samples CDetailed information relating to changes in the molecular functions MF of DEGs in PCRC and control colorectal samples D KEGGpathway analysis for DEGs E The significant module of the PPI network F The hub genes identified from the PPI network The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The overall survival Kaplan“Meier of six hub genesA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table The correlation and linear regression analysis between PCRC and relevant gene expressionPCRCMultiple linear regressionVIFODTGene symbolPearson™s correlation coefficientPvalueρaCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2Bˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’PvalueaPearson™s correlation coefficient between PCRC and relevant characteristics ρ Pearson™s correlation coefficientbMultiple linear regression analysis PCRC primary colorectal cancer P005 P001 P0001Table Receiver operator characteristic curve analysis of hub gene expression for PCRCGene symbolPCRCCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BAUC0987maxPvalue95CI““““““““““AUC area under curve max the maximum of AUC Significant variables ODT Optimal diagnostic thresholdPCRC primary colorectal cancer P0001Correlation linear regression and ROC analysisThe Pearson™s correlation coefficient was used in the correlation analysis and CLCA4 ρ ˆ’ P0001GUCA2A ρ ˆ’ P0001 GCG ρ ˆ’ P0001 SST ρ ˆ’ P0001 MS4A12 ρ ˆ’P0001 PLP1 ρ ˆ’ P0001 CHGA ρ ˆ’ P0001 PYY ρ ˆ’ P0001 VIP ρ ˆ’ P0001 and GUCA2B ρ ˆ’ P0001 were significantly correlated with PCRC Table In themultivariate linear regression model holding all other variables at a fixed value the natural logarithmic DN remainedassociated with CLCA4 GUCA2A SST MS4A12 PLP1 CHGA PYY and GUCA2B P005 Table To identify accurate thresholds for hub genes to predict PCRC we constructed ROC The expression of all hubgenes was associated with a diagnosis of PCRC AUC Pvalue0001 Table and Figure The ROCcurve of CLCA4 was shown in Figure 4A and the area under curve of CLCA4 was maximal The ROC curve ofGUCA2A was shown in Figure 4B The ROC curve of GCG was shown in Figure 4C The ROC curve of SST wasshown in Figure 4D The ROC curve of MS4A12 was shown in Figure 4E The ROC curve of PLP1 was shown inFigure 4F The ROC curve of CHGA was shown in Figure 4G The ROC curve of PYY was shown in Figure 4H TheROC curve of VIP was shown in Figure 4I The ROC curve of GUCA2B was shown in Figure 4J The ROC curves ofper hub genes are shown in Figure 4K The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure ROC curves of hub genes for PCRCA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 G CHGA H PYY I VIP J GUCA2B K ROC curves of allhub genes The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Clinicopathological variables and the expression status of CLCA4 and MS4A12CLCA4PMS4A12PLow High Low High SexAgeMaleFemale years‰¥ yearsOverall survival months‰¥ monthsPearson™s chisquared test was usedP005Figure The verification of expression and overall survival analysis for CLCA4 and MS4A12A The relative expression of CLCA4 based on PCR B The relative expression of MS4A12 based on PCR C The overall survivalof PCRC based the expression of CLCA4 D The overall survival of PCRC based the expression of MS4A12Basic information of PCRC patientsPatients™ basic information were presented in Table The mean patient age was years old range “ yearsand the median OS was months range “ monthsRTqPCR analysis validation of hub genesAs presented in the result CLCA4 P005 Figure 5A and MS4A12 P005 Figure 5B were markedly The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963downregulated in PCRC samples when compared with the adjacent normal colorectum tissues It should be notedthat the expression situation of CLCA4 and MS4A12 were consistent in above results of bioinformaticsLow expression of CLCA4 and MS4A12 in PCRC patients wereindependent prognostic factors for the poor overall survivalThe Kaplan“Meier OS curves were analyzed Low expression of CLCA4 was predictive of a shorter OS in the PCRC patients P005 Figure 5C Low expression of MS4A12 was predictive of a shorter OS in the PCRC patients P005Figure 5DDiscussionPCRC is a common digestive tract cancer which seriously affects the life expectancy and quality of life of patients Inrecent years the survey results show that the morbidity and mortality rate are on the rise [] The clinical manifestations of patients with PCRC are related to the location and pathological type of the tumor The most common typeof pathology is adenocarcinoma The primary lesion located in the colon often causes diarrhea obstruction bleedingin the rectum anemia and cachexia in the later stage of cancer patients [] The current treatment is mainly surgerycombined with chemotherapy or radiotherapy while advocated exercise to enhance the body™s immunity and preventinfection [] Gavrilas et al found that combination of dietary preparations such as curcumin and resveratrol withchemotherapeutic drugs contributed to the prognosis of PCRC [] Clinical application benefit and safety of epidermal growth factor EGFRrelated targeted therapy and PD1PDL1 immunotherapy are still to be further studied[] The investigation found that the cost of PCRC treatment is high and it takes up a lot of medical resources andthe prognosis of patients is not necessarily proportional to the input The early treatment of early treatment patientshas a relatively low total cost of treatment and a good prognosis [] Therefore to further explore the pathogenesisof PCRC to find possible therapeutic targets to achieve early diagnosis targeted therapy individualized treatmenthas important clinical value and market prospectsBioinformatics has been widely used as a new means of exploring disease mechanism and searching fordiseaserelated genetic molecules Zhang et al found genes related to hepatocellular carcinoma by bioinformaticsanalysis Further analysis confirmed the correlation between these differential genes and diseases suggesting thatthese molecules may be used as molecular targets for early diagnosis and treatment [] Zhang et al found the mostrelevant molecules of gastric cancer miR19b3p and miR165p by analyzing the genomewide miRNA microarraydata of gastric cancer patients which provided a new idea for the diagnosis and treatment of gastric cancer [] Sunfound molecules related to the pathogenesis of colorectal cancer by screening from public databases Further analysisshowed that differentially expressed genes such as PPBP CCL28 and CXCL12 are likely to be involved in the development of colorectal cancer and may be potential diagnostic and therapeutic targets [] We found genes thatwere differentially expressed in patients with PCRC by bioinformatics analysis Low expression of PLP1 VIP SSTGCG PYY MS4A12 CLCA4 GUCA2A CHGA and GUCA2B in tumor patients compared with normal subjectsAt the same time we performed survival analysis on patients with PCRC The results showed that CLCA4 GUCA2AGCG SST MS4A12 and PLP1 genes were significantly associated with the survival of patients with PCRCCLCA4 is the chloride channel accessory CLCA4 is a member of the calciumsensitive chloridetransportingprotein family involved in intracellular ion channel activity chloride ion transmembrane transport and proteolysis Members of the calcium activated chloride channel CLCA gene family are thought to have multiple functionsincluding cell adhesion and tumor suppression Ye et al found that CLCA4 is low expressed in patients with oraltongue squamous cell carcinoma through genomewide transcriptional mapping which provides ideas for diagnosisand targeted therapy [] Bundela also found multiple differentially expressed genes in oral cancer patients in Indiaand suggested that CLCA4 may be a potential therapeutic target [] Yu et al found that CLCA4 is low expressedin breast cancer patients Further analysis revealed that CLCA4 is a marker of breast epithelial differentiation andmay be involved in tumor proliferation and metastasis Clinical data analysis showed that patients with breast cancerwith low expression of CLCA4 had lower recurrencefree survival rate suggesting that it may serve as a diagnosticand therapeutic target [] Hu found that CLCA4 was low expressed in bladder cancer tissues Further analysis revealed that CLCA4 may be involved in the proliferation and invasion of bladder cancer through PI3KAKT signaltransduction suggesting that CLCA4 may be a target for diagnosis and treatment [] Liu found that CLCA4 mayinhibit epithelial“mesenchymal transition EMT by affecting PI3KATK phosphorylation thereby inhibiting cellmigration and invasion of hepatoma cells [] Yang found that patients with colorectal cancer CRC had low expression of CLCA1 and CLCA4 and further experiments confirmed that CLCA1 is involv

Thyroid_Cancer

Purpose of Review Recognize which are the elements that predict why a person is aging faster or slower and which interventionwe can arrange to slow down the process which permits to prevent or delay the progression of multimorbidity and disabilityRecent Findings Aging is a complex process that leads to changes in all the systems of the body and all the functions of theperson however aging develops at different rates in different people and chronological age is not always consistent withbiological ageSummary Gerontologists are focused not only on finding the best theory able to explain aging but also on identifying one or moremarkers which are able to describe aging processes These biomarkers are necessary to better define the agingrelated pathologies manage multimorbidity and improve the quality of life The aim of this paper is to review the most recent evidence onaging biomarkers and the clusters related to them for personalization of treatmentsKeywords Biomarker of aging Frailty syndrome Aging phenotype Quality of life Multimorbidity Life expectancy SocialneedsIntroductionœMost people don™t grow up Most people age They findparking spaces honour their credit cards get married havechildren and call that maturity What that is is aging”Maya Angelou One of the biggest megatrends impactingthe world today is population aging Aging is a topic thathas captivated both scientists and philosophers throughouthistory but aging as a population scenario emerged on aThis is part of the Topical Collection on Geriatric Oncology Beatrice Di Capuabeatricedicapuagmailcom UOC di Radioterapia Oncologica Dipartimento Diagnostica perImmagini Radioterapia Oncologica e Ematologia FondazionePoliclinico Universitario œA Gemelli IRCCS Rome Italy Dipartimento di Scienze dell™invecchiamento neurologicheortopediche e della testacollo Fondazione Policlinico UniversitarioA Gemelli IRCCS Rome Italy Moffitt Cancer Center Tampa FL USAworldwide scale for the first time in the last century Thus itis hard to really identify a definition of aging It is a decrease infitness with chronological age it is a developmental phasebeyond the normal life trajectory and it is a time of the increased risk of physical and psychological disabilities testingthe limits of resilienceAging occurs at a different rate in varying geographic regions of the worldEurope is currently the oldest region with of thetotal population aged and older However the Asia andLatin America older population is growing fast with Asia™solder population almost tripling in size from million in to million in []All these data do not consider aging as an epiphenomenonbut an individual data of the global population just a chronological number Aging is intrinsically a complex scenariocharacterized by changes that take place at different levels ofbiological systems Biological age is of course influenced bychronological age but chronological age is by itself not representative of biological age biological age is determined byphysiological reserve and functional status Assessing biological age is essential to predict life expectancy and resilience to 0c Page of Curr Oncol Rep stressors [] If any definition of aging may appear incompleteand insufficient much more difficult and complex is to findthe marker or biomarker that can identify itMany theories currently trying to explain aging processesand many biomarkers are identified to measure aging and itsevolutionary stages Theories and biomarkers are not studiedto extend life span but to guide therapeutic choices and optimize patient management and personalization of careThe purpose of this paper is not purely to list which biomarkers are able to identify the various stages of aging ratherexplain how an epiphenomenon natural and physiological isso complex [] how many factors are protagonists in its development and how many actors and characters play in maximizing its individual features taking into account social andmorbidity biomarker These factors such as frailty loss ofautonomy essential needs and comorbidities influence theaging process and are able to justify why the biological ageof a person living in a country does not correspond to the ageof another person living in a country with better sociosanitaryconditionsClinical and Biological Aging PhenotypesThe aging phenotype can be described as a complex mosaicresulting from the interaction of a variety of environmentalstochastic and genetic“epigenetic eventsstimuli impinginglifelong on our body [ ]There is no clear evidence which molecular cellular orphysiological changes are the most important drivers of theaging process andor how they influence one another [] In itsbroadest sense aging merely refers to the changes that occurduring an anisms™ life span though the rate at which thesetake place varies widely [] Despite its enormous complexityinvolving combinations of these variables a small number ofbasic molecular mechanisms underpin the aging process including a set of evolutionary highly conserved basic biological mechanisms responsible for body maintenance and repairOne of the key mechanisms is inflammation a typical featureof the aging process is the development of a chronic lowgrade inflammatory status named œinflammaging [8cid129] whichemerged as critical in the pathogenesis of major agerelatedchronic diseases such as atherosclerosis type diabetes and neuro degeneration Inflammaging plays a pivotal role in themost important geriatric conditions such as sarc ia [9cid129cid129]osteoporosis [] frailty and disability thus contributing tomortality [] Interestingly a variety of tissues adipose tissue muscle ans brain liver systems immune systemand ecosystems gut microbiota of the body indicated asœsubsystems can contribute to the onset and progressionof such a systemic inflammatory state [] by increasing theproduction of several proinflammatory mediators or loweringthat of the antiinflammatory ones [8cid129]To differentiate the innocuous changes from those leadingto increased risk of disease disability or death biogerontologists tend to use a more precise term”senescence”when describing aging [] Senescence is thereforethe progressive deterioration of bodily functions over time andnormal human aging has been associated with a loss of complexity in a wide range of physiological processes and anatomic structures [] including blood pressure [] strideintervals [] respiratory cycles [] and vision [] amongothers such as postural dynamics [] ultimately leading todecreased fertility and increased risk or mortality []Systemic consequences of aging are widespread but theycan be clustered into four domains Fig “ Changing in body composition“The balance between energy availability and energydemandSignaling networks that maintain homeostasis““ NeurodegenerationThese changes develop in parallel and affect each otherthrough many feedforward and feedback loopThe phenotype that results from the aging process is characterized by increased susceptibility to disease high risk ofmultiple coexisting diseases impaired response to stress theemergence of œgeriatric syndromes altered response to treatment high risk of disability and loss of personal autonomywith all its psychological and social consequences On theother hand all these factors influence aging itself in a dynamic and parallel way so that they can be considered as not onlya consequence of aging but also an integral part of the agingprocessTheories of AgingHuman aging is currently defined as a dynamic process involving the continual adaptation of the body to lifelong exposure tointernal and external damaging as conceptualized in the œremodelling theory of aging [21cid129cid129] Theories of aging are generallyclassified as either program or damage theories Programmedaging theories suggest that there is a deliberate deterioration withage because a limited life span results in evolutionary benefits[] This plan could be a result of œaging genes The firstdescribed mutation to yield a significant extension in the life spanof Caenorhabditis elegans was in the ageI gene which wasshown to result in a increase in mean life span and a increase in maximum life span of this anism []Evolutionary biologists may argue that aging occurs due to theabsence of natural selection at the postreproductive stage of life[] Although such aging theories are subjectively appealing asthey convey a cure for aging the accumulation of damage is aspontaneous entropydriven process [] Among the damage 0cCurr Oncol Rep Fig Systemic consequences ofagingPage of theories a prevailing idea is that of oxidative damage Reactiveoxygen species ROS are generated during metabolism throughseveral interrelated reactions The supposition that aging may becaused by ROS has been further substantiated by studies involving transgenic animals for genes encoding antioxidants The lifespan of Drosophila melanogaster has been extended by overexpression of both superoxide dismutase SOD and catalase bothantioxidant enzymes [] Since mitochondria are the major producer of ROS in mammalian cells mitochondrial DNAmtDNA is therefore particularly susceptible to oxidative damage [] Mitochondrial maintenance is therefore essential topreserve cellular homeostasis and impaired mitochondrial maintenance has been described as a shared hallmark of numeroushuman pathologies and aging [] Mitochondrial DNA varieswith age and it is commonly considered that DNA hypomethylation is a typical aspect of the aging process [] ROS are activeintermediates of DNA methylation as well as histone modification These reactive oxygen species may play a role in epigeneticprocesses physiological phenotypic variations caused by external or environmental factors that switch genes onoff throughreactions of nucleophilic substitution at the DNA levelConsequently it has been suggested that better preservation ofDNA methylation levels slower cell metabolism and improvedcontrol in signal transmission through epigenetic mechanismscould be key processes involved in human longevity Oxidativedamage to proteins is irreversible and irreparable [] and mustbe degraded by the proteasome The proteasome is the mostimportant proteolytic machinery in eukaryotic cells largely responsible for the removal of oxidized proteins and the preventionof its aggregation [] However it has been shown that theactivity of proteasome is impaired during aging leading to theaccumulation of oxidizing proteins aggresome and lipofuscinsocalled the age pigment Similarly to oxidative damage nitrosamine damage”that caused by reactive nitrogen speciesRNS such as nitric oxide”has been suggested to also contribute to agerelated diseases namely hepatic steatosis and apoptosis [] as well as functional and structural changes in the cardiovascular system [ ] sleep homeostasis [] psychological disorders [] and dementia []Most supporters of the genomic instability theory of agingrefer to telomere shortening [] and mutation in DNA mitochondrial Telomeres are the repeated DNA sequences at theends of linear chromosomes which are unable to be fullyreplicated by DNA polymerasesMutations in mtDNA cause a wide range of human mitochondrial diseases and have been implicated in agerelateddiseases and agingBiomarker FeaturesFinding the biomarker of aging is one of the most importantgoals of medicine The National Institutes of HealthBiomarkers Definitions Working Group defined a biomarkeras œa characteristic that is objectively measured and evaluatedas an indicator of normal biological processes pathogenicprocesses or pharmacologic responses to a therapeutic intervention []The American Federation for Aging Research AFAR recommends the following criteria for biomarkers of aging [39cid129]It must predict a person™s physiological cognitive andphysical function in an agerelated way independentlyof chronological age 0c Page of Curr Oncol Rep It must be testable and not harmful to test subjects forexample a blood test or an imaging technique it mustalso be technically simple to perform and it must be accurate and reproducibly without the need for specializedequipment or techniquesIt should work in laboratory animals as well as humanssince preliminary testing is always done in nonhumansubjectsFerrucci et al reviewed the biomarkers proposed as elements of a theory based on the balance between œresiliencemechanisms and œaccumulated damages where biomarkersact in reducing resilience mechanisms or increasing damages[40cid129] Tables and The pathways eligible to become biomarkers are thefollowingGenomic Instability Endogenous and exogenous agents continuously challenge the integrity of DNA when DNA repairmechanisms cannot manage the repeated damage the result isan accumulation of DNA somatic mutations This phenomenon causes dysregulation of gene expression and the production of altered proteins that lead to cellular damage Somaticmutation accumulation has been observed in skeletal musclecells neurons and lymphocytes B related to aging [“]nevertheless quantification of DNA repair capacity in humanshas yet to be finalized [“]Telomere Attrition Telomeres are the DNA sequences that areplaced at the end of the DNA chain and protect theTable Biological changesunderlying agingGenomic instabilityTelomere attritionEpigenetic alterationscid129 DNA methylationcid129 Histone modificationcid129 Noncoding RNALoss of proteostasisMitochondrial dysfunctionCellular senescenceDeregulated nutrientsensingSteam cell exhaustionAltered intercellular communicationchromosome ends from damage During each replicationtelomeres are reproduced but not completely so with agingthey become shorter and contribute to cellular senescence[“] To date different techniques are available to detecttelomere length in circulating cells however no techniqueshave been validated for evaluating aging because of the heterogeneity between different cells between individuals andhigh measurement errors that make these techniques not yetvalid in clinical practice [“]Epigenetic Alterations Epigenetics refers to those mechanismsexternal to DNA that modulate gene expression in cells theregulation of gene expression determines the phenotypic characteristics of the different cells and tissues The main mechanismsare DNA methylation histone modification and noncodingRNA While DNA methylation is easily measured in circulatingcells and seems to be correlated to aging [ ] measuringhistone modification or noncoding RNA is difficult and expensive Recent evidence correlates DNA methylation with agingand agerelated chronic diseases in humans [ ]Individuals with higher levels of DNA methylation have a higherrisk of developing several agerelated diseases and prematuremortality for all causes and cardiovascular diseases [] as wellas physical and cognitive functions [ ]Loss of Proteostasis The repair of damaged structures or theirelimination is fundamental to maintain cell integrity and function [] Studies suggest that proteostasis becomes defectivewith aging and contributes to immunosenescence [] and thatautophagy appears to be more functional in longlived peopleAccumulation of DNA somatic mutationsDysregulation of gene expressionAltered proteins productionTelomere shortening contribute tocellular senescenceAltered gene expressionRelated to agerelated chronic diseasesAccumulation of damaged structuresAltered energy productionIncreased ROS productionApoptosisprogrammed cell deathActivation of pathways leading to apoptosisProduction of SASPIncrease of life span in dietary restrictionDecline of regenerative potentialInflammagingDysfunction of endocrine neuronaland immune systems 0cCurr Oncol Rep Page of Table Measurable biomarkers classified by respective hallmarksPathways measuredMeasurable biomarkersHallmarkGenomic instabilityTelomere shorteningCellular senescencecid129 DNA repair mechanismscid129 DNA modificationscid129 Telomere lengthcid129 Markers of DNA damage responsecid129 Telomerase activitycid129 Senescent markers in blood and tissueEpigenetic changes or epigenetic clockcid129 DNA methylationcid129 Histone acetylationcid129 Noncoding RNAMitochondrialDecreased autophagy proteostasiscid129 Mitochondrial volumenumbershapecid129 Mito respirationcid129 Markers of biogenesiscid129 mtDNA copy number and haplotypescid129 Autophagy markerscid129 Chaperon proteinsStem cell exhaustionDeregulated nutrientsensingAltered intercellular communicationcid129 Proliferative capacity in vitrocid129 Resistance to stresscid129 Growth hormone GH axiscid129 Metabolism alterationscid129 Measures of inflammationcid129 yH2AX immunohistochemistrycid129Leukocyte telomere lengthcid129MIR31HGcid129 p16INK4acid129 Senescenceassociated secretoryphenotype SASP proteinscid129 Measures of DNA methylationcid129 SIRT1 SIRT2 SIRT3 SIRT6 SIRT7cid129 Dosage of circulating microRNAs miR34aMiR21 miR1263p miR151a3pmiR181a5p miR1248cid129 p31 MRI spectroscopycid129 Growth differentiating factor GDF15cid129 NADcid129 Target of rapamycin TORcid129 Protein carbamylationcid129 Advanced glycation end productscid129 Insulinlike growth factor IGF1cid129 HGBA1ccid129 IL6cid129 TNFαcid129 CRP Creactive proteincid129 TNFRII tumor necrosis factorα RII[] Measuring the loss of proteostasis mechanism could be agood biomarker but to date there are no valid techniques forthis purposeMitochondrial Dysfunction The main role of mitochondria isto guarantee energy for the cell through the production ofATP They are also involved in signaling by the productionof ROS and in apoptosisprogrammed cell deathMitochondrial dysfunction is a good biomarker of aging andis associated with disability in older persons through the reduction of muscle strength [65cid129]Many techniques are measuring oxidative phosphorylationand ROS generation that have been associated with chronicdisease [ ] nevertheless the relation with aging is notcompletely validatedCellular Senescence Genomic instability telomere shorteningand other endogenous and exogenous mechanisms can inducethe cell to activate specific pathways that lead to apoptosis[] This process is called cellular senescence and is characterized by structural and functional changes in the cell []Senescent cells produce proinflammatory cytokines andchemokines growth factors and matrix proteases called œsenescenceassociated secretory phenotype SASP [ ]which may induce some agerelated diseases [“] Thedetection of SASP has been proposed as a biomarker of aging[]Deregulated NutrientSensing Genetic mutations in growthhormone and the insulinlike growth factor have been linkedto longevity [] Moreover dietary restriction showed to increase life span in primates [ ] For these reasons thispathway has been proposed as biomarkers of agingSteam Cell Exhaustion The decline in the regenerative potential is one of the elements at the base of aging [] Despitepharmacological interventions being explored to counteractthis phenomenon [] evidences are still poor 0c Page of Curr Oncol Rep Altered Intercellular Communication With aging we also observe changes in intercellular communication as inflammatory reaction increases the other communication ways becomedysfunctional endocrine neuronal immune system []As we discussed earlier inflammation can be inappropriately increased in aging and this has been related to agerelated disease [ ]Indeed the pathways described as potential biomarkers ofaging are strongly related to inflammation for this reasonmeasuring circulating levels of cytokines is considered anew field of research [ 84cid129 ]Aging and Life ExpectancyAging and life expectancy are closely related In a broadsense determining an individual™s life expectancy is also away of schematizing his or her aging process Life expectancyis a statistical measure of the average time an anism isexpected to live based on the year of its birth LEB itscurrent age and demographic factors including gender []In the last decades life expectancy has increased in high income country the rise in human life expectancy has involveddeclines in intrinsic and extrinsic mortality processes associated respectively with senescence and environmental challenges []In association to this increased longevity there are diseasescalled agerelated that increase quadratically with age andcause a progressive loss of physical mental and cognitiveintegrities leading to impaired function and increased vulnerability to morbidity mortality [] and disability in additionto increasing care needs and agerelated burden measuredthrough the sum of disabilityadjusted life years DALYs ofthese diseases among these adults Fig Ninetytwo of the of the Global Burden of Disease causes were identified asagerelated diseases In particular cardiovascular diseaseneoplasm and chronic respiratory disorders are those withhigher agerelated disease burden []Determinants of Frailty Syndrome as AgingBiomarkerFrailty can be defined as a state of increased vulnerabilityto stressors or a loss of capacity to resolve homeostasisperturbation Frailty condition is closely related to aging[88cid129cid129] and the frailty indexes can consequently be considered biomarkers of aging themselves In frail individuals it is possible to find both changing in body composition and balance between energy availability and energydemand Moreover in the definition of frailty it is welldescribed how signaling networks maintain homeostasisand association with neurodegeneration These fouraspects all refer to the hallmarks of aging Frailty is associated with adverse clinical outcomes including falls institutionalization and death [88cid129cid129]Two principal models emerged in the last decades that areable to conceptualize and consequently measure frailty in everyday clinical practice and research the œfrailty phenotypemodel and the cumulative deficits modelThe frailty phenotype was first described by Fried and colleagues in analyzing data from the CardiovascularHealth Study CHS involving men and women aged years and older In this study it was investigated whichcharacteristics of the population were predictive of falls disability hospitalization and death Their operational definitionof frailty included a cluster of at least three of the followingvariables unintentional weight loss selfreported exhaustionlow energy expenditure slow gait speed and weak gripstrength This model does not take into consideration cognitive impairment as a cause of increased vulnerability as thiscould contribute to functional decline and adverse events inolder people [ ]The cumulative deficits model was developed byRockwood and colleagues as part of the prospectiveCanadian Study of Health and Aging CSHA involvinga cohort of older adults [] The authors identified parameters including diseases disabilities signssymptoms and laboratory values which were defined asœdeficits The sum of the deficits in a single individualallowed for the calculation of a frailty index ie thenumber of deficits divided by Frailty in this modelis not considered as a cluster of symptoms but is conceptualized as a gradable syndrome with a higher number ofdeficits implying an increased vulnerability state The twomodels of frailty show significant overlap although theycapture slightly different sides of the same problem It isimportant to notice that physical frailty is frequently associated with multimorbidity [ 93cid129 ]It has been observed that the frailty phenotype construct is intrinsically related to mobility issues Indeedin older adults physical performance measures are a robust and consistent predictor for disability hospitalization institutionalization and death both in the researchand in the clinical setting Lower physical performance isfrequently associated with loss of skeletal muscle massand quality causing reduced strength and functional impairment [95cid129cid129] This process has been called sarc iaEven though sarc ia has been long associated withaging it has to be acknowledged that it can develop muchearlier in life [] Different definitions exist for this condition for the operational definition of sarc ia both inthe clinic and for research purposes that prioritize theassessment of muscle strength over muscle mass to identity sarc ic patients Strength is more closely related tosurvival and functional decline compared with muscle 0cCurr Oncol Rep Page of CARDIOVASCULAR DISEASESAtrial fibrillaƟon and fluÆ©er endocardiƟs hypertensive heart disease intracerebralhaemorrhage ischaemic heart disease ischaemic stroke myocardiƟs nonrheumaƟc valve disease other cardiomyopathy other cardiovascular and circulatory diseases peripheralartery diseaseNEOPLASMSLeukaemia lymphoma mulƟple myeloma myelodysplasƟc syndroms and other hematopoieƟc neoplasms brain and nervous system cancer breastcancer prostate cancer larynx cancer lip and oral cavity cancer oesophagealcancer stomach cancer colon and rectum cancer liver cancer gallbladder and biliary tract cancer pancreaƟc cancer kidney cancer bladder cancer melanoma and nonmelanoma skin cancer ovarian cancer uterine cancer thyroid cancer tracheal bronchus and lung cancer mesothelioma othermalignant neoplasms other benign and insitu neoplasmsGASTROINTESTINAL ENDOCRINE AND KIDNEY DISEASESChronic kidney disease type diabetes mellitus cirrhosis due to nonalcoholicsteatohepaƟƟs pancreaƟƟs paralyƟc ileus and intesƟnal obstrucƟon pepƟculcer disease vascular intesƟnal disorders diarrhoeal diseasesSKIN AND SUBCUTANEOUS DISEASESCelluliƟs decubitus ulcer fungal skin diseases pyoderma other skin and subcutaneousdiseasesFig Agerelated diseases adapted from Chang et al []mass [95cid129cid129] According to EWGSOP criteria sarc iais defined by the presence of low muscle strength criterion and either or low muscle quantity or quality criterion or low physical performance criterion [95cid129cid129]The physical performance parameters used in the identification of frailty syndrome both integrated eg SPPB andalone walking speed handgrip strength can be used as agingperformance biomarkersDetermination of Medical and Social NeedsWhy consider medical and social needs aging biomarkersIn Robert J Havighurst said œIn considering theneeds of older people it is well first to remember that olderpeople have the needs that are common to all people andsecond that they have special needs due to the fact that theyare old people This sentence describes everything there is toknow about the need for the elderly and answers the questionbeforeIn every society and age there is what is meant by normality An elderly person in this scenario needs what is needed tomaintain this level of normalcy Activity of daily living andinstrumental activity of daily living ADL and IADL aloneremodelled according to the context and gender can identifythe minimum necessary Conducting needs assessment various areas must be considered including physical health mental health emotional care social cultural economic nutritional service security legal and educationalCHRONIC RESPIRATORY DISEASESAsbestosis chronic obstrucƟve pulmonary disease coal worker pneumoconiosis intersƟƟallung disease and pulmonary sarcoidosis other pneumoconiosis silicosis lower respiratoryinfecƟonsNEUROLOGICAL DISORDERSAlzheimer™s disease and other demenƟas motor neuron disease Parkinson™sdisease encephaliƟs pneumococcal meningiƟsAGE RELATED DISEASESENSE AN DISEASESHearing loss vision loss ex agerelated macular degeneraƟon cataract glaucoma other sense an diseases refracƟon disorders trachomaINJURIESDrowning environmental heat and cold exposure falls foreign body in other body part other transport injuries other unintenƟonal injuriesOTHER DISEASESCongenital musculoskeletal and limb anomalies digesƟve congenital anomalies endocrine metabolic blood and immune disorders other haemoglobinopathies and haemolyƟcanaemiasMany tools are used to evaluate people™s needs The majority of these tools are focused on physical performance ableto maintain autonomy few studies focus on social needs andthe costs of care In the West World of patients accountfor of total health care expenditures This is represented by older people individuals with multiple chronic conditions many medications frequent hospitalizations and limitations on their ability to perform basic daily functions due tophysical mental or psychosocial challenge []Since the health care and social needs of older adults differfrom that of other adults it is necessary to identify the needs ofthe elderly to make proper plans that will promote their healthCurrently most of the conducted studies had mainly focused on the elderly physical health needs and had neglectedto take into account other needs such as social and health careneeds Furthermore in addition to quantitative studies discovering the older adults™ œperceptions of their own health needsis also necessaryConclusionThere is a large interest of researchers in biomarkers of agingand despite some of them seem to be very promising biological biomarkers are still far from a clinical application to datethere is no technique that meets the mentioned criteria of theideal biomarker [40cid129] Moreover we know that the biologicalpathways are the final agents of aging but on one side theycan be influenced by social economic and environmental factors and on the other side they express in various disease and 0c Page of Curr Oncol Rep disabilities of the person physical and cognitive impairmentsagerelated disease systems functions sensory functions etcFig To date more than a single biomarker to assess agingwe should consider a cluster of biomarkers that comprisethe various elements that we analyzed social and educational aspects economic factors country of origin presence of agerelated disease presence of dependence indaily activities physical capability cognitive functionlung and cardiovascular function and presence of sensorydysfunctions In Table we propose several clinical andlaboratory biomarkers that can be used in clinical practiceand researchThe geriatric assessment GA can currently be considered a system capable of monitoring multiple biomarkersclinical and laboratory of aging and at the same timeable to relate them to each other Through the GA it ispossible to make a prediction of the risk of toxicity of atreatment of life expectancy of social needs and of compliance with the treatments GA is composed indeed byseveral evaluations made through standardized toolswhich examine various aspects of the person a multidimensional assessmentAlthough it seems difficult to imagine a geriatric assessment as a biomarker currently for its characteristicsand for the high predictivity it has it can be consideredthe gold standard in the management of the older individual and instrumenttoward which other biomarkersshould be evaluatedThe purpose of this paper was to evaluate the multipleaspects that distinguish the aging process Aging must noFig Mechanisms connectingdifferent clusters of biomarkerslonger be described as a simple demographic event butas a complex mosaic in which several tesserae relate toeach other some in a very evident way others often in amore subdued but all fundamental way Each aging theory has attempted to justify this process effectively however there is no single biomarker to date that has beenfound able to identify the stage of this process At thesame time clinical clusters have been added to purelybiological markers and social ones should certainly beconsidered It therefore becomes important not to consider biomarkers only as life span but to try to overcomethis link and focus on the set of factors that influencingeach other are able to guide aging in good health andgood quality of life towards a lived aging as a slowdecline At the time we are writing this paper COVID infection is reaping victims especially in Italy Thehighest mortality is observed among the older adultsbut surprisingly it seems to maintain similar values between the youngest and oldest old over yearsCurrently no plausible justification is provided for thesedata In frailty the number of comorbidities the reducedfunctional reserve was the most used reasons Indirectlythis infection is highlighting the need to use parametersthat can more easily identify the aging process regardlessof chronological ageThe studies analyzed in the literature show that if on theone hand there are physiological biomarkers able ofhighlighting some features of aging other functionalmarkers performance social and economic status somepathologies and the presence of addiction are able ofspeed it up or slow it

Thyroid_Cancer

Research letterthe COVID19An analysis of SARSCoV2 cellentry genes identifies the intestineand colorectal cancer assusceptible tissuesEditorSARSCoV2 is the causative agentforpandemicCOVID19 has necessitated rapidchangesin surgical practice andanisation through both the initialpeak and ongoing recovery period1SARSCoV2 infects cells by interacting with the host cellsurfaceprotein ACE2 and utilises TMPRSS2in viralspike protein priming tofacilitate cell entry Fig 1a2 WhilstCOVID19 is predominantly a respiratory disease approximately of patients have concurrent gastroinsymptoms3 SARSCoV2testinalRNA and live virus have been identified in stool from COVID19 patientsand SARSCoV2 readilyinfectsintestinal anoids46 Despite thesecircumstantial data gastrointestinaltransmission has not yet been formally confirmed Cancers commonlyexpress different genes from the tissueof origin and it is largely unexploredtumours can be infectedwhetherwith SARSCoV2 We soughttoexplore the expression of ACE2 andTMPRSS2 in large publicly available normaltissue and pancancerexpression data sets to understandwhether levels of these genes identifysusceptible tissuesAnalysis ofthe normaltissueGenotype Tissue Expression projectGTEx dataset showed high ACE2expression in the testis small intestinekidney heart thyroid and adipose tissue Fig S1a supporting informationTMPRSS2 levels were highest in theprostateintestinepancreas lung salivary gland kidneythyroid and liver Fig S1b supportinginformation Whilst initial analysissuggested only kidney and thyroidcoexpressed high levels of ACE2 andTMPRSS2 closer inspection of smallstomachsmall BJS Society LtdPublished by John Wiley Sons Ltdinformationintestinal and colonic samples revealedheterogeneity with a subpopulationalso coexpressing highlevels that was confirmed by Kmeansclustering Fig 1bc Fig S1cd supportingIn colonictissue high levels of both genes werefound in younger patients and greaterTMPRSS2 expression in femalesalthough neither factor defined thesubpopulation observed Fig S2ajsupporting information Biopsylocation and tissuecompartmentfrom colon samples were explored andhigher levels of both genes were foundin the mucosa and proximal colonalthough these factors also failed tofully define the high coexpressingsubpopulation Fig S2kmsupporting information These normalexpression data appear to identify aproportion of individuals who in thegastrointestinal tract express high levels of both genes known to be involvedin cell entry of SARSCoV2The Cancer Genome Atlas programTCGA is the largest pantumourcollection of genomicand transcriptomic sequencing data Havingidentified the heterogeneous geneexpression within the normal gastrointestinaltract we interrogatedTCGA and GTEx gene expressiondata to identify relative expressionof ACE2 and TMPRSS2 in tumourscompared to their tissue of originGenerally there was no correlationbetween high expression of ACE2and TPMRSS2 in normal tissue andhigh tumour expression Fig S3adsupporting information Howeverwe identified colorectal cancer asunique amongst human malignancies by coexpressing higher levels ofboth ACE2 and TMPRSS2 relativeto normal Fig 1de We exploredTCGA data to identify if molecularsubgroups of colorectal cancers specific mutations or other commonlycollectedcouldvariablesclinicaldefine tumours with varying expression of ACE2 and TMPRSS2 Therewas a subtle yet significanttrendfor higher ACE2 expression withyounger age but no association withsex Fig S4a supporting information Possession of a BRAF mutationwas found to predict lower levels oftumour ACE2 however TMPRSS2expression was unchanged Fig S4bcsupporting information StrikinglyACE2 levels were very low in tumourswith microsatellite instability MSIFig 1f Cumulatively these data identify aproportion of healthy individuals assusceptible to putative SARSCoV2intestinal infection and that patientswith colorectal cancer may be at evengreater risk of infection Further clinical studies are urgently required toexplore this mode of transmission ofCOVID19Conflicts of Interest and Sourcesof FundingThere are no conflicts of interestsScientistSJAB is supported by an AdvancedFellowshipCliniciangrantfrom Cancer Research UKC14094A27178 and core fundingfrom Wellcome and MRC to theWellcomeMRC Cambridge StemCell InstituteMahnaz DarvishDamavandi1 JamesLaycock2 Christopher Ward1 MilouS van Driel1 Mae A Goldgraben3and Simon JA Buczacki121WellcomeMRC Cambridge Stem CellInstitute University of CambridgePuddicombe Way Cambridge UK2Cambridge Colorectal Unit CambridgeUniversity Hospitals NHS Trust HillsRoad Cambridge UKand 3Department of Medical GeneticsUniversity of Cambridge Addenbrooke™sTreatment Centre Cambridge UKSCICambridgeSiBucz 101002bjs11911BJS 0cFig ACE2 and TMPRSS2 are expressed heterogeneously in the normal intestine and colorectal cancerResearch letterabdfcea Schematic demonstrating the mode of entry of SARSCoV2 into cells via interactions with the cell surface proteins ACE2 and TMPRSS2 b Scatterplot of the expression of ACE2 and TMPRSS2 across all colon GTEx samples Yellow circle highlights high coexpressing samples c Scatter plot of theexpression of ACE2 and TMPRSS2 across all small intestine GTEx samples Yellow circle highlights high coexpressing samples d Box and whisker plotof expression of ACE2 between normal large intestine Grey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQROneway ANOVA P TPM Transcripts per million e Box and whisker plot of expression of TMPRSS2 between normal large intestineGrey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQR Oneway ANOVA P TPM Transcripts permillion f Box and scatter plot of ACE2 expression levels from TCGA COADREAD data sets between tumour subtypes CIN chromosomal instabilityMSI microsatellite instability GS genome stable and POLE DNA polymerase epsilon Median IQR KruskalWallis p BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0cResearch letter S¸reide K Hallet J Matthews JBSchnitzbauer AA Line PD Lai PBS Immediate and longterm impactof the COVID19 pandemic ondelivery of surgical services Br J Surg 101002bjs [Epub ahead of print] Hoffmann M KleineWeber HSchroeder S Kruger N Herrler TErichsen S SARSCoV2 CellEntry Depends on ACE2 andTMPRSS2 and Is Blocked by aClinically Proven Protease InhibitorCell “280e8 Yang L Tu L Implications ofgastrointestinal manifestations ofCOVID19 Lancet Gastroenterol Hepatol “ Wu Y Guo C Tang L Hong ZZhou J Dong X Prolongedpresence of SARSCoV2 viral RNA infaecal samples Lancet GastroenterolHepatol “ Wang W Xu Y Gao R Lu R Han KWu G Detection of SARSCoV2in Different Types of ClinicalSpecimens Jama “ Lamers MM Beumer J van der Vaart JKnoops K Puschhof J Breugem TI SARSCoV2 productivelyinfects human gut enterocytes Science 101126scienceabc1669 [Epub ahead of print]Supporting informationAdditional supporting informationcan be found online in theSupporting Information section atthe end of the BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0c'

Thyroid_Cancer

case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies P“LGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors 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 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine„¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete„¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and “ of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in “ and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patient™s glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors™ contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci “ https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol “ https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol “ https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg “ https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model J Exp Clin Cancer Res https doi101186s1304 60180907z Hatakeyama T Kawai N Nishiyama Y Yamamoto Y Sasakawa Y Ichikawa T Tamiya T 11Cmethionine MET and 18Ffluorothymidine FLT PET in patients with newly diagnosed glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Hayashi K Jutabha P Endou H Anzai N cMyc is crucial for the expression of LAT1 in MIA Paca2 human pancreatic cancer cells Oncol Rep “ https doi103892or20121878 Huse JT Snuderl M Jones DT Brathwaite CD Altman N Lavi E Saffery R SextonOates A Blumcke I Capper D et al Polymorphous lowgrade neuroepithelial tumor of the young PLNTY an epileptogenic neoplasm with oligodendrogliomalike components aberrant CD34 expression and genetic alterations involving the MAP kinase pathway Acta Neuropathol “ https doi101007s0040 Johnson DR Giannini C Jenkins RB Kim DK Kaufmann TJ Plenty of calcification imaging characterization of polymorphous lowgrade neuroepithelial tumor of the young Neuroradiology “ https doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol “ https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med “ https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol “ https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology “ https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other epileptogenic brain neoplasms J Neurol Neurosurg Psychiatry “ https doi101136jnnp200405160 Ryall S Tabori U Hawkins C Pediatric lowgrade glioma in the era of molecular diagnostics Acta Neuropathol Commun https doi101186s4047 z Ryall S Zapotocky M Fukuoka K Nobre L Guerreiro Stucklin A Bennett J Siddaway R Li C Pajovic S Arnoldo A et al Integrated molecular and clinical analysis of pediatric lowgrade gliomas Cancer Cell “583e565 https doi101016jccell Salisbury TB Arthur S The regulation and function of the ltype amino acid transporter LAT1 in cancer Int J Mol Sci https doi103390ijms1 Schindler G Capper D Meyer J Janzarik W Omran H HeroldMende C Schmieder K Wesseling P Mawrin C Hasselblatt M et al Analysis of BRAF V600E mutation in nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma ganglioglioma and extracerebellar pilocytic astrocytoma Acta Neuropathol “ https doi101007s0040 Sumdani H Shahbuddin Z Harper G Hamilton L Case report of rarely described polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg “ https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol “ https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery “ discussion “ https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med “ https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg “ https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol “ https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol “ https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol “ https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol “ https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res “ https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and tumorigenesis Cell Rep “ https doi101016jcelre p201712002 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res “ https doi101038sjcr72901 Zhao C Zhang Y Wang J A metaanalysis on the diagnostic performance of 18FFDG and 11Cmethionine PET for differentiating brain tumors AJNR Am J Neuroradiol “ https doi103174ajnrA3718 Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou 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 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [“] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [“] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[“] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ‰¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure„¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening„¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009“ of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU “ Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE “ Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at “ A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at ×g for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturer™s instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturer™s instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope × objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ΔΔCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncan™s multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncan™s multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncan™s multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining × Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup“ BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Fir“micutesand Proteobacteria “ There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella“ Helicobacter “ and Clostridium “ were the most abundant followed byOscillospira “ Bacteroides “ andLactobacillus “Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c

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Journal of International Medical Research “ The Authors reuse guidelinessagepubcomjournalspermissions journalssagepubcomhomeimrCase ReportNivolumab plus gemcitabinedexamethasone and cisplatinchemotherapy induce durablecomplete remission inrelapsedrefractory primarymediastinal Bcell lymphomaa case report andliterature reviewGang Huang1 Ju Huang2 Zhili Zhang2Chongchong Xue1 and Yuan Liu2AbstractPrimary mediastinal large Bcell lymphoma PMBCL is an uncommon but aggressive type ofBcell lymphoma Patients with relapsed refractory PMBCL rrPMBCL have limited therapeuticoptions and usually have a relatively poor outcome Immune checkpoint blockade has become apotential treatment for this disease We report here a case of a female patient with rrPMBCLwho was treated with nivolumab plus gemcitabine dexamethasone and cisplatin GDP chemotherapy Complete remission was achieved after four cycles of combined therapy With continuednivolumab maintenance monotherapy she has remained in complete remission for longer than months This is the first report of nivolumab plus GDP chemotherapy inducing completeremission in patient with rrPMBCL This case supplements the limited literature and providesimplications for clinical trial designs regarding the potential use of nivolumab in the treatment ofrrPMBCL1Department of Hematology Yuebei People™s HospitalShantou University Medical College ShaoguanGuangdong Province China2Guangdong Women and Children Hospital GuangzhouGuangdong ChinaCorresponding authorYuan Liu Medical Genetic Centre Guangdong Womenand Children Hospital No Xingnan Rd PanyuDistrict Guangzhou Guangdong ChinaEmail yuanliu005163comCreative 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 ResearchKeywordsRelapsed refractory primary mediastinal Bcelllymphoma nivolumab checkpoint blockadegemcitabine dexamethasone cisplatin chemotherapy programmed cell death completeremissionDate received February accepted July IntroductionPrimary mediastinal large Bcell lymphomaPMBCL is an uncommon but aggressivetumor that accounts for to of nonHodgkin lymphoma1 PMBCL is distinguished from diffuse large Bcell lymphomaby virtue of distinct clinical pathologicaland genetic features2“ Recently PMBCLwas listed as a separate entity in the latestWorld Health anization classification of hematopoieticand lymphoidtumors5 PMBCL has a similar clinical presentation as classical Hodgkin lymphomacHL and PMBCL also shares certain features at the molecular level particularly9p241 alterations and programmed celldeath protein ligand 1ligand PDL1PDL2 expression6“ At present management and outcome of PMBCL are still critical and a more serious situation is faced bypeople who are diagnosed with relapsedand refractory PMBCL rrPMBCL19The optimal salvage chemotherapy andautologousforrrPMBCL are of limited efficacy19stem celltransplantRecently agents targeting programmedcell death PD1 and PDL1 have beenimmunotherapy10developed in tumorAntiPD1 therapy with monoclonal antibodies has been approved for the treatmentof several types of solid tumor and cHLThe therapeutic potential of antiPD1 therapy in other malignancies is likely to beapproved soonIn a humanizedimmunoglobulin G1 recombinant monoclonal antibody for the PD1 receptor pidilizumab was approved by the US Food andDrug Administration FDA for treatingandpediatricpatientsthatwithadultrrPMBCL11 Another agenttargetsthe PD1 receptor called nivolumab isa fully humanized immunoglobulin G4monoclonal antibody that has been grantedapproval by the US FDA for treating several solid malignancies and cHL The therapeutic efficacy of nivolumab in patientswith rrPMBCL remains unclearWe report here a patient with rrPMBCLwho received combined treatment with offlabel nivolumab and GDP chemotherapyComplete remission CR was achievedafter four cycles of such combined treatment At the time of this submission thepatient has remained in CR for longerthan months with continued nivolumabmaintenance monotherapyCase reportA 32yearold woman presented to YuebeiPeople™s Hospital with intermittent dyspneaand chest pain A positron emission tomography PET scan showed a 10cm mass inthe anterior superior mediastinum with astandardized uptake value of Themass showed unclear margins and compressed the ascending aorta and pulmonarytrunk Small pericardial and left pleuraleffusions were also observedThe mass was diagnosed as PMBCL by asubsequent biopsy Immunohistochemicalstaining showed thatlarge lymphocyteswere positive for CD20 CD79a Pax5BCL6 CD23 CD30and multiplemyeloma1 and negative for CD10 CD3CD5chromogranin Asynaptophysin 0cHuang et alterminalencodingandincludingtwocycles positiveregion inendomysialdeoxynucleotidyltransferase cytokeratin CK CK19 andandS100 Ki67 wassituEpstein“Barrhybridization was negative She was initially treated with six cycles of frontline chemotherapyofrituximab cyclophosphamide doxorubicinvincristine and prednisolone RCHOPand four cycles of doseadjusted etoposidecyclophosphaprednisolonemiderituximabDAEPOCHR were administered Thetimeline of treatment is shown in aShe received tumor resection by thoracoscopic surgery after she continued twodoxorubicinvincristinecycles ofgemcitabine dexamethasonecisplatinumetoposide and rituximabtherapy Her first CR was achieved inDecember However monthslater a PETcomputed tomography CT scan showedhypermetabolic lesions located at the leftlung and right adrenal gland but not inthe primary mediastinal site bThe patient reported no physical symptomsand received a repeat tissue biopsy whichconfirmed a relapse with PMBCL She wastreated with each cycle of a dexamethasoneifosfamide cisplatin and etoposide regimenand ibrutinib bendamustine and cytarabine therapy A chest CT scan showedFigure Summary of treatment and monitoring the tumor response a Patient™s timeline chart with thedates of treatment and monitoring the tumor response b Positron emission tomography images Upperpanel a scan of the relapsed hypermetabolic lesions located at the left lung and right adrenal gland beforecombined treatment Lower panel complete remission was achieved after four cycles of nivolumab plusGDP chemotherapyRCHOP rituximab cyclophosphamide doxorubicin vincristine and prednisolone DAEPOCHR doseadjusted etoposide prednisolone vincristine cyclophosphamide doxorubicin and rituximab GDPERgemcitabine dexamethasone cisplatinum etoposide and rituximab CR complete remission PMBCLprimary mediastinal large Bcell lymphoma DICE dexamethasone ifosfamide cisplatin and etoposideIBC ibrutinib bendamustine and cytarabine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical Researchthat the right adrenal gland lesion had partially responded while the lesions in the leftlung had progressed After those cycles ofchemotherapy the patient showed GradeIV myelosuppression and had to receiveblood transfusion treatment Moreover acerebrospinal fluid examination showedthe presence of atypical lymphocytes andno symptoms of infection of the central nervous system were observed Intrathecal chemotherapy cytarabine mg methotrexate mg and dexamethasone mg was thenadministered and no atypical lymphocyteswere detected by repeated cerebrospinalfluid analysis These findings highly suggested a potential risk of metastasis of thecentral nervous systemtreatmentBecause the disease had progressed withsevere myelosuppression and there were nostandard chemotherapy guidelines or alternative treatment options for the patientother salvage treatments of her refractorydisease needed to be considered Aftermuch discussion with the patient and herfamily she declined autologous hematopoietic stem cell transplantation and receivedcombinedgemcitabine mg dexamethasone mg and cisplatinum mg GDPchemotherapyand the offlabel antiPD1 antibody nivolumab mg After four cycles of combined treatment a repeated PETCT scanshowed thatshe had secondary CRb She received two more cyclesof combined treatment with nivolumab andGDP chemotherapy and then continuedsingle nivolumab maintenance treatment mg Since her first dose in May she received doses of nivolumab Shereported moderate fatigue and pyrexia in to days after each administration ofnivolumab Blood tests indicated normalfunction of the liver kidney and thyroidFigure She also had normal bloodlevels of creatinine albumin globulin lactate dehydrogenase aspartate transaminasetotalaminotransferaseofalaninethey2b Neutrophilbilirubin and urea nitrogen during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the first four combined therapies because of toxicity of GDPchemotherapy butrecovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2cFurthermore no adverse signs and symptoms were observed in the lungs brainand skin At the time of this submissionshe has remained in CR for longer than months with continued nivolumab maintenance therapyEthics approval was obtained from theethicalcommittee of Yuebei People™sHospital Written informed consent wasobtained from the patient for analysis ofthe samples and publicationDiscussionTreatment and outcome are critical in managing PMBCL Because there is no established standard approachthe firstlinetreatment of PMBCL is generally thesame as that for diffuse large Bcell lymphoma including RCHOP and DAEPOCHR Relapse of PMBCL usually occurs in thefirst to months after completion offrontline therapy with a lower incidenceapproximately “than diffuselarge Bcell lymphoma19 There are varioussecondlineregimens for patients with rrPMBCL includingthe rituximab ifosfamide carboplatin andetoposide regimenthe rituximab dexamethasone cytarabine and cisplatin regimen and rituximabGDP12 Because of alack of standard guidelines or treatmentoptions for PMBCL the outcome greatlydepends on the patients™ response to theregimen Thisremains poordespite these secondline salvage chemotherapies and subsequent autologous hematopoietic stem cell transplantation912immunochemotherapyresponse 0cHuang et alFigure Blood test values during the whole treatment process since the first dose of nivolumab The firstfour cycles were nivolumab plus GDP chemotherapy and nivolumab maintenance monotherapy wasadministered since the fifth cycle a Thyroxine thyrotropin FT3 and FT4 levels b Levels of creatininealbumin globulin lactate dehydrogenase aspartate transaminase alanine aminotransferase total bilirubinand urea nitrogen c Neutrophil and platelet countsFT4 free thyroxine FT3 free triiodothyronine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical ResearchIn recent years strategies focusing on thecheckpoint blockade have been developedin tumor immunotherapy10 Therapeuticantibodies targeting the PD1“PDL1 axispossess clinical activity and an acceptablesafety profile in treating a growing list oflymphomas13solid tumors and BcellBased on a clinical study of patientswith rrPMBCL pidilizumab was approvedby the US FDA for treatment of adult andpediatric patients with rrPMBCL in Another antibody nivolumab has beengranted approval for treating several solidmalignancies and cHL However studiesregarding application of nivolumab forPMBCL are limited Only five reportshave described using nivolumab for treatment of PMBCLrrPMBCL Table asfollows In a phase I study published intwo patients with PMBCL wererecruited and treated with nivolumab atdoses of or mgkg every weeks afterprevious systemic treatments14 No objective responses were observed in this previous study In another phase I study onewithrefractorypatient with PMBCL received combinedtherapy of nivolumab and ipilimumaband died during the therapeutic process15Recently two reports showed the potentialtherapeutic efficiency of nivolumab forpatientsPMBCLrrPMBCL who showed failure with conventional immunochemotherapy1617 Both ofthese two cases had immunerelated adverseeffects during the antibody treatment process One patient with highgrade neutr ia had nivolumab stopped temporarilyand was treated with intravenous immunoglobulin16 The other patient with zosterreactivation was controlled by administration of valacyclovir17 Recently Zinzaniand colleagues showed that combined treatment of nivolumab and brentuximab vedotin had promising antitumor activity and amanageable safety profile in patients withrrPMBCL18 In this phase II study patients were recruited and treated withnivolumab mgkg and brentuximab vedotin mgkg every weeks The objectiveresponse rate was and achievedTable Reports regarding application of nivolumab in primary mediastinal large Bcell lymphomarelapsedand refractory primary mediastinal large Bcell lymphomaNumberof cases DoseCombinedtreatmentAdverse events or mgkg“ mgkgIpilimumab““ResponseyesnoNoNo ofpatientsReportsLesokhin AMet al Ansell S et alWright Zet al Yassin R et alZinzani“ mgkgNoNoHighgrade neutr iaYesZoster reactivationYes mgBrentuximabNeutr iaPL et al vedotinthrombocyt iaand peripheralneuropathy of patientsPresent case“ mgkgGDPMild fatigue and pyrexiaYeschemotherapyNote “ means not indicated in the report 0cHuang et alofcyclescombinedCR and achieved partial remission Of patients of them had drugrelatedadverse events and the most common wereneutr ia thrombocyt ia and peripheral neuropathy18 In the present case weattempted several available approaches intreating the patient™s relapsed disease butfailed to control the progress of the massAfter much discussion with the patient andher family we considered an offlabel nivolumab and GDP chemotherapy as salvagetreatmentfor the patient In September her second CR was achieved afterfourtreatmentCurrently with continued nivolumab maintenance monotherapythe patient hasremained in CR for longer than monthsImmunerelated adverse events that areassociated with checkpoint blockade oftenstart within the first few weeks to monthsafter treatment but can occur any time andin any an The most common immunerelated adverse events are hypothyroidismnausea diarrhea pyrexia and fatigue1920In the present case we were concernedabout immunerelated an damage sincethe first dose of nivolumab The patientreported moderate fatigue and pyrexiaafter each administration of nivolumaband soon recovered within to daysBlood testing was performed during thewhole therapeutic process and the datawere reviewed and analyzed Blood levelsof thyroxine thyrotropin free triiodothyronine and free thyroxine indicated no thyroiditisFigure 2a Our patient alsoshowed normal metabolic data during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the first four combined therapies because of toxicity of GDPchemotherapy but they then recovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2c2b NeutrophilUnlike otherarelymphomas prognosticbiomarkersinPMBCL12 Some serum molecules such aslackinglargelyCCL17 and CD163 are considered aspotential biomarkers for predicting andmonitoring responses and detection ofrelapses in patients with Hodgkin lymphoma1221 The role of serum biomarkers inPMBCL remainsinvestigatedRadiological imaging should only be usedin patients who have new clinical symptomsor signs suggestive of relapse but not inasymptomatic patients922betoTo the best of our knowledge this is thefirst reported case of nivolumab plus GDPchemotherapy that induced CR with nosevere immunerelated an damage in apatient with rrPMBCL We also reportthe longest followup observation of successful application of nivolumab in apatient with rrPMBCLThis report supplements the limited literature of nivolumab fortreatment ofPMBCLrrPMBCL and provides implications for clinical trial design regarding thepotential use of nivolumab in treatment ofrrPMBCL Further investigation needs to beperformed for potential application of singleor combined use of nivolumab for patientswith rrPMBCL who experience failure withconventional therapeutic approachesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterestFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsorcid000000034880ORCID iDYuan LiuReferences Martelli M Ferreri A Di Rocco A et alPrimary mediastinal large Bcell lymphomaCrit Rev Oncol Hematol “ 0cJournal of International Medical Research Savage KJ Monti S Kutok JL et al Themolecular signature of mediastinallargeBcell lymphoma differs from that of otherdiffuse large Bcell lymphomas and sharesfeatures with classical Hodgkin lymphomaBlood “ Rosenwald A Wright G Leroy K et alMolecular diagnosis of primary mediastinalB cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma J ExpMed “ Mottok A Wright G Rosenwald A et alMolecular classification of primary mediastinal large Bcell lymphoma using routinelyavailable tissue specimens Blood “ Swerdlow SH Campo E Pileri SA et alThe revision ofthe World Healthanization classification of lymphoid neoplasms Blood “ XuMonette ZY Zhou J and Young KHPD1 expression and clinical PD1 blockadelymphomas Blood in Bcell“ Van Roosbroeck K Ferreiro JF TousseynT et al Genomic alterations of the JAK2and PDL loci occur in a broad spectrum oflymphoid malignancies Genes ChromosomesCancer “rearrangements Twa DD Chan FC BenNeriah S et alGenomicinvolving programmed death ligands are recurrent in primary mediastinallymphomaBlood “large Bcell Cwynarski K Marzolini MAV BarringtonSF et al The management of primary mediastinal Bcell lymphoma a British Societyfor Haematology Good Practice Paper BrJ Haematol “ Ribas A and Wolchok JD Cancer immunotherapy using checkpoint blockade Science “ HematologyOncology Cancer Approvals Safety Notifications Available online URL wwwfdagovdrugsinformationondrugsapproveddrugsucm610670htmdrugsapproveddrugsucm610670htmwwwfdagovdrugsinformationon Lees C Keane C Gandhi MK et al Biologyand therapy of primary mediastinal Bcelllymphoma current status and future directions Br J Haematol “ Goodman A Patel SP and Kurzrock RPD1PDL1 immunecheckpoint blockadein Bcell lymphomas Nat Rev Clin Oncol “ Lesokhin AM Ansell SM Armand P et alNivolumab in patients with relapsed orrefractory hematologic malignancy preliminary results of a phase Ib study J Clin Oncol “ Ansell S Gutierrez ME Shipp MA et alA phase study of nivolumab in combination with ipilimumab for relapsed or refractory hematologic malignancies CheckMate Blood “ Wright Z and Brown A Highgrade neutr ia in a patient successfully treated withnivolumab for refractory primary mediastilymphoma Blood Adv nal Bcell“ Yassin R Hajeer A Alshieban S et al HLAgenotype and response to nivolumab therapy in relapsed refractory primary mediastinal Bcell lymphoma Curr Res Transl Med “ Zinzani PL Santoro A Gritti G et alNivolumab combined with brentuximabvedotin forrelapsedrefractory primarymediastinal large Bcell lymphoma efficacyand safety from the Phase II CheckMate Study J Clin Oncol “ Postow MA Sidlow R and Hellmann MDImmunerelated adverse events associatedwith immune checkpoint blockade N EnglJ Med “ Zinzani PL Ribrag V Moskowitz CH et alSafety and tolerability of pembrolizumab inpatients with relapsedrefractory primarylymphoma Bloodmediastinal “large Bcell Jones K Vari F Keane C et al SerumCD163 and TARC as disease response biomarkers in classical Hodgkin lymphomaClin Cancer Res “for Cheson BD Fisher RI Barrington SF et alinitial evaluationRecommendationsstagingofHodgkin and nonHodgkin lymphoma theLugano classification J Clin Oncol “assessmentandresponse 0c'

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"Interferon COVID19SARSCOV2IranIn this study efficacy and safety of interferon Interferon beta1-b in the treatment of patients with severe COVID19 wereevaluatedAmong an label randomized clinical trial adult patients ‰¥ years old with severe COVID19 wererandomly assigned to the IFN group or the control group Patients in the IFN group received Interferon beta1-b mcg subcutaneously every other day for two consecutive weeks along with the national protocol medicationswhile in the control group patients received only the national protocol medications lopinavirritonavir oratazanavirritonavir plus hydroxychloroquine for “ days The primary outcome of the study was time toclinical improvement Secondary outcomes were inhospital complications and 28daymortalityBetween April and May patients were enrolled and finally patients in each group completed the study Time to clinical improvment in the IFN group was significantly shorter than the control group[“ vs “ days respectively p HR CI “] At day the percentageof discharged patients was and in the IFN and control groups respectively OR CI“ p ICU admission rate in the control group was significantly higher than the IFN group vs p The duration of hospitalization and ICU stay were not significantly differentbetween the groups Allcause 28day mortality was and in the IFN and control groups respectively p Interferon beta1-b was effective in shortening the time to clinical improvement without serious adverse events inpatients with severe COVID19 Furthermore admission in ICU and need for invasive mechanical ventilationdecreased following administration of Interferon beta1-b Although 28day mortality was lower in the IFN group furtherrandomized clinical trials with large sample size are needed for exact estimation of survival benefit of Interferon beta1-b IntroductionCoronavirus disease CoVID19 was reported from Wuhan forthe first time in late December Causing severe acute respiratorysyndrome coronavirus 2SARSCoV2 [] it rapidly spread throughoutthe world to the extent that the World Health anization WHOstated it as pandemic in March [] Until July more than million confirmed cases of CoVID19 were reported worldwideFurthermore more than deaths were recorded []Until now there is no definite antiviral treatment for CoVID19 andattempts continue for finding effective treatments worldwide Howeverfrom the beginning of the pandemic various treatments such as antiretrovirals antimalaria agents favipiravir remdesivir and corticosteroids immunoglobulin and cytokine blockers as adjunctive therapieswere suggested for the treatment of CoVID19 [] Except for the remdesivir which has had acceptable results the efficacy of other drugshas not been significant on the outcomes of the patients with CoVID19[“]Interferons IFNs have a key role in defense against viral infectionsas a component of innate immune system [] Invitro activity of IFN Ž Corresponding author at Department of Pharmacotherapy Tehran University of Medical Sciences Tehran IranEmail addresses khalilihtumsacir Khalilihsinatumsacir H Khalili101016jintimp2020106903Received July Received in revised form July Accepted August Available online August Elsevier BV All rights reserved 0cInternational Immunopharmacology prophylaxis deep vein thrombosis treatment of electrolyte disordersand antibiotic therapy were considered according to the hospital protocols The duration of the study was two weeks A 4week followupperiod was considered for all patientsPatients™ demographic data baseline diseases symptoms at the timeof disease presentation vital signs and laboratory data at the time ofhospital admission were recorded Patients were daily monitored interms of changes in the vital signs hemodynamic parameters oxygenation status laboratory data and treatment strategies Clinical statusof the patients was assessed by the sixcategory ordinal scale at days and of the randomization [] Need for supplemental oxygentherapy and also invasive or noninvasive respiratory supports wereevaluated regularly OutcomesTime to clinical improvement was considered as primary outcome ofstudy Clinical improvement was defined as improvement of at leasttwo points from the baseline status on the sixcategory ordinal scale[] This scale contains the subsequent categories death hospital admission requiring invasive mechanical ventilation hospitaladmission requiring noninvasive positive pressure ventilation hospital admission requiring oxygen hospital admission not requiring oxygen discharge Secondary outcomes were clinical statusof patients at day and ICU admission and intubation rateslength of hospitalization and ICU stay and 28day mortalitySide effects related to IFN therapy and other adverse events duringthe study period were monitored and recorded as the safety outcomesCategorization of adverse events was done according to the commonterminology criteria for adverse events CTCAE National Institutes ofHealth and National Cancer Institute Also serious complications during the hospitalization course such asacute respiratory distress syndrome ARDS nosocomial infectionsseptic shock acute kidney injury AKI and acute hepatic injury AHIwere considered Statistical analysis and randomizationContinuous variables are demonstrated as median interquartilerange IQR and categorical variables as frequencies and percentagesContinuous variables were compared between the groups by MannWhitney U test The Fisher™s exact test was applied for comparison ofcategorical variablesThe Hazard Ratio HR and CI for clinical improvement wereestimated by Cox proportional hazards regression analysis The effect ofischemic heart disease lymphocyte count Aspartate aminotransferaseAST and Creactive protein CRP on the primary outcome was evaluated by the adjusted Cox regression models as potential confoundingfactors Time to clinical improvement was estimated by KaplanMeierplot and compared with a logrank test All statistical analysis was doneby SPSS software version Time to clinical improvement was estimated to be approximately days and sample size was calculated by following equationH Rahmani et alhas been shown against severe acute respiratory syndrome coronavirusSARSCoV and Middle East respiratory syndrome coronavirus MERSCoV [“] Although IFN was used less than IFN α for the treatment of SARSCOV and MERSCoV in human studies it was effective inthe treatment of MERSCoV in retrospective studies and case series[“] The efficacy of Interferon beta1-b is being assessed in the treatment ofMERS in a randomized clinical trial [] According to the presence ofthis evidence IFN was considered as a promising option for thetreatment of CoVID19In this label randomized clinical trial efficacy and safety ofInterferon beta1-b in the treatment of patients with severe CoVID19 were assessed Materials and methods Study designThis label randomized clinical trial was designed to evaluatethe efficacy and safety of Interferon beta1-b in the treatment of patients withCoVID19 Patients with severe CoVID19 who were hospitalized duringApril to May in Imam Khomeini Hospital Center one ofthe largest referral hospitals in Tehran Iran were includedThe protocol of the study was approved by Ethics Committee ofTehran UniversityReferencenumberIRTUMSVCRREC13981053 Furthermore the study was registeredas a clinical trial register ID IRCT20100228003449N27 The studyprotocol was described for participants and written informed consentswere obtained from all patients or their firstdegree family members Eligibility criteriaof MedicalSciencesSARSCoV2 in patients™ nasopharyngeal swabs was detected usingRealTime Polymerase Chain Reaction RTPCR Total RNA extractionwas done applying Viral Nucleic Acid Extraction kit Cat No YVN50YVN100 from RBC Bioscience Taipei Taiwan The Novel Coronavirus2019nCOV Nucleic Acid Diagnostic Kit PCRFluorescence Probingof Sansure Biotech S3102E Changsha China was used for RTPCRAdult patients ‰¥ years old with positive PCR and clinicalsymptomssigns of pneumonia including dyspnea cough and feverperipheral oxygen saturation SPO2 ‰ in ambient air or arterialinspired oxygen PaO2oxygen partial pressure to fractionalFiO2 or SPO2FiO2 and lung involvement in chestimaging were included These criteria indicated severe form of thedisease [] At baseline patients with serious allergic reactions to IFNhistory of suicide thoughts and attempts alanine amino transferaseALT × the upper limit of the normal range uncontrolled underlying diseases such as neuropsychiatric disorders thyroid disorderscardiovascular diseases and also pregnant and lactating women werenot includedRecruitment was considered during the first 48hour of the hospitaladmission During the study period patients who received less than doses of Interferon beta1-b were excluded If patients were discharged beforefulfilment of the treatment course the treatment was applied at home ProceduresEligible patients were recruited in the IFN group or the controlgroup according to the permuted block randomization Patients in theIFN group received Interferon beta1-b along with the national protocol medications while in the control group patients received only the nationalprotocol medications Interferon beta1-b Ziferon® Zist Daru Daneh Co Iranwas administrated as mcg subcutaneously every other day for twoconsecutive weeks The national protocol consisted lopinavirritonavir mg BD or atazanavirritonavir mg daily plushydroxychloroquine mg BD in first day and then mg BD for“ days Other supportive cares such as fluid therapy stress ulcernkzz1122211222nn121512005015Z19612Z1041 0cH Rahmani et alInternational Immunopharmacology Fig Consort flowchart of the studyAccording to the above equation at least patients in each groupwere expected to make a difference of days in time to clinical improvement with power of Patients were randomly recruited to the IFN group or the control group The method of randomizationwas the permuted block randomization patients per block A biostatistician who was not involved in patients™ care did this process Results PatientsA total of patients were screened Of them patients did nothave the eligibility criteria of study and patients were referred fromanother hospital Three and four patients withdrew the consent duringthe study in the IFN group and control groups respectively Four patients did not adhere to IFN injection after second or third dose Alsothree patients in the control group were enrolled in another trialFinally patients in each group completed the study Fig The median IQR age of patients was “ years and of them were male No significant difference in terms of the patients™demographic data was detected between the groups The most commoncomorbidities were hypertension diabetes mellitus and ischemic heartdisease Dyspnea fever and cough were the most frequent symptoms atthe time of hospital admission The median IQR time from onset of thesymptoms to hospital admission was “ and “ days in the IFNgroup and control groups respectively The time from onset of thesymptoms to randomization was not statistically significant betweenthe groups All of patients required respiratory support at the time ofrandomization Oxygenation through facemask was required for morethan percent of patients None of the patients in both groups wereintubated at baseline Table Vital signs and laboratory data of patients at the time of recruitment were comparable between the groupsTable During the hospitalization course oxygen saturation droppedin and of patients in the IFN and control groups respectively All of those patients were intubated At least one antibioticwas administrated for and of patients in the IFN groupand control groups respectively Methylprednisolone was administeredfor of patients in the IFN group and of patients in the4Power085 0cH Rahmani et alTable Baseline characteristics of patientsParameter Median IQR or n AgeSexMaleFemaleComorbid conditions nHypertensionDiabetes mellitusIschemic heart diseaseAsthmaCOPDMalignancyTransplantationSymptoms at admission nDyspneaFeverCoughChillsDuration of symptoms before admissionmedian IQR daysTime from symptom onset torandomization median IQR daysSix category scale at day of intervention3hospital admission requiring highflownasal cannula or noninvasivemechanical ventilationsupplemental oxygen hospital admission requiringInterferon groupn “ ““Control groupn “ ““control group The dose of methylprednisolone was mg daily for days Methylprednisolone was considered during the cytokine orhyperinflammation phase days “ of onset of the symptoms Approximately and of patients in the INF and control groupsneeded vasopressors during the hospitalization course respectivelyTable Primary outcomesThe time to clinical improvement in the IFN group was significantlyshorter than the control group [“ vs “ days respectivelyp ] Table Moreover the Cox proportional hazards regression analysis showed that time difference to clinical improvement wasstatistically significant between the groups HR CI“ Fig Then the model was adjusted for the confoundingInternational Immunopharmacology Interferon groupn Control groupn Table Respiratory support and medicationsParameter n Respiratory supportNasal cannulaFace maskNIPPVIMVAntibiotics mer em piperacillintazobactam ceftriaxone FQsvancomycin azithromycin andColistin n CorticosteroidsVitamin CVasopressorsDiphenhydramineCardiovascular drugsStatinsARBsBetablockersACEIsNIPPV noninvasive positive pressure ventilation IMV invasive mechanicalventilation FQs fluoroquinolones ARB Angiotensin Π Receptor Blocker ACEIangiotensin converting enzyme inhibitorfactors and similar results were seen HR CI “ Secondary outcomesAccording to the six category scale and of patientswere discharged in the IFN and the control groups at day respectivelyOR CI “ p Only one patient in thecontrol group died at day Also at this time and patients wereintubated in the IFN and control groups respectively At day thepercentage of discharged patients reached to and in theIFN and control groups respectively OR CI “p Furthermore the number of deaths increased to and patients the IFN and control groups respectively Finally at day ofinclusion the proportion of discharged patients were in the IFNgroup and in the control group OR CI “p At this time ICU admission rate in the control group wassignificantly higher than the IFN group vs p Moreover more patients in the control group needed invasive mechanical ventilation compared with the IFN group but the rate was notstatistically different p Although length of hospitalization wasTable Patients™ vital signs and laboratory data at the time of hospital admissionParameter Median IQRTemperature °CHeart rate beats minuteRespiratory rate breathsminSystolic blood pressure mm HgSPO2 Laboratory dataWhite Blood Cell cells μlAcute Lymphocyte count cellsμlHemoglobin gdlPlatelet count cells × 103μlBlood Urea Nitrogen mgdlCreatinine mgdlAspartate aminotransferase ulAlanine aminotransferase ulAlkaline phosphatase ulTotal bilirubinmgdlCreactive protein mgdlErythrocyte sedimentation rate mmhLactate dehydrogenase ulInterferon group n ““““““““““““““““““Control group n ““““““““““““““““““ 0cH Rahmani et alInternational Immunopharmacology Table Outcomes and complicationsParameter Median IQR or n Time to clinical response medianIQR daysICU admission n Intubation requirementLength of stay in ICU days median IQR daysLength of stay in hospital days median IQR daysAllcause mortality at day Six category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeSix category scale at day of intervention Death Hospital admission requiring invasive mechanical ventilation Hospital admission requiring highflow nasal cannula or noninvasive mechanical ventilation Hospital admission requiring supplemental oxygen Hospital admission not requiring supplemental oxygen DischargeInterferon group n “““Control group n “ ““pvalueOR95 CI“““shorter [ “ days in the IFN group vs “ days in thecontrol group p ] but length of ICU stay was not significantlydifferent between the groups Allcause 28day mortality was and in the IFN and control groups respectively p Table Safety outcomesA total of and common adverse events were recorded duringthe study period in the IFN and control groups respectively Moreovernumber of serious adverse events was in the IFN group and in thecontrol group The incidence of grade or of adverse events washigher in the control group than the IFN group As it was expected IFNrelated common adverse effects injection site reactions and flulikesyndrome occurred only in the IFN group More patients in the controlgroup experienced ARDS secondary infections septic shock AKI andAHI compared with patients in the IFN group Table Nosocomial infections were detected in patients and patientsin the INF and control groups respectively Bloodstream infection withstaphylococcus aureus was detected in a patient in the INF group Threepatients in the control group experienced ventilator associated pneumonia with klebsiella pneumonia in two patients and acinetobacterbaumannii in another patient Other patients in the control group hadbloodstream infection with staphylococcus aureus DiscussionThis is first randomized clinical trial that evaluated efficacy andsafety of IFN subtype 1b in patients with severe COVID19 In thisstudy Interferon beta1-b as addon therapy significantly shortened the time toclinical response increased the discharge rate at day and decreasedneed for ICU admission in these patients However duration of hospitalization intubation rate length of ICU stay and allcause 28daymortality were not significantly changed Incidence rates of commonand serious adverse events were higher in the control group comparedwith the IFN group The sample size was calculated to assess effect ofInterferon beta1-b on time to clinical improvement in hospitalized patients withCOVID19 However the sample size might not have enough power todifferentiate effects of Interferon beta1-b on the secondary endpointsIFN is a subtype of the type INFs that is released by the lymphocytes as the first cytokine following exposure to viruses It activatesinterferonstimulated genes ISGs after binding to the receptors Theantiviral effects of IFNs are regulated through these genes InadequateIFN response caused uncontrolled viral replication raised viral load andled to poor outcomes in SARSCoV infection A strong IFN responsefollowing infection with SARSCoV2 was detected [“] Expressionof ISGs significantly increased in patients with CoVID19 [] In evaluation of transcriptional responses in various models in vitro ex vivoand in vivo BalancoMelo et al showed that the levels of IFN I andIFN III decreased in SARSCoV2 infection In in vitro model expressions of IFN I and IFN III were not detected in A549 cells as adenocarcinomic human lung cell line infected with SARSCoV2 Of notemoderate increase in the expression of ISGs was observed Next stepthe cells were treated by IFN that caused substantially reduction inthe viral replication Furthermore in ex vivo model the levels of IFN Iand IFN III were undetectable following infection of human bronchialepithelial cells with SARSCoV2 Finally in vivo assessment was considered Postmortem lungtissue samples were extracted from patientswith COVID19 and related transcriptional responses were comparedwith samples from the healthy individuals Similar to previous modelsmodest expressions of ISGs were detected but not about IFNs It is interesting that in all of the models robust cytokine and inflammatoryresponses were noticed []In the study of Yuan et al the antiviral activity of agents including hostbased IFNs IFN 1a Interferon beta1-b pegylated IFN α2a andIFN γ1B and virus targeting antivirals remdesivir and lopinavir wereassessed EC50 of these agents was determined according the plaquereduction assay The most potent IFNs were Interferon beta1-b EC50 IUml and IFN 1a EC50 IUml The EC50 values for remdesivirand lopinavir were determined as and µM respectively TheCC50 values of IFNs remdesivir and lopinavir were IUml µM and µM respectively Among IFNs the most reductive effects on viral load belonged to IFN 1a and Interferon beta1-bHowever Interferon beta1-b showed highest potency and selectivity indexagainst SARSCOV2 []In a randomized clinical trial and patients were recruited in 0cH Rahmani et alInternational Immunopharmacology Fig KaplanMeier plot for estimation of time to clinical improvementthe combination and control groups respectively Patients in the combination group received Interferon beta1-b lopinavirritonavir and ribavirinwhile those in the control group received only lopinavirritonavir Theprimary outcome was defined as the time to reach a negative RTPCR ofrespiratory secretions for SARSCoV2 The time to resolution of thesymptoms was considered as one of the secondary outcomes Themedian time to achieving a negative RTPCR was significantly shorterin the combination group compared to the control group vs days Moreover resolution of the symptoms occurred notably fasterin the combination group than the control group vs days []Similar with our study Interferon beta1-b was started in the viral phase ofCOVID19 ie within first days of onset of the symptoms In our studymedian time from onset of the symptoms to randomization was daysIn both studies first dose of Interferon beta1-b was administered within to h of hospital admission Initiation of antiviral agents as soon aspossible following onset of the symptoms is critical in control of viralreplication and prevention of tissue viral invasion The efficacy of antivirals significantly decreased after establishment of the cytokines release phase in COVID19 [] Due to resource limitations evaluation of viral clearance was not possible in our study No patient died inHung et al study while in our study approximately and ofpatients died in the IFN and control groups respectively Regardingcomparison of the results it should be considered that Hung et alevaluated Interferon beta1-b efficacy in patients with mild to moderate COVID while in our only study patients with severe COVID19 were included Moreover considering severity of the disease incidence rates ofthe serious complications during the hospitalization course were muchhigher in our studyEstebanez et al evaluated the efficacy of Interferon beta1-b in patientswith COVID19 Of them and patients were assigned to the IFNand control groups respectively Inhospital mortality was considered asthe primary outcome of study The mortality rate was statistically significant in the control group than the IFN group vs [] Retrospective design and lack of matching of the groups in termsof receiving other antivirals should be considered when interpreting theresultsIn a case series characteristics and outcomes of five patients withsevere COVID19 who were treated with Interferon beta1-b lopinavirritonavirand hydroxychloroquine were described The antiviral regimen appliedfor these patients was similar to our study Treatment was successful in patients while clinical status of patients deteriorated during thetreatment course All patients received corticosteroids Furthermore allpatients were initially admitted in another hospital and later transferred to the referral hospital [] Clinical outcomes of patients mighthad been affected during lag time of the transfer Moreover patientswere different in terms of the clinical presentations and managementstrategies So definite role of Interferon beta1-b in treatment of these patientscannot be assessedPayandemehr et al evaluated the efficacy of IFN 1a in patientswith moderate to severe COVID19 during a singlearm labelclinical trial All patients received IFN 1a along with hydroxychloroquine lopinavirritonavir and oseltamivir In this study only No at riskInterferon 0Control 0cH Rahmani et alTable Summary of the adverse events during the study periodParameter n Control group n Interferon groupn Any gradeAny gradeGrade or Common adverse events““““““““““““““Grade or “Leukocytosis“Leuk iaLymph ia“Thrombocyt ia“Thrombocytosis“AnemiaHyperkalemia“Hypokalemia“Hyponatremia“Increased creatinine“Increased aspartateaminotransferase“Nausea“Diarrhea“Abdominal pain“Injection site reaction“Flulike syndromeSerious adverse eventsARDS“Nosocomial infection“Septic shock“Acute kidney injury“Acute hepatic injuryARDS acute respiratory distress syndromepatients needed ICU admission and only one death occurred in thehospital Fifteen of the discharged patients were followed for days Noside effects were detected while in our study some patients experiencedcommon adverse effects such as injection site reactions and flulikesyndrome It might be due to receiving concomitant antipyretics andanalgesics that masked these reactions Furthermore main outcomes ofthe study were not welldefined in the method section Duration of thefollowup was only days []The efficacy of IFN 1a in patients with COVID19 was assessed inanother study In this noncontrolled prospective trial patients wereenrolled Five doses of mcg of IFN 1a were administrated subcutaneously on alternate days for these patients The patients also received hydroxychloroquine and lopinavirritonavir for days Theprimary outcome of the study was symptom alleviation during 14dayperiod Within days all patients became afebrile The resolution ofother symptoms gradually occurred [] The oxygenation status andtypes of respiratory supports were not exactly defined In general highflow nasal cannula was applied for most patients and three patientsreceived noninvasive positive pressure ventilation NIPPV No seriousadverse events were detected and none of the patients died Rate of ICUadmission and requirement for invasive mechanical ventilation werenot reported in this study Accounting these limitations absence ofcontrol group and small sample size the interpretation of the resultsshould be done with cautionIn another study efficacy and safety of IFN 1a were evaluated inpatients with severe COVID19 in an label randomized clinicaltrial Fortytwo and patients were recruited to the IFN and controlgroups respectively Time to clinical response based on the six ordinarycategory scale was primary endpoint of this study Following twoweektreatment with IFN 1a time to clinical response was not statisticallydifferent between the groups On day the numbers of dischargedpatients were significantly higher in the IFN group compared with thecontrol group vs Early administration of IFN 1asignificantly reduced the mortality rate compared with late administration [] Absence of followup PCR and chest imaging along withthe small sample size were the major limitations of the studyOur study suffered from some limitations Follow up chest imagingInternational Immunopharmacology or virological assessment was not possible due to resources limitationstherefore the effect of Interferon beta1-b on viral clearance was not determinedSmall sample size did not allow accurate estimation of survival benefitof Interferon beta1-bIn conclusion Interferon beta1-b was effective in shortening the time toclinical improvement without serious adverse events in patients withsevere COVID19 Furthermore ICU admission rate and need for invasive mechanical ventilation significantly reduced by administrationof Interferon beta1-b Although compared with the control group Interferon beta1-b reduced duration of hospitalization length of ICU stay intubation rateand 28day mortality were not statistically different between thegroups Further randomized clinical trials with enough sample size areneeded to accurately estimate survival benefit of Interferon beta1-bCRediT authorship contribution statementHamid Rahmani Data Curation Formal analysis InvestigationWriting original draft Effat DavoudiMonfared Data CurationAnahid Nourian Data Curation Hossein Khalili ConceptualizationMethodology Supervision Writing review editing NooshinHajizadeh Project Administration Narjes zarei Jalalabadi ProjectAdministration Mohammad Reza Fazeli Resources MonirehGhazaeian Resources Mir Saeed Yekaninejad Formal analysisDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influence the work reported in this paperAcknowledgementWe would like to thank the nurses and other staffs of ImamKhomeini Hospital Complex for their kind supports and also Ms AvaKhalili for English proofreading the manuscriptFundingThe authors did not receive any fund for this workAppendix A Supplementary materialSupplementary data to this article can be found online at 101016jintimp2020106903References[] L Wang Y Wang D Ye Q Liu Review of the novel coronavirus SARSCoV[] JH Beigel KM Tomashek LE Dodd AK Mehta BS Zingman AC Kalil based on current evidence Int J Antimicrob Agents 101016jijantimicag2020105948[] D Cucinotta M Vanelli WHO Declares COVID19 a Pandemic Acta Biomed “ 1023750abmv91i19397[] Johns Hopkins Coronavirus Resource Center Home Page July coronavirusjhuedumaphtml[] JM Sanders ML Monogue TZ Jodlowski JB Cutrell Pharmacologic treatmentsfor coronavirus disease COVID19 a review JAMA 101001jama20206019E Hohmann HY Chu A Luetkemeyer S Kline DL de Castilla RW FinbergK Dierberg V Tapson L Hsieh TF Patterson R Paredes DA SweeneyWR Short G Touloumi DC Lye N Ohmagari MD Oh GM RuizPalaciosT Benfield G Fatkenheuer MG Kortepeter RL Atmar CB Creech J LundgrenAG Babiker S Pett JD Neaton TH Burgess T Bonnett M GreenM Makowski A Osinusi S Nayak HC Lane Remdesivir for the treatment ofCovid19 preliminary report N Engl J Med 101056NEJMoa2007764DK Manson C Kubin RG Barr ME Sobieszczyk NW Schluger Observationalstudy of hydroxychloroquine in hospitalized patients with Covid19 N Engl JMed “ 101056NEJMoa2012410[] J Geleris Y Sun J Platt J Zucker M Baldwin G Hripcsak A Labella[] B Cao Y Wang D Wen W Liu J Wang G Fan L Ruan B Song Y Cai M Wei 0c[] Z Zhou L Ren L Zhang J Zhong Y Xiao Z Jia L Gou J Yang C Wang 101016jantiviral2020104791S Jiang D Yang G Zhang H Li F Chen Y Xu M Chen Z Gao J Yang J DongB Liu X Zhang W Wang K He Q Jin M Li J Wang Heightened innate immuneresponses in the respiratory tract of COVID19 patients

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

Overexpressed EphB4 conduce to tumor development and is regarded as a potential anticancer target HomoharringtonineHHT has been approved for hematologic malignancies treatment but its effect on hepatocellular carcinoma HCC has notbeen studied This study elucidated HHT could restrain the proliferation and migration of HCC via an EphB4catenindependent manner We found that the antiproliferative activity of HHT in HCC cells and tumor xenograft was closelyrelated to EphB4 expression In HepG2 Hep3B and SMMC7721 cells EphB4 overexpression or EphrinB2 Fcstimulation augmented HHTinduced inhibitory effect on cell growth and migration ability and such effect wasabrogated when EphB4 was knocked down The similar growth inhibitory effect of HHT was observed in SMMC and EphB4SMMC7721 cells xenograft in vivo Preliminary mechanistic investigation indicated that HHTdirectly bound to EphB4 and suppressed its expression Data obtained from HCC patients revealed increasedcatenin expression and a positive correlation between EphB4 expression and catenin levels HHTinducedEphB4 suppression promoted the phosphorylation and loss of catenin which triggered regulation of catenindownstream signaling related to migration resulting in the reversion of EMT in TGFinduced HepG2 cellsCollectively this study provided a groundwork for HHT as an effective antitumor agent for HCC in an EphB4catenindependent mannerIntroductionGlobally hepatocellular carcinoma HCC is one of themost fatal malignancies with poor prognosis and anincreasing incidence1 Although the major therapeuticapproaches such as surgical resection radiation therapyand chemotherapy have advanced clinical applicationsthe 5year survival rate of HCC remains less than Most patients still suffer from tumor recur invasivenessand metastasis At present sorafenib a multiple tyrosinekinase inhibitor is one of the most representative optionsfor advanced HCC butlimited andaccompanied with reduced sensitivity and severe adversesometimesisCorrespondence Yanmin Zhang zhang2008mailxjtueducn1School of Pharmacy Health Science Center Xi™an Jiaotong University No Yanta Weststreet Xi™an Shaanxi PR ChinaEdited by B Zhivotovskyevents34 Therefore much effort is needed on this frontto uncover new antiHCC therapeutic strategies5Erythropoietinproducing hepatocytereceptor B4EphB4 is a member of the tyrosine kinase family andplays a pivotal role in tumor progression6“ Activatedby its corresponding ligand EphrinB2 EphB4 controlscell“cell interactions angiogenesis tumor growth andmetastasis910 Studies on the expression of EphB4 innumerous cancer types have shown overexpressed levelin breast colorectal lung and blood cancers correlatingwith poor prognosis11“ It has been reported that highEphB4 expression enhanced the growth and migrationof pancreatic colorectal and papillary thyroid carcinoma and such effect could be reversed by EphB4knockdown making EphB4 a promising target for cancer treatment14“ Our previous study has confirmed The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT exhibited a growth inhibitory effect on HCC cells in vitro and in vivo a The chemical structure of HHT b Effects of HHT on cellproliferation in Hep3B HepG2 SMMC7721 Bel7402 and Bel7404 cells were determined by MTT assay p comparedto the IC50 of HepG2 cells Cells were treated with increased gradients of HHT for h n cultures for each dose c Protein expression of EphB4 inHep3B HepG2 and cells d Quantification of c n independent experiments e Effects of HHT on colony formation in HepG2cells The upper row the colony formation picture the lower row the individual colony picture — magnification f Photographs of control andHHTtreated group tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor inhibitory rate n mice g h data represent mean ± SEM p p compared to vehicle controls i Inhibitory rate of HHT on tumor mass n micethe high expression of EphB4 in HCC17 and its functionin HCC migration remains poorly understoodHomoharringtonine HHT Fig 1a is a compoundextracted from traditional Chinese medicine and has beenapproved for the treatment of leukemia by Food and DrugAdministration18 Previous studies indicated that HHTcould suppress protein synthesis essentialfor cancersurvival and induce apoptosis by upregulating the proapoptotic protein Bax and inducing caspase3mediatedcleavage of PARP19 In addition to hematologic tumorsHHT also demonstrated its effectiveness in renal cellcarcinoma colon rectal cancer and nonsmall cell lungcancer20“ However the effect of HHT on HCC and theunderlying EphB4related mechanism of action have notbeen studied In this study HHT was found to suppressthe proliferation and migration of HCC cells through anEphB4catenin dependent mannerResultsHHT exhibited a growth inhibitory effect on HCC cellsin vitro and in vivoTo determine the effect of HHT on the cell viability ofHCC cells several different HCC cells HepG2 Bel7402Hep3B and SMMC7721 were treated with an increasedgradient of HHT for h The results showed that HepG2cells were most sensitive to HHT treatment with an IC50value of μM while the IC50 values of Bel7402Hep3B Bel7404 and SMMC7721 cells were and μM respectively Fig 1b Immunoblotting analysis showed that HepG2 cells exhibitedhigher EphB4 expression Fig 1c d suggesting thepositive correlation between the inhibitory effect of HHTand EphB4 expression Similar results were obtained fromthe colony formation assay HHT significantly reduced thecolony size and the number of HepG2 cells at a doseOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig The inhibitory effect of HHT on HCC cells was associated with EphB4 expression a EphB4 expression analysis of EphB4siRNA or EphB4overexpression OE HepG2 cells b Effects of HHT on cell proliferation in wildtype EphB4siRNA EphB4OE or EphrinB2 Fc stimulated HepG2 cellsn cultures for each dose p compared to the IC50 of HepG2 cells c EphB4 expression analysis of EphB4OE Hep3B cells d Effects of HHTon cell proliferation in wildtype and EphB4OE Hep3B cells n cultures for each dose p compared to the IC50 of Hep3B cells e EphB4expression analysis of wildtype and EphB47721 cells f Photographs of control and HHTtreated group of tumors and EphB47721tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor mass n mice i Body weight ofcontrol and HHTtreated group mice n g“i data represent mean ± SEM p compared to vehicle controlsdependent manner in comparison to the control groupFigs 1e and S1a Moreover xenografts model of HepG2cells confirmed the antitumor effect of HHT in vivo HHTgavage groups showed remarkable reduction in tumrowth Fig 1f“i and the inhibitory rate reached and at the mgkg mgkg and mgkg inHHT gavage groups respectivelyThe inhibitory effect of HHT on HCC cells was associatedwith EphB4 expressionTo evaluate whether the proliferation inhibitory effectof HHT on HCC cells was related to EphB4 expressionEphB4 siRNA or plasmid was utilized to transfect theHCC cells Figs 2a and S1b and EphrinB2 Fc was used tostimulate the HCC cells As is shown in Fig 2a b HepG2cells with EphB4 knockdown were less sensitive to HHTwhereas HepG2 cells with EphB4 overexpression EphB4OE demonstrated elevated sensitivity to HHT treatmentcompared with wild type HepG2 cells HepG2 cells following EphrinB2 Fc stimulation showed a drug responsecurve that was similar to that of EphB4 OE subline Fig2b Meanwhile following transfection with EphB4 plasmid Hep3B cells harboring high expression ofEphB4 showed less cell viability after HHT treatmentcompared with wild type Hep3B cells Fig 2c d Forin vivo test an EphB4overexpressing SMMC7721EphB47721 cell line was established Figs 2e and S1cand the antitumor effect of HHT on xenograft model ofOfficial journal of the Cell Death Differentiation Associationcellsand EphB4wild type SMMC7721 cells was investigated HHT has an enhanced inhibitory effect on EphB47721 tumor growth comparedwith that on wild type tumor Fig 2f“h And therewas no obvious body weight and spleen index reductionduring the test Figs 2i and S1dThe suppression of HHT on SMMC7721 cells migrationwas associated with EphB4Migration assay and wound healing assay were conducted to investigate the effect of HHT on HCC cellmigration The results showed that HHTtreated wide typeSMMC7721 cells had decreased migration as comparedwith controls whereas both of EphB4 overexpression andEphrinB2 Fc stimulation in SMMC7721 cells strikinglyenhanced migration restraint effect of HHT Fig 3a cSimilar result was observed in wound healing assay whichdemonstrated that both transfection with EphB4 andexogenous stimulation with soluble EphrinB2 Fc inSMMC7721 cells delayed the closure of wound gapsfollowing HHT treatment Fig 3b d These results indicated that the suppression of HHT on HCC cells migration was closely associated with EphB4 expressionHHT suppressed HepG2 cell migration induced by TGFstimulationTGF stimulation could induce EMT and increase themigration of tumor cells We next investigated the effect 0cZhu Cell Death and Disease Page of Fig The suppression of HHT on SMMC7721 cell migration was associated with EphB4 a Transwell assays were conducted to observe themigratory cells in HHTtreated wide type EphB4OE or EphrinB2 Fc stimulated cells Scale bars μm b The migration rate of HHTtreated EphB4OE or EphrinB2 Fc stimulated cells observed through woundhealing assays Scale bars μm c Quantification of a n Leftp compared to the migrated cell number of cells Right p and p compared to the migration rate of cells atindicated concentration of HHT d Quantification of b n p compared to HHTtreated cells All data represent mean ± SEMof HHT on HCC cells migration after TGF stimulationby transwell migration assay and wound healing assay Asshown in Fig 4a c although the higher number ofmigration cells was observed in the TGF inducedHepG2 cells as compared to controls the addition ofHHT reduced the migrated cells Importantly concurrenttreatment with HHT and NVPBHG712 a small molecule EphB4 kinasespecific inhibitor had a greaterrestraint effect on the migration of TGF inducedHepG2 cells Wound healing assay showed similar resultsthat HHT could delay the closure of wound gaps in TGF induced HepG2 cells whereasthe addition ofEphB4 siRNA impaired such effect Fig 4b d Theseresults indicated that TGF induced the migration abilityin HepG2abrogated byEphB4 suppression of HHTcells which could beHHT bound to EphB4 and suppressed its expressionWe further evaluated the regulation of HHT on EphB4expression The results showed decreased EphB4 proteinexpression after HHT treatment both in HepG2 cells andtumor tissues Figs 5a c and S2a b Exogenous stimulation with soluble EphrinB2 Fc increased EphB4 proteinexpression while in HepG2 cells treated with EphrinB2 Fcand HHT the protein levels of EphB4 were strikinglydecreased Figs 5b and S2c HHT treatment resulted in aremarkably reduced EphB4 mRNA level at a dosedependent manner Figs 5d and S2d We treatedHepG2 cells with NVPBHG712 HHT or both to evaluate the change of EphB4 expression The results indicated that coadministration of HHT and NVPBHG712produced an even greater decrease in the expression levelof EphB4 in HepG2 cells than by either alone Figs 5e andS2e Given these findings a molecular docking assay wasconducted to confirm the affinity of HHT bound to theactive site of EphB4 The results revealed that HHToccupied in the active site of EphB4 through five hydrogen bonds which were associated with amino acid residues LYS647 GLU664 TYR736 ASP758 and THR Fig 5f The molecular docking results indicated thatHHT fit well with EphB4EphB4 was positively correlated with catenin in HCCpatients and HHT inhibited the phosphorylation andnuclear translocation of cateninEpithelial to mesenchymal transition is a prerequisitefor cell migration and lies downstream of catenin23Although previousstudies have reported that EphOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT suppressed HepG2 cell migration induced by TGF stimulation a Transwell assays were conducted to observe the migratory cellsin control and TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 treated HepG2 cells Scale bars μm b The migrationrate of control and TGF TGF HHT or TGF HHT EphB4 siRNA treated HepG2 cells observed through woundhealing assays Scale bars μm c Quantification of a n d Quantification of b n All data represent mean ± SEM p p and p compared tothe indicated groupsreceptor is conducive to EMT progression in hepatomacells24 the relationship between EphB4 and catenin hasnever been shown before To investigate the role ofcatenin in HCC we analyzed the mRNA level ofcatenin in HCC patients using The Cancer GenomeAtlas TCGA database RNASeq data from this databaseshowed that catenin expression was significantly higherin carcinoma tissue compared with paracarcinoma tissueFig 6aImmunohistochemistry was used to detectcatenin expression in pairs of HCC and noncarcinoma tissues The results showed that cateninexpression was remarkably increased in carcinoma tissuescompared with noncarcinoma tissues Fig 6b c whichwas consistent with the findings in TCGA database Todelineate the possible relationship between EphB4 andcatenin Spearman™s correlation analysis was conductedand the results revealed that catenin expression waspositively correlated with EphB4 levels in HCC tumortissues Fig 6dOfficial journal of the Cell Death Differentiation AssociationWe next analyzed the regulation of catenin in HepG2cells exposed to HHT Western blot analysis indicatedthat HHT could downregulate catenin expression andupregulate the phosphorylation of catenin level both inHepG2 cells and xenograft tumors Figs 6e f and S2f gThese results were also observed in immunohistochemicalassay for xenograft tumors Fig 6g And a remarkablyreduced catenin mRNA level was also observed inHHTtreated HepG2Immunofluorescence staining was used to examine the distribution of catenin in HepG2 cells exposed to HHT Theresults in Fig 6h demonstrated that HHT restrained thelevel of catenin in the nucleus as well as in the cytoplasm Figure 6eshowed that phosphorylation ofcatenin was obviously increased at and nM ofHHT which has been reported to contribute to process ofcatenin degradation25 These data indicated that HHTsuppressed nuclear translocation of catenin and promoted its phosphorylationS2hcellsFig 0cZhu Cell Death and Disease Page of Fig HHT bound to EphB4 and suppressed its expression a Western blot analysis of HepG2 cells EphB4 expression after HHT treatmentb Western blot analysis of EphB4 expression in HepG2 cells treated with HHT orand EphrinB2 Fc c Immunochemistry analysis of EphB4 expression inHepG2 tumors after HHT treatment n — magnification d RTPCR analysis of HepG2 cells EphB4 expression after HHT treatment n Alldata represent mean ± SEM p compared to vehicle controls e Western blot analysis of HepG2 cells EphB4 expression after HHT NVPBHG712or both treatments f Molecular docking analysis of the EphB4 protein and HHTEcadherin was overexpressed in HCC patients and HHTregulated EMTrelated moleculesGiven the positive correlation of EphB4 and cateninin HCC patients the Ecadherin expression in HCCpatients was examined As shown in Fig 7a b lowerEcadherin protein was observed in carcinoma tissueswith higher expression in the noncarcinoma tissue groupBased on the result that Ecadherin was reduced in HCCpatients and HHT could restrain the migration of HCCcells we next analyzed the effect of HHT on EMTrelatedmolecules by western blotting and immunohistochemistryassay Promotion of Ecadherin and inhibition of Snailwere observed in HHTtreated HepG2 cells and tumorsFigs 7c d g and S3a b Furthermore the results in Figs7e“g and S3c d indicated that the essential members ofMMPs family MMP2 MMP3 and MMP9 were suppressed by HHT both in HepG2 cells and in the tumortissues of xenograft models And the mRNA level ofMMP2 and MMP9 were reduced in a dose depensentmanner in HepG2 cells exposed to HHT Fig S3eHHT repressed catenin and EMTrelated moleculesthrough EphB4 suppressionNext the expression changes of EphB4 catenin andEMTrelated molecules after HHT administration for thedifferent time points were evaluated by western blottingThe results in Figs 8a and S4a“c demonstrated that theprotein level of EphB4 was significantly decreased afterHHT treatment within h and the expression of othermolecules was unchanged attime point ThethisOfficial journal of the Cell Death Differentiation Associationexpression and phosphorylation of catenin wereremarkably changed within h of HHT administrationIncreased Ecadherin expression and decreased SnailMMP2 and MMP9 expression were observed within hof HHT treatment These findings indicated that HHTmight regulate the expression of catenin and EMTrelated molecules by targeting EphB4 receptor NVPBHG712 was utilized to investigate the changes in thesemolecules after EphB4 suppression The results in Figs 8band S4e demonstrated that both HHT and NVPBHG712could suppress catenin expression and promote itsphosphorylation level Furthermore the upregulation ofEcadherin and downregulation of Snail MMP2 andMMP9 were also seen in HHT or NVPBHG712 monotherapy These effects exerted by a single administrationof HHT or NVPBHG712 were significantly augmentedby the combination of the two moleculesEMTrelated molecules in HepG2 cells following TGF stimulation was also investigated by western blot assayand the results were shown in Figs 8c and S5a b Theexpression of Ecadherin was downregulated and theprotein levels of Snail MMP2 and MMP9 were upregulated by TGF and these effects could be reversed by theaddition of both HHT and NVPBHG712 And concurrent addition of HHT and NVPBHG712 furtheraugmented the effect of monotherapyDiscussionContinuous stimulation of proliferative signals andmaladjustment of related monitoring mechanisms are 0cZhu Cell Death and Disease Page of Fig EphB4 was positively correlated with catenin in HCC patients and HHT inhibited the phosphorylation and nuclear translocation ofcatenin a mRNA expression of EphB4 in HCC carcinoma tissue and paracarcinoma tissue in the TCGA database p b Representativefigures of immunohistochemical analysis of catenin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCC patients respectively — magnification c Quantification of b n p d The positive correlation between the expression ofcatenin and EphB4 e Protein expression of catenin and pcatenin in HepG2 cells treated with HHT for h f Protein expression of cateninand pcatenin in HepG2 tumor EphB4 expression after HHT treatment g Immunochemistry assay of catenin and pcatenin in HepG2 tumortissues — magnification h Immunofluorescence analysis of the catenin protein in HepG2 cells treated with HHT catenin green DAPI bluestaining and merged images indicate the nuclear translocation and expression of catenin Scale bars μm All data represent mean ± SEMimportant causes of tumor formation The growth factorreceptor can be activated by growth factors to generateintracellular cascade signals to regulate the proliferationof tumor cells EphB4 is an important member of thereceptor tyrosine kinase family which is overexpressedand conduces to tumor growth and migration in variouscancers61315 Our previous study has confirmed theoverexpression of EphB4 in the tumor tissues of HCCpatients emphasizing EphB4 a potential target for HCCtreatment17 However there is no drugs targeting EphB4on the market In this study we found the inhibitory effectof HHT on HCC cell proliferation and migration in anEphB4 dependent manner and the underlying preliminarily mechanism was clarifiedHHT has been proved effective in the treatment ofleukemia butin HCC inhibition wasunknown We revealed the antiproliferative ability ofits potentialHHT on several HCC cell lines In particular HepG2cells with the highest EphB4 protein expression were themost sensitive to HHT treatment demonstrating thatthe inhibitory effect of HHT on HCC cells might berelated to EphB4 expression Xenograft models in nudemice confirmed the inhibitory effect of HHT on HepG2cell growth in vivo For in vitro experiments EphB4overexpression and EphrinB2 Fc stimulation increasedthe sensitivity of wild type HepG2 or Hep3B cells toHHT while transient transfection with EphB4 siRNAdecreased such effect in HepG2 cells Similar resultswere drawn from in vivo experimentsthat HHTexhibited enhanced inhibitory effect in xenograft ofEphB47721 cells compared to xenograft of wild type cells The results above indicated that EphB4might play an indispensable role in the suppression ofHCC cell proliferation by HHTOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Ecadherin was overexpressed in HCC patients and HHT regulates EMTrelated molecules a Representative figures ofimmunohistochemical analysis of Ecadherin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCCpatients respectively — magnification b Quantification of a n p All data represent mean ± SEM c Protein expression of Ecadherin and Snail in HepG2 cells treated with HHT for h d Protein expression of Ecadherin and Snail in HepG2 tumor EphB4 expression after HHTtreatment e Protein expression of MMP2 MMP3 and MMP9 in HepG2 cells treated with HHT for h f Protein expression of MMP2 MMP3 andMMP9 in HepG2 tumor tissues after HHT treatment g Immunochemistry assay of Ecadherin MMP2 and MMP9 in HepG2 tumor tissues —magnificationInvasion and migration are the main causes of tumormetastasis and the critical juncture of tumor staging inHCC2627 Since EphB4 has been reported with promotingcell migration potentialin both normal and malignantcells7 we investigate the role of EphB4 in cell migrationsuppression in HHTtreated HCC cells Our results indicated that both EphB4 overexpression and exogenous stimulation with soluble EphrinB2 Fc exacerbated theantimigratory ability of HHT on SMMC7721 cells both inwound healing and transwell migration assay FurthermoreTGF a multifunctional cytokine was used to stimulatethe migration ability of HepG2 cells28 The obtained resultsdemonstrated that HHT restrained the migration of HepG2cells stimulated by TGF while EphB4 knockdown bysiRNA impaired such inhibitory effect Combined HHT andNVPBHG712 treatment significantly augmented the antiin TGFstimulated HepG2 cells asmigratory effectcompared to either agent alone Our further investigationconfirmed that HHT was able to bind to EphB4 withhydrogen bonds and suppress its expression both in vitroand in vivo These results indicated that HHT could inhibitcell migration by regulating EphB4 in HCCOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT repressed catenin and EMTrelated molecules through EphB4 suppression a Protein expression of catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either HHT nM NVPBHG712 μM or the combination of both b Proteinexpression of EphB4 catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with HHT nM for the indicated durationc Protein expression of EphB4 Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either vehicle TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 d Schematic diagram of HHT inhibited the migration of HCCIt has been reported that Eph receptor could mediateEMT progression and adhesion to conduce migratory andmetastatic processes in hepatoma cells24 There is a wideacceptance that EMT is a prerequisite for cell migrationand catenin can trigger EMT2329 yet whether EphB4could regulate catenin remains unknown catenin wasthe key molecule of the Wntcatenin pathway and thenuclear translocation of which could not only promotethe expression of matrix metalloproteinases MMPs butalso suppress Ecadherin expression3031 In this studyboth TCGA database and our own HCC patient samplesanalysis demonstrated that catenin was significantlyoverexpressed in HCC patients at protein and mRNAlevels We also analyzed the expression data of EphB4 andcatenin in TCGA database and the results indicated thatthe mRNA level of the two molecules in HCC was significantly correlated suggesting that catenin might playa critical role in HCC migration suppression by HHT Weexamined the regulation of HHT on catenin and theresults showed that HHT strikingly inhibited cateninexpression at protein and mRNA level and promoted itsphosphorylation in vitro and in vivo Moreover the resultof immunofluorescence assay showed that the nucleartranslocation of catenin was restrained by HHTAs a key molecule of tumor migration Ecadherincould be regulated by catenin and the loss of Ecadherin is the critical marker of EMT2329 Wecompared the protein expression of Ecadherin in thecarcinoma tissues of HCC patients and the noncarcinoma tissues The resultindicated that Ecadherin level was prominently decreased in the carcinoma tissues compared to that in the noncarcinomatissues HHT treatment upregulated the protein levelof Ecadherin both in HepG2 cells and xenografttumors Furthermore Snail is a transcription factorand its expression is increased during the process ofOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of EMT We found that Snail expression wassignificantly downregulated in HHTtreated cells andtumors Most of the primary tumor cells are polarepithelial cells and connected to each other by intercellular adhesion molecules As the tumor progressesthe intercellular adhesion molecules are degraded byMMPs15 Tumor migrationrelated molecules MMPsare the downstream signaling of the Wntcateninpathway and could be regulated by catenin Thisstudy indicated that the expression of MMPs including MMP2 MMP3 and MMP9 was significantlysuppressed by HHT in vitro and in vivoThe obtained data showed that HHT could targetEphB4 and suppress its expression The expression ofEphB4 and catenin in HCC was positively correlatedaccording to TCGA data analysis HHT treatmentregulated the expression of catenin and its downstream signaling such as Ecadherin and MMPs Nextwe focused on the relationship between the effect ofHHT on EphB4 and catenin and the downstreamsignaling We investigated the protein levels in HepG2cells exposed to HHT for different duration and theresults confirmed that catenin might be the downstream signaling of EphB4 receptor EphB4 specificinhibitor NVPBHG712 could suppress the proteinlevel of catenin and promote its phosphorylationThe expression of Ecadherin Snail and MMPs wasalso significantly changed after EphB4 was suppressedby NVPBHG712 And the regulating effect on EphB4catenin and its downstream cascades was remarkablycoadministration of HHT and NVPBHG712 In addition the increased expression of Snail and MMPs anddecreased expression of Ecadherin confirmed thatTGF induced EMT in HepG2 cells Both HHT andNVPBHG712 could reverse the regulating effect ofTGF and such effect was enhanced by combinedHHT and NVPBHG712 treatment These findingsindicated that HHT could reverse the EMT of HepG2cells by restraining EphB4 expression the suppressionof which further inhibited the nucleus translocation ofcatenin and regulated the expression of EMT related molecules including Ecadherin Snail MMP2and MMP9in HepG2augmentedcellsafterIn conclusion we discovered a positive correlation ofEphB4 and catenin in HCC patients and that EphB4 wasinvolved in HCC cell migration progression by regulatingcateninmediated EMT HHT suppressed EphB4expression and further led to catenin loss resulting inthe regulation of Ecadherin Snail and MMPs to preventEMT progression in HCC cells Fig 8d Our researchmay provide new insight into the migration mechanism ofEphB4 in HCC and HHT possesses great potential in thedevelopment of antiHCC drugsOfficial journal of the Cell Death Differentiation AssociationMaterials and methodsReagentsHHT HPLC ‰¥ was obtained from Baoji Herbest Biotech Co Ltd Shaanxi China NVPBHG712Purity ‰¥ was purchased from MedChemExpressCo Ltd Dulbecco™s modified Eagle™s mediumDMEM RPMI medium and PBS were fromHyClone Logan UT USA Fetal bovine serum FBSwas purchased from ExCell Bio Co Ltd ShanghaiChina MTT powder RNase and propidium iodidewere from Sigma“Aldrich St Louis MO USADimethyl sulfoxide DMSO was from Tianjin KemiouChemical Reagent Co Ltd Tianjin China OptiMEM medium was purchased from Gibco CaliforniaUSA Trypsin and PenicillinStreptomycin solutionwere obtained from Beijing Solarbio Science Technology Co Ltd Beijing China Lipofectamine reagent was purchased from Invitrogen Carlsbad CA USA RIPA Lysis Buffer and BCA proteinassay reagent kit were purchased from Pioneer Biotechnology Co Ltd Protease and phosphatase inhibitors were obtained from Roche TechBaselSwitzerland Ultra Signal Enhanced Chemiluminescent ECL Reagent kit was purchased from 4A Biotech Co Ltd Beijing China EphB4 catenin andpcatenin rabbit mAb were obtained from CellSignaling Technology Boston MA USA Ecadherin Snail GAPDH rabbit mAbs and goat antirabbitIgG were purchased from Protein technology GroupChicago Illinois USA MMP2 MMP3 and MMP9rabbit mAb were from ABclonal Boston MA USAThe EphB4 bacterial strain was from VectorBuilderPatientsAll the patients who were eligible underwent surgery atthe First Affiliated Hospital of Xi™an Jiaotong UniversityFifteen HCC tissues from HCC patients and fifteenhepatic tissues from nonHCC patients were obtainedfrom consenting patients and used with permission ofbiomedical ethics committee of Xi™an Jiaotong UniversityHealth Science Center Project No Cell cultureHuman hepatocellularcarcinoma HepG2 Hep3BSMMC7721 Bel7402 and Bel7404 cells werepurchased from the Shanghai Institute of Cell Biology atthe Chinese Academy of Sciences Shanghai Chinawithout mycoplasma contamination The HepG2 andHep3B cells were cultured in DMEM with FBS and Penicillin and Streptomycin solution SMMC7721Bel7402 and Bel7404 cells were cultured in RPMI1640with FBS and Penicillin and Streptomycin solution Allthe cells were incubated in a humidifiedatmosphere incubator of CO2 at °C 0cZhu Cell Death and Disease Page of Cell viability assayMTT method was used to analyze cell viability Thegrowing cells were seeded in 96well plates overnightThen the cells were treated with increased concentrationof HHT for h followed by incubation with the mixtureof serum free medium and MTT solution for “ h Themixture was replaced by DMSO After min shakingthe plates were determined using EPOCH BioTekInstruments Inc USA at a wavelength of nmColonyforming assayThe growing cells were seeded in 12well plates at adensity of cells per well Following h incubationthe cells were exposed to HTT for h followed bycultured in fresh complete medium for weeks Afterwashed twice with PBSthe colonies were fixed bymethanol and stained using crystal violet Imageswere obtained via an inverted fluorescence microscopeMigration assayThe HCC cells were cultured into the upper chamber at adensity of — cells per well accompanied by μLcomplete medium in the lower chamber Following incubation for h for EphB4 p

Thyroid_Cancer

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so 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 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"EPMA celebrated its 10th anniversary at the 5th World Congress in Pilsen Czech Republic The history of theInternational Professional Network dedicated to Predictive Preventive and Personalised Medicine PPPM 3PM is rich inachievements Facing the coronavirus COVID19 pandemic it is getting evident globally that the predictive approach targetedprevention and personalisation of medical services is the optimal paradigm in healthcare demonstrating the high potential to savelives and to benefit the society as a whole The EPMA World Congress Supplement highlights advances in 3P medicineIntroductionEuropean Association for Predictive Preventive and PersonalisedMedicine has been created in In the historical 1stEPMA World Congress took place in Bonn GermanyIn the EPMA celebrated its 10th anniversary at the 5thWorld Congress in Pilsen Czech Republic The decadeoldprofessional history of the EPMA is rich in achievementsHerewith we briefly highlight some of themGeographic distribution of the 3PMrelevant expertise underthe EPMAumbrella started with approximately countries in currently the EPMA is represented in countries University Hospital in Pilsen Medical Faculty in Pilsen CharlesUniversity Prague Czech RepublicEuropean Medical Association Brussels BelgiumEuropean Association for Predictive Preventive and PersonalisedMedicine EPMA Brussels BelgiumThe historical 1st EPMA World Congress in former Bundestag BonnGermany September Declarations The authors declare that they have no competing interest Permissions by responsible ethic commissions for correspondingcontributions have been received and thoroughly check prior topublishing the EPMA World Congress Supplement Research involving human participants andor animals was performedin accordance with international regualtions All the patient investigations conformed to the principles outlined in theDeclaration of Helsinki Informed consent was obtained from all individual participants included in the corresponding study Olga GolubnitschajaOlgaGolubnitschajaukbonndePredictive Preventive and Personalised 3P Medicine Departmentof Radiation Oncology University Hospital Bonn RheinischeFriedrichWilhelmsUniversitt Bonn VenusbergCampus Bonn Germany 0cworldwide who actively promote 3PM concepts in biomedicalsciences and practical medicine strongly benefiting patients andhealthcare systemsThe first issue of the EPMA Journal Springer Nature wasreleased in March In the journal received its firstIF in it reached Nowadays the EPMA J is ahighly recognised international forum for 3P medicine operating in a hybrid subscription access modus ScopusCiteScore of the EPMA J is wwwscopuscomsourceid19700201201originsourceInfozonerefpointranktabs1 thereby Scopus ranks the EPMA Jamongst the top in the category œHealth Policy due tohighly requested and wellcited strategic papers created bymultiprofessional groups of EPMA experts such as“ General report recommendations in predictive preventive and personalised medicine white paper ofthe European association for predictive preventive andpersonalised medicine 1011861878“ Medicine in the early twentyfirst century paradigm andanticipation “ EPMA position paper 101186s1316701600724SCImago topranks the EPMA J in all three categoriesnamely œHealth policy œMedical Biochemistry and œDrugdiscoverywwwscimagojrcomjournalsearchphpq19700201201tipsidIn Springer Nature awarded the belowmentioned the status of an œ with a potential to change thew o r l d  i n t h e c a t e g o r y œ M e d i c i n e a n d P u b l i cHealth wwwspringernaturecomgpresearcherscampaignschangetheworldmedicinepublichealth“Pregnancy Associated Breast Cancer The Risky StatusQuo and New Concepts of Predictive Medicine EPMA J s1316701801297œAdvances in Predictive Preventive and PersonalisedMedicine is a very successful EPMASpringer Nature bookseries which educates both professionals and the general population in 3P medicine Since book volumes havebeen released dedicated to a whole spectrum of PPPM relatedaspects such as digital health information technology framework application of artificial intelligence in healthcare drugdelivery systems liquid biopsy and multilevel diagnosticsamongst othersœHorizon  is the main European Scientific Programmewhich EPMA experts have contributed to with 3PMrelatedprotocols as well as with the topexpertise provided byRepresentatives and Members of the association involved inthe evaluation panelsEPMA JournalEPMA AWARD for EXCELLENCE in BIOMEDICALSCIENCES was created in and the 1st EPMA awardwas given to Prof Dr Josef Flammer University of Basel forphenotyping of the œFlammer Syndrome which the EPMAinternational jury panel valued as being of great clinical utilityœYoung professionals in PPPM Award was created bythe EPMA in Atthe international workshopslinked to the biannual EPMA World Congresses thepresentations made by young professionals get evaluated by an international jury panel The best presentations and smart 3PM concepts receive awards thateffectively promote the careers of young professionalsin innovative biomedical fieldsEPMA World Congress in Pilsen Czech Republicattracted 3PM experts from countries The congress wasdedicated to innovation in a broad spectrum of biomedicalfields with a specific focus on the concepts of predictive diagnostics targeted prevention and personalisation of medical services in œCancer œMetabolic DisordersœCardiovascular Disease œNeurological Neurodegenerativeand Neuropsychiatric Disorders œInflammatory DisordersœDentistry œBiobanking and Screening ProgrammesœMultiomics œMicrobiome Immune Pre andProbiotics and œInnovative Technologies among othersFurther there were several new topics presented at the congress among others these were œImplementation of 3PMConcepts in Plastic Surgery œApplication of ArtificialIntelligence in Medicine “ 3PM strategies and œMedicalUse of Cannabis The latter topic was discussed in the EUParliament in and the EPMA position has been elucidated by the EPMA Representatives for more information seethe below linkhttpwwwepmaneteulatestevents2019epmapositiononmedicaluseofcannabispresentedattheeuparliamentOral and poster presentations provided valuable information regarding pilot projects towards personalisedhealthcare eg awarded by ICPerMedindividualisedpatient profiles multilevel biomarker panels patientstratification creation and application of innovative ITtools ethical issues doctorpatient collaboration optimalthe modern hospitalstructure and anisation ofambitioned to practically implementthe paradigmchange from reactive to predictive preventive andpersonalised medicineWorld First 3P Medical Unitthe historically first worldwide unitIn March dedicated to Predictive Preventive and Personalised3P Medicine led by SecretaryGeneral of the EPMAProf Dr Olga Golubnitschaja was created in Germanyatthe Department of Radiation Oncology UniversityHospital Rheinische FriedrichWilhelmsUniversittBonn 0cEPMA Journal3PM vision and strategiesPPPM for Twentyfirst Century Biosensing PainlessPersonalised PointofCare Monitoring with Wearableand Implantable DevicesAndrews RACorresponding author Nanotechnology Smart Systems NASA Ames Research Center Moffett Field CA USA email rjarusselljandrewsKeywords Artificial intelligence Biosensors Blood pressuremonitoring BraincomputerBrainmachine interfaceContinuous monitoring Diabetes ElectrocardiogramElectroencephalogram Epilepsy Fall detection Gait disorders Glucose monitoring Implantable sensors Ingestible sensors Internet of things Iontophoresis Interstitial fluidNanosensors Neurotechnology Pressure monitoring Salivamonitoring Seizure detection Smart contact lenses Smartmouthguards Smart patches Smart skin Smart watchesSmartphone apps Skin patches Sweat monitoring Tear monitoring Temperature monitoring Tissuedevice interfaceWearable sensors Wireless monitoringIntroductionMany people do not realize they already have adopted wearable devices for medical monitoring”smartwatches Typicalstories of smartwatches providing lifesaving diagnostic information include the following A smartwatch alarming allnight regarding abnormal heartrate alerted the wearer to seekmedical attention for what proved to be atrial fibrillation [] A hiker”lost as nightfall approached”stumbled and fellon difficult terrain Unbeknownst to the hiker the fall triggered his smartwatch to automatically call the emergencyphone number in the USA thereby avoiding what couldhave been a tragic outcome Smartphones with accelerometerand GPS capabilities have apps for people with epilepsy whomay require emergency medical assistance []Medical monitoring has not always been so painless persistent and unobtrusive Atrial fibrillation required attachingelectrodes to the skin with a conductive gel in turn connectedto a device”possibly portable but certainly obtrusiveMonitoring of blood glucose by diabetic patients required repeated fingersticks”painful intermittent and obtrusivePhases of Biofluid MonitoringDiagnostic techniques for biofluids eg blood urine salivaand cerebrospinal fluid CSF have evolved over the pastseveral decades Fig [] The first phase”extendingfrom the twentieth century to the present”entailsobtaining a sample from the patient an invasive procedurefor blood and CSF and sending it to a laboratory for analysisResults are not available for hours to days for samples obtained from outpatients and minutes to hours for inpatientsFig The four technological waves of biochemical monitoring reference []with permissionThe second phase began about two decades ago with pointofcare POC monitoring where the laboratory comes to thepatient ie to the recently obtained sample rather thantransporting the sample to the laboratoryThe third phase more recently available consists of wearabledevices This is the epitome of POC monitoring since the patientand the device are inseparable Smartwatches can do this forpulse and blood pressure patches applied to the skin for continuous blood glucose monitoring The patches eg for glucosemonitoring typically monitor the analyte concentration in interstitial fluid ISF which closely parallels blood glucose [“]The line between the third and fourth phases”wearable andimplantable devices”is blurred Part of this is due to expansion of the fluids monitoring from blood or ISF to sweatsaliva and tears Most would call a mouthguard to monitor salivaa wearable device”but what about a œsmart contact lens tomonitor tears Truly implantable devices eg inserted subcutaneously by a minor surgical procedure can monitor analytes suchas glucose for months potentially longer rather than the days toa week or so of most patches []Power to the Patient”Digitizing Biofluid MonitoringDuring the sampletolab and POC phases urine was the idealbiofluid”noninvasively obtained and relatively easilytransported Blood required an invasive procedure a needlestickSweat and tears were not easily obtained in a manner guaranteeing uniformity and saliva could vary greatly depending on timeof sampling eg after a drink or a mealWearable devices have transformed those problems into oneconsisting of a tissuedevice interface TDI challenge 0cEPMA JournalContinuous biofluid monitoring is a reality sampling urineblood or other biofluids continuously was not practical previously outside a hospital setting with an indwelling catheterfor urine or blood or even CSF A second problem in phasesone and two was obtaining continuous diagnostic informationfrom the biofluidThe smartphone and smartwatches plus machine learning andartificial intelligence AI have allowed not only continuousbiofluid monitoring but also continuous realtime interpretation of that monitoring information in a precise and personalized manner”œdigital biomarkers [] This can answer thequestionœWhat does this biofluid monitoring value mean for this particular patient at this precise momentOnce answered that information can guide realtime precisepersonalized treatment eg continuous feedbackguided orclosedloop insulin release in diabetes The patient if desiredcan have control over when the biofluid monitoring information is gathered or processed or transmitted eg to adatabank The patient can remove the patch or the smartwatchor turn off the smartphone containing the app transferring thedataBlood Sweat Tears and SalivaAlthough the primary target has been a wearable monitor ofblood glucose for diabetic patients other biological signalsthat can be measured through the skin include chemicals beyond glucose”potassium chloride lactate electrical electrocardiogram ECG electroencephalogram EEG electromyogram EMG and physical temperature pressure lightsound [“] Additionally non or minimally invasive monitoring has included measures ranging from respiratory rate tojoint movement to gait [ “] This review is primarilylimited to the TDI for biofluidsWearable skin patches depend on knowledge of thestructure of human skin [ ] œSmart skin exhibitsmany technological advances as illustrated in Fig [] Skin patches usually monitor ISF concentrations of thechemical of interest relatively straightforward for ISF glucoseas a surrogate for blood glucose Sweat however poses adifferent problem since sweat is not continuously availablefor monitoring In the typical skin patch for sweat the patchincorporates an electrode to deliver a cholinergic agent such ascarbacholinto the skin for stimulation of sweationtophoresis [ ]Fig Recent research trends in smart skin from four viewpoints First the structures of smart skins are advancing from stretchable toultrathin to breathable sensors resulting in enhancement of biocompatibility and reduced burden of sensor attachment Second multimodality is expanding from electrical to physical to chemical sensors Third more advanced functions such as stimulation drug deliveryand displays are being incorporated in addition to sensing functions Fourth novel materials such as selfhealing conductors intrinsicallystretchable semiconductors and photoactive materials are being developed reference [] with permission 0cEPMA JournalMonitoring tears is challenging the rate of tearing is notuniform the device must be acceptable to the patientTearbased biofluid sensors include smart contact lenses anddevices placed in the lower eyelid Fig 3A [ ]Fig 3A Tearbased biosensors a Contact lens sensor previously under development by Google and Novartis to measure tear glucose concentrationPrototype platform contained integrated electronics for sensor response processing and wireless transmission b Multifunctional wearablelens for monitoring both glucose in tears and intraocular pressure using enzymesmart sensor system incorporated onto a contactfunctionalized graphenesilver nanowire hybrid nanostructures c A wireless glucose sensor incorporated into a contactlens platformwith wireless power transfer circuitry and display pixels for a fully integrated and transparent platform that does not hinder vision dWearable contact lens tear glucose biosensor applied to an artificial eye with schematic representation of smartphonebased quantification of glucose levels through reflection of incident light by the photonic microstructure within the lens The smart contact lens systemintegrated with a glucose sensitive hydrogel monitors changing glucose concentrations in vitro without complicated fabrication proceduresand allows rapid response time for continuous measurements e NovioSense electrochemical tear glucose sensor A small springlikesensing device is designed to be placed within the conjunctival fornix for continuous access to tear glucose reference [] withpermission Saliva is readily available but suffers from analyte variabilityeg temperature and concentration resulting from the presence of liquids of varying temperatures over time in the oralcavity hot vs cold drinks [] Patient acceptance of a devicein the oral cavity”given that some saliva biofluid sensors aremouthguards or otherwise bulkyobtrusive”is another issueFig 3B [] 0cEPMA Journal Fig 3B Salivabased biosensors a Mouthguardbased wearable salivary uric acid biosensing platform with integrated wireless electronics andanalysis of salivary uric acid concentrations b Mouthguardbased sensor for glucose monitoring in saliva with onbody applicationand analysis of increasing glucose concentrations c Onbody depiction and crosssectional configuration of radio frequency trilayertoothmounted sensor for wireless monitoring of food consumption This dielectric sensor fabricated with biocompatible materials iscapable of being mounted onto tooth enamelto detect foods and fluids during ingestion when functionalized with analytesensitivelayers Projected uses were for detection of sugars alcohol salinity pH and temperature d Operational principles and electronicsconfiguration of a wireless usercomfortable sensing platform for longrange oral monitoring of sodium intake during hypertensionmanagement reference [] with permissionSometimes It Takes Guts to MonitorConfirmation of ingestion of prescribed medications particularly in unreliable patients eg dementia is another biosensing challenge One solution is the œsmart pill”a capsulecontaining a microsensor that is swallowed monitoringwhether the medication is present in the stomach [] Theœsmart pill communicates with a skin patch which not onlydocuments that the pill was swallowed and when but also ifdesired blood pressure pH and temperatureFor continuous monitoring a sensor can be stationed in thegut most likely the stomach Such monitoring could includemedication ingestion pH controlled drug delivery and imaging of the gut lining An ingestible sensor that is selfpowered by stomach acid in contact with zinc and copper electrodes on the sensor surface is being developed[] Another ingestible capsule under development usesa microneedle that inserts into the stomach wall to deliver a drug eg insulin [] 0cEPMA JournalWear Your Heart on Your Sleeve Wear Your Brain onYour HatThe topic of brain biomonitoring”from EEG to nextgeneration brainmachine interfaces BMIs”is beyond the scope of this but has been recentlyreviewed [] An area of concern regarding brainbiomonitoring is directtoconsumer DTC marketingof devices that are of undocumented value or possiblerisk [“] Brain biomonitoring information obtained through DTC marketing raises questions of bothpersonal privacy and ultimate use of such data bymarketers Increasing DTC availability of brain electrical stimulation eg via a skullcap notably transcranial direct current alternating current and randomnoise stimulation techniques raises questions of safety [] Ethical considerations regarding DTC brainbiomonitoring and biostimulating remain unresolved[“]Conclusions and Expert RecommendationsThe field of wearable and implantable biosensors is evolvingso rapidly that no review truly reflects the œstateoftheartAdvances in the TDI and AI promise that such devices willnot only enhance diagnostic capabilities but also provide awealth of information for improved treatmentsSpecific recommendationsIncorporating the latest technology into biosensors”fromnanotechniques to microfluidics”is essential Asmartphone from ten years ago would be unacceptablein the consumer marketplace outdated diagnostic techniques in medicine are similarly unwarranted Similarly the latest AI is necessary to analyze the hugeamounts of data that wearable and implantable biosensorsprovide The consumerpatient must be involved in device development from the outset What may appear wonderful inthe lab or the boardroom may prove a failure in the marketplace and social media Consumerpatient acceptanceCPA is crucial for widespread adoption Flexibility is key Some patients may prefer a patch forcontinuous glucose monitoring others a smart contactlens and others an implanted device requiring a minorprocedure for implantation but not frequent replacementWhat works in a highincome country such as Belgiummay not work in a lowincome country such as BurkinaFaso Legislation and safeguards regarding the huge amounts ofpersonal medical data generated by wearable and implantable biosensors is essential since data collection and storage systems can be hacked This is especially crucial withregard to biomodulating devices eg cardiac pacemakers brain stimulation and controlled drug deliverysystems Given the vulnerability to hacking wearable and implantable biosensors require the same caution as other widespread threats to population health eg toxins both liquid and aerosol biological warfare agents and radiationReferences Weichert W ˜My watch kept on alarming all night aboutmy heart rate™ Oxford Med Case Rep “ Seizario detecting seizures and falls Available seizariohealthhappycom [Accessed Apr ] Heikenfeld J Jajack A Feldman B Granger SWGaitonde S Begtrup G et al Accessing analytes inbiofluids for peripheral biochemical monitoring NatBiotechnol “ Guk K Han G Lim J Jeong K Kang T Lim EK et alEvolution of wearable devices with realtime diseasemonitoring for personalized healthcare Nanomaterials Khan S Ali S Bermak A Recent developments in printable flexible and wearable sensing electronics forhealthcare applications Sensors Kim J Campbell AS EstebanFernandez de Avila BWang J Wearable biosensors for healthcare monitoringNat Biotechnol “ Someya T Amagai M Toward a new generation ofsmart skins Nat Biotechnol “ Waltz E Sweet sensation Nat Biotechnol“ McCarthy A The biomarker future is digital ClinicalOMICS 2020JanFeb24“ Massaroni C Nicolo A Lo Presti D Sacchetti MSilvestri S Schena E Contactbased methods for measuring respiratory rate Sensors Faisal AI Majumder S Mondal T Cowan D Naseh SDeen MJ Monitoring methods of human body jointsstateoftheart and research challenges Sensors McDonnell S Ingestible sensors powered by stomachacid Tech Briefs 2018Aug45“Jarchum I To the stomach and beyond Nat Biotechnol“ Frank JA Antonini MJ Anikeeva P Nextgenerationinterfaces for studying neural function Nat Biotechnol“Ienca M Haselager P Emanuel EJ Brain leaks and consumer neurotechnology Nat Biotechnol “ 0c Wexler A Separating neuroethics from neurohype NatBiotechnol “Jarchum I The ethics of neurotechnology NatBiotechnol “The Navarra Genomes Project NAGEN Benefits for Predictive Preventive and PersonalizedMedicinePasalodos S1 Salgado J1 Miranda M1 Maillo A1Matalonga L2 Beltr¡n S2 Carmona R3 P©rezFlorido J3Etayo G4 Lasheras G4 Bernad T4 G³mezCabrero D1Angel Gonz¡lez L5 Brennan P6 Gut I2 Dopazo J3Pinillos I4 Lasa I1 Alonso A11Navarrabiomed Complejo Hospitalario de NavarraUniversidad Pºblica de Navarra UPNA IdiSNAPamplona Spain2Centro Nacional de An¡lisis Gen³mico CNAGCRGCenter for Genomic Regulation Barcelona Institute ofScience and Technology BIST Barcelona Spain3rea de Bioinform¡tica Fundaci³n Progreso y Salud Nodode Gen³mica Funcional INBELIXIRes Bioinform¡ticade ER BiERCIBERER CDCA Hospital Virgen delRoc­o Sevilla Spain4Navarra de Servicios y Tecnolog­a NASERTIC Spain5AVANTIA Pyramide Asesores Spain6NENC NHS Genomic Medicine Centre Newcastle uponTyne UKCorresponding author Dr Angel Alonso GenomicMedicine Unit Navarrabiomed Complejo Hospitalario deNavarra Universidad Pºblica de Navarra UPNA IdiSNAEPMA JournalCIrunlarrea Pamplona Spain emailangelalonsosancheznavarraesKeywords predictive preventive personalized medicinegenomics next generation sequencing NGS whole genome sequencing WGS rare diseases eHealth bioinformatics big data ICPerMed multiomicsBackgroundIn the past few years extraordinary developments in the fieldof next generation sequencing NGS technologies such aswhole genome sequencing WGS have made it possible forclinicians to have access to a huge amount of biological information which could potentially explain complex genetic diagnoses genetic predisposition to severe diseases reproductiverisks and inappropriate responses to certain medicationsThese advances herald a new era of predictive preventive personalized medicine PPPM although incorporation into clinical practice has proved to be challenging [] œNAGEN is a Spanish regional pilot study to implement recentadvances of cutting edge genomic research technology intoreal clinical practiceGoal materials and methodsNAGEN ™s main goal is the implementation of the wholegenome sequencing WGS derived information as a clinicaltool for the development of PPPM in the Public Health ServiceA scientific implementation approach was used to identify andcategorize both the local barriers and facilitators to acceleratethe incorporation of translational genomics intohealthcare see Fig Fig Local barriers for genomic medicine implementation in Navarra NAGEN project 0cEPMA JournalKey Actions for this implementation Subjects NAGEN is recruiting patients and theirrelatives affected with one condition from a list of nearly rare diseases RD Albeit rare joint RD™s prevalence ishigh “ with a very high social impact wide multidisciplinary medical coverage and a high rate of identifiablegenetic causes These features make it possible to involve themedical community raise population awareness and offergood support to evidencebased medicine practice The rateof genome per inhabitants facilitates a wide participation from patients and health professionals Results and incidental findings Pertinent findingsexplaining the referral condition secondary findings onpersonal and reproductive risks of severe inherited diseases and pharmacogenomic variants determining drugsdose and toxicity are reported based on patient™s choiceproviding the necessary evidence of the effectiveness ofmedical interventions based on genomic medicine Newgenetic counselling interventions variant validation andreporting pathways have been put in place for the bestprovision of services Electronic health record EHR adaptation The existingEHR has been modified to host a newly designed recruitment tool which enables and guides the identification andimmediate referral of patients from any point in theNavarre health system network An additional development also makes it possible that clinically actionable genomic results are available for participants™ doctors withall other clinical information across the system Clinical research A number of new exciting genomicresults potentially providing new insights into the geneticbasis of RDs and additional information on populationgenomics are being produced by NAGEN offeringexceptional material to support new research It is a maingoal of the Project to ensure an adequate data harmonization which enables data sharing for research under anappropriate regulatory and legal framework Optimized use of preexisting public infrastructures Inorder to overcome the lack of local facilities NAGEN externalizes WGS sequencing services to CNAGCRG the Spanish world leader public centre for genomicanalysis Bioinformatic analysis also relies primarily onCNAGCRG through the RDConnect GenomePhenome Analysis Platform which was deployed for theproject to store analyse and interpret the genomic datamaking use of the phenotypes encoded with the HumanPhenotype Ontology HPO and the experts from theBioinformatics Platform of the Rare Diseases Spanishnetwork CIBERer through the Interactive VariantAnalysis IVA tool based on the genome browserGenome Maps but expertise in this field has graduallybeen transferred to the newly created local TranslationalBioinformatics Unit during the course of the ProjectICT New ICT solutions have been adopted for NAGEN allowing the storage and high performance managing of massive genomic data through an innovative partnership with NASERTIC a local company providing dataanalysis infrastructures such as the new IBM POWER processor which build on crossdisciplinary collaborationin research and development with the local industry ELSI While genetic data protection is widely regulated forclinical and research purposes within the NAGEN project the local Health Research Authority has specificallyresolved that the massive genomic information resultingfrom WGS will also be part of the patient™s medical recordand it will accordingly be protected and stored In order toenable the use of genomic data for research the constitutionof a œGenomic Library has been proposed which wouldaccept specific research enquiries on anonymized genomicsequences upon pertinent EC approval This scenario requires a new regulatory legal framework which has alsobeen explored through a specific partnership with Avantia from the Pyramide group a local consulting companywith wide experience in data protectionResultsKey results to date Clinical and preclinical results Around patients have todate followed through the aboveoutlined pathway and of the families have now found the longawaited geneticcause for their previously unexplained condition and nowhave hope of an improvement of their clinical care based onthese findings Remarkably of these diagnoses wereattributed to genes previously unknown to cause a humandisease or causing different phenotypes than those previously described Fig Additionally of participants carriedgenetic predispositions to severe diseases had reproductive risks and had pharmacogenomic actionable variants influencing prescription Table Further candidategenomic variants potentially explaining patients™ diseaseshave been identified in an additional of participatingfamilies which provides an extended base for new collaborative research projects Interestingly about differentmedical specialities have referred patients to NAGEN indicating a desirable multidisciplinary involvementin this implementation initiative Healthcare workforce education and public empowerment Monographic NAGEN symposiums hospitalbriefings clinical sessions and face to face meetings havebeen anized ing the participation to all medicalprofessionals in the region Moreover the designatedspecialities œphysician champions especially commissioned to facilitate recruitment help with the clinical 0cinterpretation of genomic variants and to spread the wordreceived category and CME credits from a NAGEN tailored genomics education programme Public involvement has also been possible through a press conference which was widely covered by national general andmedical press and social media conferences at theœScience Week and œRare Diseases Day a specificwebsite wwwnagen1000navarraes and communicationsto national and international congresses Sustainability After deducting marginal costs due to theTranslational Bioinformatics Unit establishment and ICTinfrastructures the costeffectiveness analysis CEA recEPMA Journalognized a full running costs of ‚¬ per RD diagnosisprior to familial cascade genetic testing and including duoand trio studies costs when necessary compared with‚¬ average cost per diagnosis estimated for thestandard of care pathway [] Considering that costbenefit analysis CBA outperforms CEA for RDwe conducted a survey of all participants whichshowed that more than of them would be willing to pay more than ‚¬ for the genomic information they received after their participation inNAGEN regardless of whether their diagnosiswas ultimately achieved or notFig Pertinent clinical findings a Piechart showing the performance of genomic diagnoses achieved by NAGEN and of strong and mild candidates genomic variants b Table listing OMIM codes and diagnoses in red cases with no OMIM codificationTable Clinical Actionable Incidental FindingsClinical Actionable Incidental FindingsTypeConsentN of patients of casesDisease PredispositionReproductive RiskPharmacogenomicpatients and it was awarded as the Best Practice in PersonalisedMedicine by ICPerMed in Significantly it resulted in setting the new Genomic Medicine Unit of Navarrabiomed and itsNAGEN strategy which has now raised ‚¬ million for RDprojects on PM over the past years NAGEN is an exemplarpractice for the Spanish Senate Initiative for a National Strategyon Genomics and PM and has given rise to the launch ofthe Navarra Government Strategy on Personalised Medicine announced in November Conclusions and expected impactsGenomics has become a major contributor to multiomics andPPPM related approaches in management of major and fatal pathologies such as cancer diabetes and stroke [“] NAGEN illustrates how translational research and innovation in thefield of genomics and PPPM is already delivering real benefits toAcknowledgements This study will always be in debt to all participa

Thyroid_Cancer

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

Thyroid_Cancer

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

"Trophoblast cell surface antigen TROP2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion The association between TROP2 expression and occurrence anddevelopment of oral squamous cell carcinoma OSCC remains to be understoodMethods We investigated the role of TROP2 in OSCC patients using a combination of biophysical approaches Atotal of OSCC patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry KaplanMeier survival curve analysis and atomic force microscopy to analyzeTROP2 expression morphology and mechanical properties of OSCC tissuesResults TROP2 was overexpressed in of poorly differentiated OSCC samples High levels of TROP2 wereassociated with survival rate lower than and patient age odds ratio [OR] P confidence interval [CI “] tumor size OR P CI [“] and TNM stageOR P CI [“] Average surface roughness of low medium and highly differentiatedOSCC tissues were ± ± and ± nm respectively The Pearson coefficient revealed anegative association between tumor stiffness and TROP2 expression r ˆ’ P Conclusion Overexpression of TROP2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics Taken together our findings demonstrated that TROP2 may be an indicator of OSCCdifferentiation leading to the altered mechanical properties of OSCC tissuesKeywords Oral squamous cell carcinoma TROP2 Tissue stiffness Differentiation SurvivalBackgroundOral squamous cell carcinoma OSCC is a commonsubtype of head and neck and other malignant tumors[ ] The past few decades have shown increased incidence of OSCC that is expected to rise further in the future [] Thereforeimperative to determineisit Correspondence zhangkllzu163com Baoping Zhang Shuting Gao and Ruiping Li contributed equally to thiswork1Department Hospital of Stomatology Lanzhou University Donggang westRoad Lanzhou Gansu ChinaFull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of OSCCHuman trophoblast cell surface antigen TROP2 alsocalled tumorassociated calcium signaltransduction2TACSTD2 is a surface glycoprotein encoded by TACSTD that has extracellular domains a single transmembrane domain and a short tail [ ] TROP2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal [] pancreatic [] and laryngealcancers [] Inhibiting TROP2 expression has shownpromise in clinical applications [ ] TROP2 regulates 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 0cZhang BMC Cancer Page of tumorigenic properties including cancer cell adhesion invasion and migration Tang [] have recentlyshown that TROP2 impacts growth and metastasis byactivating PI3KAKT signaling This phenomenon hasalso been observed in gallbladder cancer [] Amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ “] This has shed light on the biological functions of TROP2 and its use as a prognostic biomarker for OSCCvarious biochemical mechanismsAtomic force microscopy AFM is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesAFM has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast [] liver [] and lung [] Parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion[] Advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesThis study aims at investigating the correlation between TROP2 expression and clinicopathological characteristics of OSCC We have demonstrated the tissuemorphology and mechanics of OSCC samples duringtumor development using AFM We believe our findingswill help develop TROP2 in accurately diagnosing OSCCin tumors with different grades of differentiationMethodsTissue preparationThe protocols in this study were approved by the researchethics committee of Lanzhou University Tumor sampleswere collected from patients after obtaining written informed consent A total of patients with oral squamous cell carcinoma OSCC were registered atthesecond hospital of Lanzhou University between January and March Among these samples sampleseach showed high moderate and low levels of differentiation The experimental group comprised males and females aged “ years average years All patientswere diagnosed with OSCC based on surgery andFig Paraffin pathological sections of tissues a d g — 4fold b e h — 10fold c f i — 40fold 0cZhang BMC Cancer Page of Fig Immunohistochemical staining was performed to detect the expression of TROP2 at different stages of OSCCpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery Pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded Cancer and cervical lymph nodetissues were collected after maxillofacial surgery All specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding Among them and Fig Average optical density of TROP2 poorly differentiated squamous cell carcinoma showed high expressionP 0cZhang BMC Cancer Page of Table Correlation between TROP2 expression and clinicopathological characteristicsCharactersnTROP2 expressionLow or noTotalGenderMaleFemaleAge‰¥ LocalizationmucosaTongueDifferentiationwellModeratePoorTumor sizeT1 ‰ cm cmT2 ‰ cmT34cmT4bLymph node metastasesN0NXDistant metastasesM0M1TNM stageI IIIII IVPerineural infiltrationNoYesVascular invasionNoYesPearson x2P value Highpatients exhibited no and cervical lymph node metastasesrespectively Clinical TNM staging was performed according to the 7th edition TNM staging classification standardjointly developed by the International Union for CancerControl and American Joint Committee on Cancer []and World Health anization guidelines []Hematoxylin and eosin HE stainingOSCC tissues were fixed overnight using neutralformalin Solarbio Beijing China paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol The sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing SolarbioBeijing China Sections were visualized and imagedusing the Olympus BX53 at magnifications of — and sealing and neutral 0cZhang BMC Cancer Page of ImmunohistochemistryHE sections were subjected to the SP method to detectTROP2 expression The sections were incubated overnight with the primary antibody against TROP2 Abcam USA at °C followed by incubation withbiotinlabeled goat antirabbitIgG AbcamUSA at °C for h The sections were then developed using DAB Beijing Zhongshan Golden BridgeBiotechnology China dehydrated transparentizationand film and neutral resin sealed The prepared sections were visualized using microscopy OlympusBX53 JapanAFMFixed tissues were placed in Petri dishes containingphosphatebuffered saline All analyses for mechanical properties were performed using the biologicalatomic force microscope BioAFM NanoWizard IIIBruker USA Silicon AFM probes from the Pointprobe®constant of Nmcoating NanoWorld USA wereCONTRreflexused The spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm AFM was performed using theseries with a kHzforcetheresonancefrequencyofcontact model and a scan rate of Hzs in airForcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms [] Six random sites wereselected for each sample and each site was measured times We used the modified Hertz contact modelto analyze forcedistance curves [] and calculateYoung™s modulus and roughness of OSCC tissueswith varying differentiationStatistical analysisStatistical analyses were performed using SPSS Statistical Product and Service Solutions IBM Forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the TukeyKramerHSD test for pairwise comparisons Pearson Chisquaretest was used to analyze clinical features and TROP2 expression based on the calculated odds ratios ORs and confidence intervalCI Survival was evaluatedusing KaplanMeier curves and the difference was analyzed using the logrank test P was consideredstatistically significantFig TROP2 total survival curve using KaplanMeier survival curves low blue line high green line 0cZhang BMC Cancer Page of ResultsTissue morphology and TROP2 expression across theclinical stages of OSCCTumor cells from poorly differentiated OSCC samples exhibited characteristic atypia poor differentiation and irregular morphology Fig Howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated OSCC as compared to those inpoorly differentiated cells TROP2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells We observed that low differentiation and high malignancy of OSCC was associated with higher TROP2 expression Fig Theaverage optical density of TROP2 among the lowmedium and highly differentiated OSCC tissues were ± ± and ± respectively Fig Table TROP2 expression risk factors with clinicopathological featuresCharacteristicsnTROP2 expressionLow or noTotalGenderMaleFemaleAge‰¥ LocalizationMucosaTongueDifferentiationWellModeratePoorTumor sizeT1 ‰ cm cmT2 ‰ cmT34cmT4bLymph node metastasesN0NXDistant metastasesM0M1TNM stageI IIIII IVPerineural infiltrationNoYesVscular invasionNoYesNote a Well vs Moderate b Moderate vs Poor c Well vs PoorP valueOR CIHigh 005a 0001b 0001c 0cZhang BMC Cancer Page of Association between TROP2 expression and clinicalcharacteristics of OSCCWe analyzed the clinicopathological characteristics of patients with OSCC with varying degree of differentiationDifferential expression of TROP2 was associated with patient age tumor differentiation tumor size TNM stagepercutaneous nerveinvasionTable P Patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high TROP2 expressionP However there was no association between theexpression of TROP2 and patient gender tumor locationlymph node metastasis or distant metastases P and vascularfiltrationTROP2 expression and patient survivalUsing KaplanMeier survival curves we observed that anincrease in TROP2 expression negatively correlated withthe overall survival of patients Fig And lowno ofTROP2 expression group™s 3years survival rate was a for high expression group and 5years ratewere and respectively TROP2 expression wasassociated with patient age P OR CI[“] tumor differentiation Well vs ModerateP OR CI [“] Moderate vsPoor P OR CI [“]Well vs Poor P OR CI [“] tumor size P OR CI[“] TNM stage P OR CI[“] vascular invasion P OR CI [“] and peripheral nerve invasionP OR Table High TROP2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher TNM staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival Thus TROP2 may be a prognostic indicator for survival in OSCC patientsFig Surface morphology of OSCC tissue sections via AFM detection irregular morphology appeared in the low differentiation 0cZhang BMC Cancer Page of Surface morphology and roughness of OSCC tissuesThe surface morphologies of OSCC tissues with varying degrees of differentiation were analyzed directtopographical imaging using BioAFM Figure showsthe representative image from each tissue acquiredduring the cantileverbased AFM nano indentationtest The tissue interface varied with tumor differentiationindicating that highly differentiated OSCC tissues had a regular and flat morphology OSCC tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization Figure summarizes the roughness ofOSCC tissues with varying differentiation The average surface roughness of low medium and highly differentiated OSCC tissues were ± ± and ± nm respectively Roughness ofthe tissue surface was enhanced with increasing differentiation of OSCC tissuesYoung™s modulus of OSCC tissuesWe used BioAFM to determine Young™s modulusbased on the mechanical properties of OSCC tissues with varying degrees of differentiation We randomly selected six contact points from each slice andeach contact point was measured times Forcedistance curves were generated for each slice and theJPK Data Processing software version was usedto calculate Young™s modulus Figure shows theaverage variation in stiffness within individual tissuesin the range of “ kPa In the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples P Fig Surface roughness results are express as mean ± SEM nm 0cZhang BMC Cancer Page of Fig AFM test average tissue stiffness Young™s modulus E was thus confirmed to be a parameter of cell hardness for various cells and tissuePa P Thus tissue differentiation was positively associated with its stiffness Fig Association between mechanical properties and TROP2expression in OSCCThe Pearson coefficient showed a negative associationbetween the stiffness of OSCC tissues and TROP2 expression Fig r ˆ’ P Thus we detectedan increase in stiffness with varying differentiation in thetumor samplesDiscussionTROP2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines Studieshave shown that overexpression of TROP2 is associatedwith tumorigenesis and malignancy [“]In thisstudy TROP2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness The relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaImmunohistochemical analysis revealed that the expression of TROP2 in poorly differentiated OSCC tissueswas significantly higher than that in welldifferentiatedOSCC tissues Additionally TROP2 upregulation wascorrelated with tumors of advanced TNM III IV staging and poor differentiation than that in tumors withlow TNM I II staging Thus the abnormal expressionof TROP2 may be associated with the occurrence anddevelopment of tongue malignancies Furthermore highTROP2 expression predicted low survival as comparedto that in the tumors with low TROP2 expression Previous research has also demonstrated the correlation between shorter patient lifespan and high levels of TROP2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of TROP2 [] TROP2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis[] Upregulated TROP2 correlates with the poor prognosis of thyroid papillary carcinoma [] colon cancer[] liver cancer [] and other malignanciesThere have been an increasing number of studies on thebiological role of TROP2 at the molecular level TROP2induces the downregulationloss of PTEN thereby stimulating PI3KAKT signaling and tumor development []PTEN is a wellknown tumor suppressor that is a phosphatase [] and affects the PI3KPKBAKT signaling axisduring the dephosphorylation of PIP2 and PIP3 []PI3K signaling is important in regulating tumor cell proliferation migration and invasion [ ] Thus PTEN is anegative regulator of cancer [ ] Li have shownthat TROP2 activates epithelialmesenchymal transitionvia PI3KAKT signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer [] Similarly TROP2 expression stimulates the proliferation migration and invasion of osteosarcoma cells [] Hou et aldemonstrated that TROP2 regulates JAK2STAT3 signaling in glioblastoma cells [] 0cZhang BMC Cancer Page of Fig Correlation analysis between changes in mechanical stiffness of OSCC tissues and TROP2 expression Note changes have statisticalsignificance P and show a certain negative correlation r ˆ’ Functional differentiation oftissues influences themicromorphology and mechanical stiffness of OSCCcells We detected low surface roughness on OSCC tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion Poorlydifferentiated OSCC tissues are œsofter than highly differentiated OSCC tissues PI3K is an important celladhesion molecule TROP2 triggers the synthesis of proteins with homologous domains such as pleckstrinRAC Tiam and Vav Tiam and Vav activate RAC thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion []The underlying mechanisms involved in the alterationof micromechanical properties of OSCC samples and occurrence development metastasis and invasion ofOSCC tumors remain to be elucidated HE staining isthe gold standard for tumor diagnosis With the development of biomechanics in the past two decades [] the mechanical properties of tissues need to be investigated based on biomedical and physical parametersIn this study we have assayed the changes in mechanicalproperties at the micronanometer level using AFM anddetermined the association between the TNM grademetastasis and stiffness of tumor samplesIn conclusion we have demonstrated the association between differential expression of TROP2 and patient agetumor differentiation tumor size TNM stage percutaneousnerve filtration and vascular invasion Moreover high levelsof TROP2 correlated with poor overall survival in patientsHighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness Lastly high TROP2 expression resulted in reduced tumor stiffness However thisstudy had some limitations First the cohort used in thisstudy was relatively small Second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay Thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developTROP2 as a specific and efficient prognostic biomarker forOSCCConclusionThese findings could promote new methods for the earlyOSCC diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity More detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseAbbreviationsOSCC Oral squamous cell carcinoma TROP2 Trophoblast cell surfaceantigen AFM Atomic force microscopyAcknowledgementsWe thank the individual who participated in this studyAuthors™ contributionsBZ SG and RPL are responsible for conception and design Data wascollected by YTL RC JYC and YMG Data was analyzed by EW and YH KLZrevised the All authors have read and approved the manuscriptFundingThis work was supported by the Fundamental Research Funds for theCentral Universities No lzujbky2020cd03 Baoping Zhang Doctoralmaster 0cZhang BMC Cancer Page of students of the Second Hospital of Lanzhou University sdkygg17 Lan Yangand Key Laboratory of Mechanics on Disaster and Environment in WesternChina The Ministry of Education of China No “ Kailiang ZhangAvailability of data and materialsThe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateWritten informed consent was obtained from each participant before samplecollection The study was approved by the Committee for Ethical Affairs ofSchool of Stomatology Lanzhou UniversityConsent for publicationNot applicableCompeting interestsThe authors have no conflicts of interestAuthor details1Department Hospital of Stomatology Lanzhou University Donggang westRoad Lanzhou Gansu China 2Institute of Biomechanics andMedical Engineering Lanzhou University Lanzhou ChinaReceived April Accepted August ReferencesIyer S Thankappan K Balasubramanian D Early detection of oral cancerscurrent status and future prospects Curr Opin Otolaryngol Head Neck Surg“Caldeira PC Soto AML de Aguiar MCF Martins CC Tumor depth of invasionand prognosis of earlystage oral squamous cell carcinoma a metaanalysisOral Dis Online ahead of printKim Y Kim JH Increasing incidence and improving survival of oral tonguesquamous cell carcinoma Sci Rep McDougall AR Tolcos M Hooper SB Cole TJ Wallace MJ Wallace Trop2from development to disease Dev Dyn “Guan GF Zhang DJ Wen LJ Yu DJ Zhao Y Zhu L Prognostic value ofTROP2 in human nasopharyngeal carcinoma Int J Clin Exp Pathol “Stewart D Cristea M Antibodydrug conjugates for ovarian cancer currentclinical development Curr Opin Obstet Gynecol “Liu J Yang D Yin Z Gao M Tong H Su Y A novel human monoclonalTrop2IgG antibody inhibits ovarian cancer growth in vitro and in vivoBiochem Biophys Res Commun “Zhao W Jia L Kuai X Tang Q Huang X Yang T The role andmolecular mechanism of Trop2 induced epithelialmesenchymal transitionthrough mediated betacatenin in gastric cancer Cancer Med “Zhao W Jia L Zhang M Huang X Qian P Tang Q The killing effect ofnovel bispecific Trop2PDL1 CART cell targeted gastric cancer Am JCancer Res “Jordheim LP Chettab K CrosPerrial E Matera EL Dumontet C Unexpectedgrowthpromoting effect of oxaliplatin in excision repair crosscomplementation group transfected human colon cancer cellsPharmacology ““ Nishimura T Mitsunaga M Sawada R Saruta M Kobayashi H Matsumoto N Photoimmunotherapy targeting biliarypancreatic cancer withhumanized antiTROP2 antibody Cancer Med “ Wang XD Wang Q Chen XL Huang JF Yin Y Da P Trop2 inhibitionsuppresses the proliferation and invasion of laryngeal carcinoma cells viathe extracellular signalregulated kinasemitogenactivated protein kinasepathway Mol Med Rep “ Wanger TM Dewitt S Collins A Maitland NJ Poghosyan Z Knauper VDifferential regulation of TROP2 release by PKC isoforms through vesiclesand ADAM17 Cell Signal “Tang G Tang Q Jia L Chen Y Lin L Kuai X TROP2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the PI3KAkt signaling pathway Int J Mol Med“Trerotola M Li J Alberti S Languino LR TROP2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinRACK1 axis J Cell Physiol“Li T Su Y Yu X Mouniir DSA Masau JF Wei X Trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury Cell Transplant “Stoyanova T Goldstein AS Cai H Drake JM Huang J Witte ON Regulatedproteolysis of Trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling Genes Dev “Sun X Xing G Zhang C Lu K Wang Y He X Knockdown of Trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via AKTcatenin pathway Cell Biochem Funct Lee H Jang Y Seo J Nam JM Char K Nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityACS Nano “Kruse SA Smith JA Lawrence AJ Dresner MA Manduca A Greenleaf JF Tissue characterization using magnetic resonance elastographypreliminary results Phys Med Biol “Kaneko TS Pejcic MR Tehranzadeh J Keyak JH Relationships betweenmaterial properties and CT scan data of cortical bone with and withoutmetastatic lesions Med Eng Phys “ Goetz JG Minguet S NavarroLerida I Lazcano JJ Samaniego R Calvo E biomechanical remodeling of the microenvironment by stromalcaveolin1 favors tumor invasion and metastasis Cell “Edge SB Compton CC Compton the American joint committee on Cancerthe 7th edition of the AJCC cancer staging manual and the future of TNMAnn Surg Oncol “ Barnes L Eveson JW Reichart P Sidransky D Pathology Genetics Headand Neck Tumours Lyon Barness p “ Zhang B Li L Li Z Liu Y Zhang H Wang J Carbon ionirradiated hepatomacells exhibit coupling interplay between apoptotic signaling andmorphological and mechanical remodeling Sci Rep Yan JF Huang GY A doublehertz model for adhesive contact betweencylinders under inclined forces Philos Trans A Math Phys Eng Sci Kowalsky CA Faber MS Nath A Dann HE Kelly VW Liu L Rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing J Biol Chem “ Zeng P Chen MB Zhou LN Tang M Liu CY Lu PH Impact of TROP2expression on prognosis in solid tumors a systematic review and metaanalysis Sci Rep Calvo A Xiao N Kang J Best CJ Leiva I EmmertBuck MR Alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinP inmouse and human tumors Cancer Res “Ju X Jiao X Ertel A Casimiro MC Di Sante G Deng S VSrc oncogeneinduces Trop2 proteolytic activation via Cyclin D1 Cancer Res “ Cubas R Li M Chen C Yao Q Trop2 a possible therapeutic target for latestage epithelial carcinomas Biochim Biophys Acta “ Zargari N Mokhtari M Evaluation of diagnostic utility ofimmunohistochemistry markers of TROP2 and HBME1 in the diagnosis ofthyroid carcinoma Eur Thyroid J “ Zhao P Zhang Z TNFα promotes colon cancer cell migration and invasionby upregulating TROP2 Oncol Lett “Sin STK Li Y Liu M Yuan YF Ma S Guan XY Downregulation of TROP2predicts poor prognosis of hepatocellular carcinoma patients HepatolCommun “ Zhang Y Zhang R Luo G Ai K Long noncoding RNA SNHG1 promotes cellproliferation through PI3KAKT signaling pathway in pancreatic ductaladenocarcinoma J Cancer “Sai J Owens P Novitskiy SV Hawkins OE Vilgelm AE Yang J PI3Kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antiPD1 responses Clin Cancer Res “ Chen X Pang B Liang Y Xu SC Xin T Fan HT Overexpression of Zhang XR Wang SY Sun W Wei C Isoliquiritigenin inhibits proliferation andEpCAM and Trop2 in pituitary adenomas Int J Clin Exp Pathol “metastasis of MKN28 gastric cancer cells by suppressing the PI3KAKTmTOR signaling pathway Mol Med Rep “ 0cZhang BMC Cancer Page of Wise HM Hermida MA Leslie NR Prostate cancer PI3K PTEN and prognosisClin Sci Lond “ Yuan B Zou M Zhao Y Zhang K Sun Y Peng X Upregulation of miR130b3p activates the PTENPI3KAKTNFκB pathway to defense againstmycoplasma gallisepticum HS Strain infection of chicken Int J Mol Sci pii E2172Li JW Wang XY Zhang X Gao L Wang LF Yin XH Epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of thePTENPI3KAKT pathway Mol Med Rep “Li X Teng S Zhang Y Zhang W Zhang X Xu K TROP2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating PI3KAKT pathway and inducing EMT Oncotarget “ Gu QZ Nijiati A Gao X Tao KL Li CD Fan XP TROP2 promotes cellproliferation and migration in osteosarcoma through PI3KAKT signalingMol Med Rep “ Hou J Lv A Deng Q Zhang G Hu X Cui H TROP2 promotes theproliferation and metastasis of glioblastoma cells by activating the JAK2STAT3 signaling pathway Oncol Rep “ Rivard N Phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function Front Biosci Landmark Ed “ Pankova D Jiang Y Chatzifrangkeskou M Vendrell I Buzzelli J Ryan A et alRASSF1A controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma EMBO J 20193813e100532 Wullkopf L West AV Leijnse N Cox TR Madsen CD Oddershede LB et alCancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential Mol Biol Cell “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12˜¯ Yuwei Duan34˜¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina˜¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [“] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the œsignature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patient™s ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time “ ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 “ Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers™ informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ˚C for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ˚C for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturer™s instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ˚C min at ˚C and cycles of s at ˚C min at ˚C using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ΔΔCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ˚C overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ˚C After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables “Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ˚CAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the body™s own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells™ insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter [“]PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [“] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [“] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patient™s cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting “ original draft Hongying Kuang Yuwei Duan Wenxia AiWriting “ review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] “LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] “ Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun “ 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes

Thyroid_Cancer

thyroid cancer diagnosis by Raman spectroscopyMarco Sbroscia15 Michael Di Gioacchino1 Paolo Ascenzi1 Pierfilippo Crucitti2 Alessandra di Masi1 Isabella Giovannoni3 Filippo Longo2 Davide Mariotti1 Anda Mihaela Naciu4 Andrea Palermo4 Chiara Taffon3 Martina Verri3 Armida Sodo1 Anna Crescenzi3 Maria Antonietta Ricci1Over the last years the incidence of human thyroid cancer disease has seen a significative increment This comes along with an even higher increment of surgery since according to the international guidelines patients are sometimes addressed to surgery also when the fine needle aspiration gives undetermined cytological diagnosis As a matter of fact only of the thyroid glands removed for diagnostic purpose have a post surgical histological report of malignancy this implies that about of the patients have suffered an unnecessary thyroid removal Here we show that Raman spectroscopy investigation of thyroid tissues provides reliable cancer diagnosis Healthy tissues are consistently distinguished from cancerous ones with an accuracy of ˆ¼ and the three cancer typology with highest incidence are clearly identified More importantly Raman investigation has evidenced alterations suggesting an early stage of transition of adenoma tissues into cancerous ones These results suggest that Raman spectroscopy may overcome the limits of current diagnostic toolsDetection of nodular thyroid pathology is increasing worldwide paired with an increased number of thyroid cancer diagnosis1 Whether this œepidemic spread is real or is due to an exaggerated use of imaging tools in clinical practice is object of a vivid debate within the scientific community Anyway management of patients with thyroid nodule causes a clinical problem and a social burden as well2 To avoid overdiagnosis and overtreatment of these patients physicians need additional information about the biology of each thyroid nodule In particular there is need for a tool able to distinguish between benign and malignant follicular lesions and between indolent and aggressive tumors3 The genomic profile of thyroid tumors has been extensively investigated4 and several immunocytochemical and molecular markers have been proposed for a more accurate classification of thyroid nodules5 However none of these markers reaches a reliable sensibility and specificity to be routinely used in clinical setting6 Moreover although the major genetic alterations of thyroid tumors are known only little information if any is available about changes in protein lipids and other cellular constituents due to thyroid oncogenesisTo correctly manage patients with thyroid nodules and to avoid unnecessary surgery we strongly need a method of analysis that overcomes the limits of current diagnostic tools by identifying specific tumoral markers possibly with high spatial resolution and low impact on the patient Cells and tissues are characterized by a specific biochemical composition Each pathology or cellular abnormality is accompanied by biochemical molecular changes Optical and spectroscopic techniques that correlate the biochemical composition molecular structure and their variations with the morphological alterations represent a powerful diagnostic and potentially clinical tool8Among the experimental techniques available for chemicalphysical studies of matter at the molecular level Raman spectroscopy RS is a good candidate for our needs This technique offers a wealth of molecular information while being not invasive and achieving spatial resolutions as high as a few μm in microRaman apparatuses RS probes the vibrational dynamics of the sample through a fast and not destructive analysis It does not require any sample preparation and can consequently be applied in a0vivo For these characteristics it has been recently applied on a variety of biological and medical samples to identify specific metabolic states of cells tissues and 1Dipartimento di Scienze Universit¡ degli Studi Roma Tre Rome Italy 2Unit of Thoracic Surgery Campus BioMedico University Rome Italy 3Pathology Unit Campus Biomedico University Hospital Rome Italy 4Unit of Endocrinology and Diabetes Campus BioMedico University Rome Italy 5Present address Dipartimento di Fisica Sapienza Universit¡ di Roma Rome Italy email armidasodouniroma3itScientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Raman spectra A Typical Raman spectra of the examined thyroid tissues labelled according to the histology report Stars label the Raman characteristic peaks of cytochrome c triangles those of carotenoids The black vertical dashed lines allow an easier comparison among the spectra B Centroids Raman spectra for the four identified clusters by Kmeans analysisbacteria and in particular to differentiate between healthy and cancer tissues9“ Preliminary studies on thyroid tissues by RS suggest that this technique may have high impact in thyroid cancer diagnosis13“In a RS experiment a laser beam is focalized on the sample and the scattered light is analyzed in frequency Typical spectra after background subtraction are shown in Fig a01A These are characterized by peaks and bands with frequency centered at the vibrational frequency of the intramolecular bonds characteristic of the main sample componentsWe have applied RS augmented by cluster analysis to investigate histological samples from human thyroid Assignement of the RS peaks to carotenoids or cytochrome c in different amounts has allowed to distinguish healthy tissues from cancerous ones and to discriminate for the first time among the three categories of carcinoma with larger incidence namely Papillary carcinoma PTC follicular variant of Papillary carcinoma FVPTC and Follicular carcinoma FC We have also analyzed samples labelled after histology as follicular adenoma FA We found that these samples are spread over four categories reported above by the cluster analysis Subsequent more specific analyses of protein expression have confirmed an anomalous level of tumoral or benign molecular markers in the adenoma samples labeled in the non healthybenign categories by RS All the samples analyzed by RS are reported in a table as Supplementary informationResultsFigure a01A shows from bottom to top the Raman fingerprint region ˆ’ cmˆ’ of four samples with a clear histology diagnosis namely HealthyBenign average of the spectra of samples Hea49 and Hea64 PTC average of the spectra of samples TIR51 and TIR58 FVPTC sample TIR56 and FC average of the spectra of samples TIR47 and TIR55 carcinoma tissues Each spectrum has been normalized to its integrated intensity We notice that each tissue has its own characteristic spectral lineshape According to Rau et a0al15 and references therein this spectral range shows the bands ascribed to proteins lipids and aminoacids Here we will not report a detailed assignment of each band to a particular biomolecule as this is sometimes tricky and not relevant to our aim We will instead put the attention on the distinctive set of peaks which characterize each histological sample We notice that the spectrum of the healthy tissues is dominated by an intense broad and structured band centered around ˆ¼ cmˆ’ possibly due to the presence in the sample of amide I and lipids19 The sharpest structure of this band falls at cmˆ’ and along with the bands at · and cmˆ’ Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Agglomerative hierarchical clustering analysis Dendrogram of the Raman spectra of human thyroid tissues as extracted from the AHCA analysis Individual samples are represented by the label Hea for healthy or TIR for not healthy followed by the patient anonymous ID code Those plotted in Fig a01A are labelled healthy FC FVPTC and PTC respectively Dashed squares identify the four clusters namely healthybenign orange Follicular carcinoma or FC black follicular variant of papillary carcinoma or FVPTC magenta and papillary carcinoma or PTC blue The arrows indicate the adenoma samples All samples within the light blue shaded area are carcinomasrepresets the fingerprint of cytochrome c20 This is a protein localized in the mitochondrial intermembrane space under normal physiological conditions The spectrum of a classical papillary carcinoma labeled PTC in Fig a01A is totally different and unambigously shows the presence of carotenoids bands at and cmˆ’ not present in healthy tissues as already reported in the literature10 In the follicular carcinoma spectrum labeled FC in Fig a01A we observe a marked enhancement of the cytochrome c bands As a matter of fact the broad band at ˆ¼ cmˆ’ that dominates the HealthyBenign spectra is barely visible in this case after spectral intensity normalization Interestingly the spectrum of the Papillary Follicular carcinoma labeled FVPTC in Fig a01A looks like a superposition of the PTC and FC spectra showing the fingerprints of carotenoids along with a clear enhancement of the bands ascribed to cytochrome c with respect to the spectra of HealthyBenign tissuesThe presence of carotenoids in both PTC and FVPTC carcinoma is peculiar and worthy of interest The cellular process of carotenoids uptake not directly synthetized by human anisms and their proper identifications is under biochemical investigationAs Raman spectroscopy is able to detect the differences between healthy and cancerous tissues it may be considered as a promising tool for in a0situ cancer diagnosis We have analyzed a total of histological samples of human thyroid tissues samples diagnosed as adenomas including one inadequate TIR59 for RS and not further considered for statistical analysis healthy ones and carcinomas ones namely PTC FVPTC FC and Hyalininzing Trabecular Tumor HTT All samples are labelled as TIR or Hea followed by the patient anonymous ID codeWithin each histology class spectra may differ for noise background or fluorescence contributions although keeping unaltered the characteristic fingerprints commented above This observation suggests that the experimental spectra can be analyzed on quantitative grounds by cluster analysis after backgrounds and fluorescence subtraction This aims at classifying each Raman spectrum into homogeneous groups based on its characteristic features in other words on the presence and relative intensity of specific bands The analysis has been performed following two distinct approaches see œMethodsThe first approach called agglomerative hierarchical clustering analysis AHCA builds a dendrogram of the entire data set by iteratively selecting pairs of the closest spectra or groups of spectra as a function of their Euclidean distance The results of this analysis are reported in Fig a0 where each sample is represented by means of the label Hea or TIR depending on whether the tissue has received a healthy or not healthy histological diagnosis followed by a progressive number This figure tells us that our spectra are consistent with a classification of the dendrogram into clusters assuming a distance threshold of compared to a maximum distance of The four clusters have been labelled according to the diagnosis of the four spectra shown in Fig a01A a priori selected as the most representative of each histology report namely the less noisy with a clear and confirmed diagnosis HealthyBenign and PTC tissues are very well distinct each other and have the smallest dispersion in terms of Euclidean distance within each cluster Indeed the smaller is the dispersion of the Euclideian distances the better is the homogeneity of the group in the present case HealthyBenign and PTC tissues are the most homogeneous The other two clusters FC and FVPTC are more dispersed although equally distinguishable Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Kmeans analysis Samples distribution within the four KM clusters as a function of their Euclidean distance from the associated centroid The same labels and colours as in Fig a0 have been used Black triangles refer to adenomasCluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster Table Euclidean distance between the cluster centersTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples healthy and carcinomas tissues This shows that RS has identified False negative diagnosis among the negative Medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisfrom the others The FC cluster is the closest to the HealthyBenign one as both are characterized by the presence of cytochrome c and distinguished only on the basis of its Raman fingerprint relative intensity The FVPTC tissues fall between FC and PTC clusters as predictable from copresence of carotenoids and citochrome c peaks in their Raman spectraThe second unsupervised statistical approach considered is called Kmeans KM its results are reported in Fig a0 This confirms that four clusters properly describe our data set Clusters contain samples from the histology reports of PTC HealthyBenign FVPTC and FC respectively The agreement between the clustering results of the two approaches is very good as we notice only one difference concerning sample TIR60 which switches from a Raman diagnosis of PTC to that of FVPTC Fig a0 shows that healthybenign tissues spectra have on the average the shortest distance from their centroid and the smallest dipersion On the contrary the spectra of FVPTC tissues are the most dispersed possibly due to a different balance of concentration between carotenoids and cytochrome c in each sample Table a0 reports the distance matrix among the clusters centroids Cluster showing the largest centroid distance from the others is readily isolated its Raman spectrum is dominated by the carotenoid fingerprint while discrimination among the others requires consideration of more spectral detailsKM analysis returns also the Raman spectrum calculated for each cluster centroid these are reported in Fig a01B for direct comparison with the prototypical spectra of the four classes of tissues The similarity of the spectra reported in Fig a01AB is astonishing and proves that our clustering is solidBased on these results we have computed a confusion matrix providing the accuracy of our analysis in discriminating between healthybenign and pathological tissues regardless of the specific pathology As reported in Table a0 our accuracy is of ˆ¼ Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomasThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisFigure a0 Immunohistochemistry images Microscopic pictures of A TIR43 sample stained with immunohistochemistry for Galectin3 high power field Hematoxylin counterstained and B TIR64 sample stained with immunohistochemistry for HBME1 medium power field Hematoxylin counterstained Panel A shows a mosaic pattern with negative cells intermingled with cells showing positive reaction in cytoplasm as well as in nuclear matrix Panel B shows strong positive reaction due to a microfocus of papillary carcinoma within the follicular adenomaTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomas after immunohistochemistry revisionThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and that the number of false positive diagnosis goes down to Finally we have applied the same analysis including the spectra of the samples classified as adenoma by the histological diagnosis Their position is highlighted by arrows in the AHCA dendrogram of Fig a0 and in the distributions of Fig a0 These figures show that the spectra of adenoma tissues spread over all four clusters defined by healthy and cancerous tissues This is a quite unexpected result as adenomas are classified as benign tissues and therefore should fall into the healthybenign cluster Accordingly the confusion matrix after inclusion of adenoma samples reported in Table a0 returns an accuracy of the statistical analysis of After the RS analysis four adenoma samples TIR43 TIR44 TIR64 TIR71 falling within the cancerous clusters have been in depth reanalyzed as far as their expression of benignity and cancerous markers are concerned by immunohistochemistry test The lowering of benignity markers CD56 or sometimes the onset of tumoral markers Galectin3 HBME1 are revealed in all these cases The immunohistochemistry images of TIR43 and TIR64 samples are shown in Fig a0 as representative of the all four cases These results could be symptomatic of early stages of progression of adenomas into a specific type of carcinoma Such type of transformation has never been demonstrated and our Raman observation s a new light about the malignant potential of thyroid FA and about the possibility to assess which of these lesions may constitute a clinical concern Interestingly the confusion matrix after this revision gives an accuracy of ˆ¼ A few anomalies visible in Figs a0 and a0 deserve a comment Sample Hea56 and sample Hea63 apparently fall into a wrong cluster namely the FVPTC cluster but interestingly very close to their TIR counterpart FVPTC and TIR63respectively This may happen when the Raman spectrum of the healthy portion is collected from a tissue region too close to the pathologic one where normal and altered cells are almost intermingled giving the superposition of healthy and cancerous Raman spectra In particular the case of sample Hea72 falling within the FVPTC cluster may be due to the diffusion of carotenoids in normal parenchyma at the border Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Biochemical analysis Expression levels of cytochrome c have been assessed in thyroids of five patients for each patient the healthy Hea and pathological TIR slices have been analysed A Levels of cytochrome c have been normalized to actin Data represent the mean values ± SDs derived from three replicates normalized to healthy counterparts Student™s t test P a0 a0 P a0 a0 compared with control B Exemplificative images of filters blotted with cytochrome c and actin primary antibodies Images have been gathered at the same timeof the PTC nodule as already observed in previous Raman chemigram map of thyroid carcinomas15 This gives in the Raman spectrum the superposition of the fingerprint of carotenoids with fingerprint of healthy tissues containing cytochrome c thus explaining the œwrong assignment obtained by Raman analysisAdditionally levels of cytochrome c have been further evaluated by biochemical analysis Fig a0 on a few cases Indeed only for patients and PTC and and FC a sufficient amount of thyroid tissue was available to perform immunoblot analyses As shown in Fig a0 cytochrome c levels significantly increase in TIR45 134fold induction compared to its healthy counterpart Hea45 P a0 a0 TIR46 27fold induction compared to Hea46 P a0 a0 and TIR47 456fold induction compared to Hea47 P a0 a0 In agreement with Raman results cytochrome c levels are similar in TIR and Hea slices of patients and In conclusion this study demonstrates the capability of Raman spectroscopy as diagnostic tool for thyroid cancer Indeed through a multivariate statistical analysis of the spectra it is possible to readily separate healthy from cancerous tissues and in the majority of the cases to discriminate among the most common cancer typologies see Supplementary information for details on the individual cases Last but not least our findings highlight the possible usefulness of Raman spectroscopy as a tool for in a0situ and early diagnosis of thyroid pathologyScientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cMethodsStudy enrollment From January to January we enrolled consecutive subjects females and males with thyroid nodular pathology referred to the Endocrinology Unit of Campus BioMedico University Hospital Their age distribution is centered at a0years with a standard deviation of a0years All the study population had received a cytological diagnosis of indeterminate suspicious or malignant lesion with a formal indication to surgery total thyroidectomy according to the international guidelines24 After signing the informed consent these patients underwent total thyroidectomy at the Surgical Unit of the same InstitutionNeck ultrasound and clinical evaluation All subjects were submitted to thyroid US evaluation using a frequency range of “ MHz on a MyLab Esaote Genova Italy US scan of thyroid gland and neck area were performed by experienced endocrinologists at the Endocrinology Unit Nodules were classified according to ACR TIRADS risk stratification criteria25 without prior knowledge of the cytological results When there was a disagreement the endocrinologists conducted a separate session to reach an unified consensus We collected demographic data including age gender family history of thyroid cancer and using the medical records we evaluated the thyroid function and the presence of autoimmune thyroiditisThyroid tissues preparation At the time of surgery the removed specimens were immediately submitted unfixed to the Pathology Unit in an appropriately labelled container The pathologist valued the gross anatomy of the samples and a tissue slice of about cm wide — cm length — mm of thickness was cut including both healthy and neoplastic areas avoiding surgical margins The slice was snap frozen on a metallic coldplate inside a cryostat A μm section was stained with haematoxylineosin in order to confirm the presence of healthy and neoplastic tissues After this step four consecutive cryostatic sections were cut at μm of thickness collected on separate slides and stored at ˆ’ —¦C until the Raman evaluation Our Raman study was exclusively performed on these frozen unfixed samples The residual slices were defrosted formalin fixed and paraffin embedded with the paired surgical samples for definitive histology Final diagnosis was reported in agreement with current edition of WHO classification of endocrine tumours26 Immunohistochemical analysis for Galectin3 Gene Tex CD56 Agilent and HBME1 Agilent was performed in each case on paraffin sections from neoplastic areas using an automatized immunostainer Omnis AgilentNodules with diameter wider than cm were submitted for biochemical analysis In such cases at the time of gross dissection further slices of about — a0cm were cut from the surgical specimen sampling both healthy and neoplastic tissue Slices were snap frozen and stored at ˆ’ —¦C until biochemical evaluationRaman spectra collection Raman spectra have been collected by means of a Renishaw InVia MicroRaman spectrometer In this setup unpolarised spectra are collected through a Leica DM2700 M confocal microscope equipped with a Leica — LWD and an Olympus — objective The required highcontrast rejection for the elastically scattered light is provided by an holographic edge filter A diffraction grating with a0groovesmm disperses the Raman inelastic scattering contribution providing a spectral resolution of about cmˆ’ A Peltier cooled — pixel CCD detector collects the dispersed light A solidstate diode laser source at nm with a nominal output power of nearly mW has been used as excitation source To prevent photodamage neutral density filters have been used for lowering the laser power at the sample The spot size has been shrink down to few microns when necessary to isolate the contribution of cells about ten microns in size from the surrounding cytoplasm thus reducing spectral interference Spectra have been collected in the extended scan mode covering the “ cmˆ’ frequency shift range and accumulating scans with an equivalent integration time of nearly s per scan Measurements have been collected on points per sample Wire Renishaw software has been used to collect the raw spectra Wire and LabSpec software has been used to perform the preliminary data reduction eg background and fluorescence subtraction while Origin OriginLab Corporation software has been employed to perform the statistical analysis After background and fluorescence subtraction all the spectra have been normalised to their integral in order to avoid any bias due to fluctuations of the experimental conditionsCluster analysis of Raman spectra In order to gain better confidence on the clustering analysis we have selected two unsupervised multivariate statistical approaches to analyze the Raman spectra the agglomerative hierarchical clustering analysis AHCA and the Kmeans one KM Both methods are based on the Euclidean distance among spectra as a measure of their similaritydissimilarity In detail in these analyses spectra are represented by their distance with respect to an origin thus generating a dissimilarity matrix Among the possible choices for the spectral distance we have chosen the Euclidean one since it maximizes the similarities among the spectra The choice to use these two methods was made to exploit their complementarity of hierarchical and nonhierarchical approaches and consequently to give solidity to the obtained results13 “The AHCA method aims at classifying observations each Raman spectrum into homogeneous groups the clusters based on the measured characteristics Euclidean distance associated to each Raman spectrum In particular we have used the completelinkage algorithm in order to enhance differences among clusters It works by pairing the most similar spectra those showing the lowest dissimilarity value in the matrix and then looking for the largest distance between such pair and the rest of the data By iteratively repeating such an approach a classification of spectra into wellseparated groups is obtained and visualised as a dendrogramThe KM method minimizes the variance within each cluster with respect to an a priori number of centroids randomly distributed This protocol starts by associating each data entry Euclidean distance associated to each Raman spectra to the cluster with the closest centroid The centroids are iteratively updated towards convergence The number of clusters k is an input of the routine In order to choose the proper value of k the most Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Spread of the individual Kmeans clusters as a function of their number Data are reported by using the same colors as in Fig a0 for the individual clusters Notice that four clusters give the largest spread among the clustersused strategy is the œelbow curve based on the explained variance within the entire dataset This analysis in our case gives only two centroids one for samples containing mainly carotenoids and one for all other cases Thus following the suggestion of the histology diagnosis we have set k a0 a0 In order to verify the reliability of this choice we have calculated the spread of each cluster with respect to its centroid as a function of k Intestingly we find that k a0 a0 is the highest kvalue with the lowest spread as shown in Fig a0We have tested the predictive accuracy of both AHCA and KM methods by generating a confusion matrix This reports the comparison between the medical and spectroscopic diagnosis The accuracy is defined asAccuracy True negative True positivenumber of total samplesBiochemical analysis Frozen thyroids have been weighted homogenized and sonicated in lysis buffer mM TrisHCl pH mM NaCl mM EDTA glycerol vv Triton X100 vv and protease inhibitors Twentyfive micrograms of protein extracts previously quantified with the Bradford assay BioRad Hercules CA have been resolved by SDSPAGE and transferred onto PVDF membranes BioRad After blocking with BSA wv dissolved in TBS buffer Tween20 vv membranes have been probed overnight at —¦C with anticytochrome c mouse mAb sc13560 Santa Cruz Biotechnology Dallas TX USA and antiactin mouse mAb sc47778 Santa Cruz Biotechnology primary antibodies Membranes have been then incubated for h at room temperature with antimouse HRPconjugated secondary antibody BioRad All the experiments have been repeated trice Blots have been acquired and processed using the ChemiDoc Imaging system BioRad Cytochrome c quantification has been performed using the Image Lab software version BioRad Laboratories Results are shown as mean ± standard deviation SD the statistical significance of differences has been tested using the Student™s t test GraphPad Prism version ethics statement The study was approved by the Ethical Committee of the University of Rome œCampus Biomedico UCBM prot TS ComEt CBM and prot PAR ComEt CBM The informed consent was collected from patients before surgery Enrolled patients were recorded in a codified file with an anonymous ID code which was registered in the software database of the Pathology Unit of the UCBM All experiments were performed in full accordance with the principle of Good Clinical Practice GCP and the ethical principles contained in the current version of the Declaration of HelsinkiReceived March Accepted July References Vaccarella S et al Worldwide thyroidcancer epidemic The increasing impact of overdiagnosis N Engl J Med “ Udelsman R Zhang Y The epidemic of thyroid cancer in the United States the role of endocrinologists and ultrasounds Thyroid Rusinek D et al Current advances in thyroid cancer management Are we ready for the epidemic rise of diagnoses Int J Mol 101056NEJMp “ 101089thy20130257 Sci Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0c TCGAR Network Integrated genomic characterization of papillary thyroid carcinoma Cell “ 101016jcell201409050 Cantara S et al Molecular signature of indeterminate thyroid lesions current methods to improve fine needle aspiration cytology fnac diagnosis Int J Mol Sci 103390ijms1 Haugen B R et al American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer Thyroid “ 101089thy20150020 Paschke R et al European thyroid association guidelines regarding thyroid nodule molecular fineneedle aspiration cytology diagnostics Eur Thyroid J “ 10115900046 Krafft C Popp J Raman4clinics the prospects of Ramanbased methods for clinical application Anal Bioanal Chem Kallaway C et al Advances in the clinical application of Raman spectroscopy for cancer diagnostics Photodiagn Photodyn Ther “ 101007s0021 “ 101016jpdpdt Li Z et al Surfaceenhanced Raman spectroscopy for differentiation between benign and malignant thyroid tissues Laser Phys Lett 1010881612201111404560 Krafft C Popp J The many facets of Raman spectroscopy for biomedical analysis Anal Bioanal Chem “ 101007s0021 Palermo A et al Raman spectroscopy applied to parathyroid tissues a new diagnostic tool to discriminate normal tissue from adenoma Anal Chem “ 101021acsanalc hem7b036 Harris A T et al Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines Teixeira C S B et al Thyroid tissue analysis through Raman spectroscopy Analyst “ 101039B8225 Rau J V et al Raman spectroscopy imaging improves the diagnosis of papillary thyroid carcinoma Sci Rep “ Head Neck Oncol 78H 101038srep3 Rau J V et al Proofofconcept Raman spectroscopy study aimed to differentiate thyroid follicular patterned lesions Sci Rep “ 101038s4159 MedeirosNeto L et al In a0vivo Raman spect

Thyroid_Cancer

"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI “ In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI “ Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI “Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [“] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast 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 0cWang BMC Cancer Page of [“]cancer However the relationship between the two diseases remains controversial [“] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorœhypothyroidism or œHT and œthyroid diseases orœbreast cancer or œBC or œbreast neoplasms or œmammarmy cancer and œrisk orœincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionœthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first author™s surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ‰¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ‰¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ “ “] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI “P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI “ P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI “In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI “109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ‰¥ ± ‰¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ‰¤ “ “ “ “ “ “ “ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092“ P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanilli™s research may causeheterogeneity [] Cristofanilli™s research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanilli™s researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies™ followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors™ contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer 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in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol “ httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol “ httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat “ httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol “ httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg “ httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA “ Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis“ httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol “ httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent “Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab “ httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer “ httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent “Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110“ httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer “ httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol “ httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat “ httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res“ Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401“Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer “ httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet “Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol “ httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol ““httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol “httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol “ httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog “ httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine levels are positively associatedwith breast cancer risk in postmenopausal women Breast Cancer Res 123R33 httpsdoi101186bcr2587Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

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

Thyroid_Cancer

"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

Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the ‚uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these findings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insufficient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social financialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreflect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Specifically procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and specificity[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical efficacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the ‚uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBGž level ‰¥ mmolling mgdl or h blood glucose ‰¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ‰¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushing™s syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may ‚uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coefficients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Student™s ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe Mann“Whitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ””””””” ± ± ± ± ” ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r ˆ’P There was a significant negative correlationbetween OC or CTX and TG rs ˆ’ P rs ˆ’ P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs ˆ’ P OC rs ˆ’ P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Thesefindings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH ˆ’ P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC ˆ’ P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars “P lmgp HTPlmgn XTC𝛽r “P HbA1c brs “P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearson™s correlation coefficient rs Spearman™s correlation coefficient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coefficients Standardized coefficients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the ‚uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [“] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ‰¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These findings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ‚ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ‚ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ‚ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ‚ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen fibres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and confirmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliflozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliflozin dapagliflozin and empagliflozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the ‚uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an ‚uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third the‚uence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespecific alkalinephosphatase BAP vitamin D or steroids all of which ‚uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its specificity for bone metabolism is lacking [] BAPis a more bonespecific marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high specificity in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreflects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These findings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DM‚uencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneficial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientific Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang œIDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and  Diabetes Research and Clinical Practicevol pp “ [] A V Schwartz D E Sellmeyer K E Ensrud œOlderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp “ [] L Forsen H E Meyer K Midthjell and T H Edna œDiabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp “ [] Y Fan F Wei Y Lang and Y Liu œDiabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp “ [] M Janghorbani R M Van Dam W C Willett and F B HuœSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp “ [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides œVitamin D and glycemic control in diabetes mellitus type  Therapeutic Advances in Endocrinologyand Metabolism vol no pp “ [] H Miyake I Kanazawa and T Sugimoto œAssociation ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalcified Tissue International vol no pp “ [] E S Siris R Adler J Bilezikian œThe clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp “ [] M B Greenblatt J N Tsai and M N Wein œBone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp “ [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell œMarkers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp “ [] H W S Cabral B F G Andolphi B V C Ferreira œTheuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp “ [] P Iglesias F Arrieta M Pi±era œSerum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp “ [] J M Wishart A O Need M Horowitz H A Morris  andB E C Nordin œEï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp “ [] P Szulc P Garnero F Munoz F Marchand and P D Delmas œCrosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp “ [] K G M M Alberti P Z Zimmet and WHO ConsultationœDefinition diagnosis and classification of diabetes mellitusand its complications Part diagnosis and classification ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp “ [] J StarupLinde and P Vestergaard œBiochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp “ [] L Achemlal S Tellal F Rkiouak œBone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp “ [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin œAssociation of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp “ [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira œMetabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp “ [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco œBone turnover markers in men andwomen with impaired fasting glucose and diabetes CalcifiedTissue International vol no pp “ [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann œLower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp “ in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu œSignificant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp “ [] A Tan Y Gao X Yang œLow serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp “ [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang œThe relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp “ [] Y Chen Q Zhao G Du and Y Xu œAssociation betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

Breast cancer BC is the most common malignancy among women Emerging studies have demonstrated that circular RNA circRNA zinc finger RNA binding protein circZFR serves as a crucial regulator in many human cancers However the role and mechanism of circZFR in BC tumorigenesis remain unclearMethods The levels of circZFR miR578 and hypoxiainducible factor 1α HIF1A were detected by quantitative realtime polymerase chain reaction qRTPCR or western blot Cell viability colony formation apoptosis migration and invasion capacities in vitro were determined by using the Cell Counting Kit8 CCK8 standard colony formation flow cytometry and transwell assays respectively Glucose uptake lactate product and adenosine triphosphate ATP levels of cells in vitro were measured using the commercial human assay kits Targeted relationships among circZFR miR and HIF1A in BC cell lines were verified by dualluciferase reporter and RNA pulldown assays Animal studies were performed to assess the effect of circZFR on tumor growth in vivoResults Our data indicated that circZFR was overexpressed in BC tissues and cells and the increased circZFR level predicted poor prognosis of BC patients CircZFR silencing or miR578 overexpression repressed BC cell viability colony formation migration invasion and glycolysis and enhanced cell apoptosis in vitro CircZFR silencing also hampered tumor growth in vivo Mechanistically circZFR acted as a sponge of miR578 and circZFR silencing hindered BC cell malignant behaviors by miR578 HIF1A was a functional target of miR578 in regulating BC cell viability colony formation migration invasion glycolysis and apoptosis in vitro Furthermore circZFR modulated HIF1A expression through sponging miR578Conclusion Our findings first identified that the silencing of circZFR suppressed BC malignant progression in vitro via the regulation of the miR578HIF1A axis providing evidence for the crucial involvement of circZFR in BC pathogenesisKeywords Breast cancer BC circZFR miR578 HIF1A Malignant progressionCorrespondence qiuxinguang2020163com Department of Thyroid Surgery The First Affiliated Hospital of Zhengzhou University No1 Jianshe East Road Erqi District Zhengzhou Henan ChinaFull list of author information is available at the end of the BackgroundBreast cancer BC remains the most commonly diagnosed cancer and the leading cause of cancerassociated death among females in [] Although the therapeutic methods have greatly improved over the past two decades effective treatment against metastatic BC is still limited [ ] Therefore a deeper understanding of what drives this disease is the first step to design innovative interventions 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 0cChen a0et a0al Cancer Cell Int Page of Circular RNAs circRNAs are covalently closed endogenous RNAs that have crucial noncoding functions in human physiologic and pathologic processes [] Work in biological functions has demonstrated the roles of circRNAs as microRNA miRNA sponges [] Dysregulation of circRNAs has recently implicated in the pathogenesis of BC [] For example Yuan et a0al uncovered that hsa_circ_0068033 a downregulated circRNA exerted a repressive impact on BC malignant progression via sequestering miR659 [] Cao and colleagues demonstrated that hsa_circ_0087784 functioned as a potential promoter in BC development through sponging miR487a [] Xu et a0al reported that circTADA2As attenuated BC progression and metastasis by the regulation of miR203a3p [] Moreover hsa_circ_001783 and circABCB10 were reported as oncogenic regulators in BC through functioning as specific miRNA sponges [ ] As for circRNA zinc finger RNA binding protein circZFR hsa_circ_0072088 it has been identified as an oncogenic modulator in many human cancers such as renal carcinoma bladder cancer and nonsmall cell lung cancer [“] Nevertheless the biological roles of circZFR in BC tumorigenesis remain largely unknownMiRNAs modulate gene expression but are frequently dysregulated in human cancers including BC [] Danza et a0al reported that miR578 was underexpressed in BRCABC and it regulated tumor angiogenesis [] However the precise function of miR578 in BC progression is still undefinedHere we undertook to examine the biological effect of circZFR in the malignant progression of BC in a0 vitro and in a0 vivo We identified that circZFR a prominently upregulated circRNA in BC controlled BC progression in a0vitro via targeting the miR578hypoxiainducible factor 1α HIF1A axisMaterials and a0methodsClinical tissues and a0cellsIn this study we enrolled BC patients admitted to The First Affiliated Hospital of Zhengzhou University from April to June The clinicopathological features of these patients were provided in Table a0 Seventy pairs of primary tumor tissues and matched healthy breast tissues were collected and stored at ˆ’ a0°C to detect the expression levels of circZFR miR578 and HIF1A Accession NM_1810543 These patients were followedup for at least a0 months and the followup information was obtained by telephone calls every a0 months The study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University and written informed consent was provided by all participantsTable Correlation and a0the a0clinicopathological features of a0BC patientsbetween a0circZFR expression CharacteristicsNumbercircZFR expressionPHighLowAge years ‰¥ Distant metastasis Present AbsentTumor size cm ‰ TNM stage I II III IVHER2 status Positive NegativePR status Positive NegativeER status Positive NegativeHER2 human epidermal growth factor receptor2 PR progesterone receptor ER estrogen receptorP P BC cell lines MCF7 ATCC ®HTB22 BT549 ATCC ®HTB122 MDAMB231 ATCC ®HTB26 and MDAMB453 ATCC ®HTB131 American Type Collection Culture ATCC Manassas VA USA were cultured in RPMI1640 medium supplemented with fetal calf serum FCS and antibiotic solution all from cell line ATCC ®CRL10317 ATCC was maintained in HyClone Logan UT USA The immortalized MCF10A Dulbecco™s modified Eagle™s mediumNutrient Mixture F12 DMEMF12 with FCS a0ngmL epidermal growth factor a0 μgmL hydrocortisone and a0 μgmL insulin all from HyClone All cells were cultured in a CO2 incubator at a0°CQuantitative real‘time polymerase chain reaction qRT‘PCRThe expression levels of circZFR HIF1A and miRNAs were gauged by qRTPCR Complementary DNA cDNA synthesis was done using total RNA a0 ng 0cChen a0et a0al Cancer Cell Int Page of isolated by Isogen Nippon Gene Tokyo Japan from tissues and cell lines The levels of circZFR and HIF1A were quantified using the TaqMan Gene Expression Assays with the indicated primers and mature miRNAs were assayed using the TaqMan MicroRNA Assays with TaqManspecific primer probes as recommended by the manufacturers Applied Biosystems Rotkreuz Switzerland qRTPCR was run in triplicate on the iCycler iQ5 device BioRad Munich Germany using the PCR conditions as previously reported [] Results were normalized to the expression of glyceraldehyde3phosphate dehydrogenase GAPDH or U6 internal control using the ˆ’ΔΔCt method [] Primer sequences for circZFR forward ²ATG GTC TGC AGT CCT GTG TG3² were and reverse ²TGG TGG CAT GTT TTG TCA TT3² for HIF1A were forward ²TTC CCG ACT AGG CCC ATT C3² and reverse ²CAG GTA TTC AAG GTC CCA TTTCA3² for miR578 were forward ²GTG CAG GGT GTT AGGA3² and reverse ²GAA GAA CAC GTC TGGT3² for miR944 were forward ²GAG TAG GCT ACA TGT TAT TAAA3² and reverse ²GTG CAG GGT CCG AGGT3² for miR5323p were forward ²ATC CTC CCA CAC CCA AGG ² and reverse ²GTG CAG GGT CCG AGGT3² for GAPDH and U6 were described previously []Lentivirus transduction and a0transient transfectionCircZFR knockdown in MCF7 and BT549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shRNAs specific to circZFR shcircZFR1 shcircZFR shcircZFR2 and shcircZFR3 Fulengen Guangzhou China and nontarget shRNA lentiviruses shNC were used as the negative control Vectortransduced cells were selected by puromycin Solarbio Beijing China at a final concentration of a0μgmL at least a0h MiR578 overexpression and knockdown cell lines were generated using the synthetic miR578 mimic a0nM Ribobio Guangzhou China and inhibitor antimiR578 a0nM Ribobio respectively with a corresponding nontarget oligonucleotide miRNC mimic or antiNC Ribobio as the negative control To elevate HIF1A expression in BC cell lines a recombinant overexpressing plasmid for HIF1A HIF1A a0 ng Ribobio or negative control plasmid vector Ribobio was transiently transfected into cell lines using Lipofectamine reagent Invitrogen Breda The Netherlands as per the manufacturers™ protocols Each experiment was performed in triplicateCell viability colony formation and a0apoptosis assaysshcircZFRinfected or shNCtransduced cell lines were transfected with or without antiNC or antimiR578 and MCF7 and BT549 cell lines were carried out the indicated transfections followed by the incubation for a0h at a0°C Cell viability assay was done using the Cell Counting Kit8 CCK8 Abcam Cambridge UK assay as per the manufacturer™s guidance Cell colony formation was tested using standard colony formation protocols as previously reported [] Cell apoptosis measurement was performed by flow cytometry using doublestaining with fluorescein isothiocyanate FITClabeled Annexin V and propidium iodide PI based on the directions of manufacturers Invitrogen events were analyzed by a flow cytometer and the apoptotic cells were determined by calculating the sum of early Annexin VPIˆ’ and late Annexin VPI apoptotic cells All experiments were done in triplicateTranswell migration and a0invasion assaysCell migration and invasion were detected by the transwell assay using modified Boyden chambers in 24transwell plates a0 μm pores Corning Amsterdam The Netherlands Chambers of cell invasion assays consisted of Matrigelprecoated membrane inserts Corning After transfection BC cell lines were seeded into the top chamber of a 24transwell insert and medium containing FCS was used as a chemoattractant in the lower chamber a0h later the well was stained with crystal violet Solarbio and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope Nikon Shinagawa Tokyo Japan at × magnification Each experiment was performed in triplicateMeasurement of a0glucose uptake lactate product and a0adenosine triphosphate ATP levelThe Colorimetric Glucose Uptake Assay Kit LLactate Assay Kit and ATP Assay Kit all from Abcam were used to determine the levels of glucose uptake lactate product and ATP according to the recommendations of manufacturers Briefly the lysates of transfected cells were prepared using the Assay buffer and incubated with standard protocols for the indicated time point followed by the measurement of the absorbance with a microplate reader Invitrogen at OD a0nm for glucose uptake a0nm for lactate product and a0nm for ATP level All assays were done in triplicate 0cChen a0et a0al Cancer Cell Int Page of Animal studiesThe xenograft models were constructed to assess the role of circZFR on tumor growth in a0vivo Animal studies were implemented in accordance with a protocol approved by the Ethics Committee on Animal Use and Care of the First Affiliated Hospital of Zhengzhou University Twelve “ a0weeks BALBc female mice Shanghai Animal Laboratory Center Shanghai China were used and randomly divided into two groups n per group shNC and shcircZFR Approximately × shNCinfected or shcircZFRtransduced BT549 cell line was subcutaneously injected into the dorsal flank of the mice One week later tumor size was measured every week with callipers and tumor volume was calculated using the following formula volume × length × width2 All mice were sacrificed at a0days after implantation and tumor tissues were collected for weightBioinformatics dual‘luciferase reporter and a0RNA pulldown assaysAnalysis for the targeted miRNAs of circZFR was performed using the online web CircInteractome https circi ntera ctome nianihgovindex html and CircBANK httpwwwcircb ankcnsearc hCirc html The putative targets of miR578 were predicted by TargetScan v7 online software httpwwwtarge tscan vert_71Targeted relationships among circZFR miR578 and HIF1A were confirmed by dualluciferase reporter and RNA pulldown assays In dualluciferase assays the fragment of circZFR containing the miR578binding sites and HIF1A ²UTR were individually cloned into pmirGLO vector Promega Madison WI USA to construct corresponding wildtype reporters circZFRWT and HIF1A3²UTRWT The TaKaRa MutanBest Kit was used to construct the corresponding mutations circZFRMUT and HIF1A3²UTRMUT as per the insturctions of manufacturers TaKaRa Dalian China Each reporter construct a0ng and a0nM of miR578 mimic or miRNC mimic were cotransfected into BC cell lines Cell line extracts were prepared with RIPA buffer TaKaRa a0h posttransfection and the ratio of Renilla to firefly luciferase was detected using the Promega Dualluciferase Assay In RNA pulldown assays cell lysates were incubated with the biotinlabeled miR578 mimic BiomiR578 Ribobio or nontarget control sequence BioNC Ribobio for a0 h at a0 °C before adding to the streptavidin beads SigmaAldrich for a0 h The beads were collected and total RNA was extracted for circZFR quantification Each experiment was performed in triplicateWestern blot for a0HIF1A expressionWestern blot was used to determine the expression of HIF1A using standard protocols The preparation of cell line extracts was done using RIPA buffer with proteinase inhibitors Roche Charente France Proteins a0 μg were resolved on a SDS“polyacrylamide gel electrophoretically blotted onto nitrocellulose membranes GE Healthcare Little Chalfont UK and probed with antibody against HIF1A ab51608 a0μgmL Abcam or GAPDH MA515738 a0 μgmL Invitrogen Following the incubation with horseradish peroxidasecoupled IgG secondary antibody ab97051 a0 μgmL Abcam the signals were visualized by Cheniluminescence GE Healthcare as recommended by the manufacturers All experiments were done in triplicateStatistical analysisData were shown as the mean ± standard deviation from at least three independent assays Pairwise comparisons were done using a twotailed Student™s t test Mann“Whitney U test or analysis of variance ANOVA with SPSS version software SPSS Chicago IL USA For survival analysis the Kaplan“Meier survival curve and logrank test were used Correlations among circZFR miR578 and HIF1A expression levels in BC tissues were determined by the Spearman correlation test All tests were considered statistically significant at pvalue ResultsOverexpression of a0circZFR predicted poor prognosis of a0BC patientsAs demonstrated by qRTPCR circZFR was significantly upregulated in BC tissues and cell lines compared with their counterparts Fig a01a b To determine its clinical relevance we preliminarily examined the link between circZFR level and the prognosis of BC patients Kaplan“Meier survival curves showed that the patients in low circZFR level group had a longer survival time than those in high circZFR group Fig a0 1c Additionally circZFR expression was remarkably associated with the distant metastasis and TNM stage of these patients Table a0Silencing of a0circZFR hindered BC cell viability colony formation and a0enhanced apoptosis in a0vitro and a0weakened tumor growth in a0vivoTo study the biological role of circZFR in BC progression the lossoffunction experiments were carried out using shRNAs against circZFR shcircZFR1 shcircZFR2 and shcircZFR3 In contrast to the negative control the transfection of the three shRNAs prominently reduced circZFR expression in both MCF7 and BT549 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR was overexpressed in BC and associated with poor prognosis CircZFR expression by qRTPCR in pairs of tumor tissues and adjacent normal tissues a MCF10A MCF7 BT549 MDAMB231 and MDAMB453 cell lines b c Analysis for the overall survival of BC patients in high n or low n circZFR level group divided by the median of circZFR expression in BC tissues using Kaplan“Meier survival analysis and logrank test P cell lines Fig a02a b Notably shcircZFR1 also named shcircZFR caused the most significant downregulation in circZFR expression so we used it for further analyses Functional experiments data revealed that the silencing of circZFR led to a striking inhibition in cell viability Fig a02c colony formation Fig a02d e as well as a strong promotion in cell apoptosis Fig a02f gTo determine whether circZFR regulated BC tumor development in a0vivo we performed the xenograft model assays When we infected BT549 cell line with shcircZFR tumor growth was remarkably blocked compared with the shNC control Fig a02h iSilencing of a0circZFR suppressed BC cell migration invasion and a0glycolysisWe also asked whether circZFR regulated BC cell migration invasion and glycolysis in a0 vitro Transwell assays showed that in comparison to the control group cell migration Fig a0 3a and invasion Fig a0 3b were significantly repressed by circZFR knockdown Moreover in both cell lines circZFR silencing resulted in decreased glucose uptake Fig a0 3c lactate product Fig a0 3d and ATP level Fig a0 3e demonstrating the suppression of circZFR knockdown on cell glycolysisCircZFR directly interacted with a0miR‘ by a0binding to a0miR‘To further understand the role of circZFR in BC pathogenesis we performed a detailed analysis for its targeted miRNAs The two online algorithms CircInteractome and CircBANK collectively revealed that circZFR harbored a putative complementary sequence for miR578 miR944 and miR5323p Fig a04a The data of qRTPCR showed that the silencing of circZFR led to a striking overexpression in miR578 and miR5323p levels but miR expression was not affected by circZFR knockdown in the two BC cell lines Fig a0 4b c Previous work had reported that circZFR regulated colorectal cancer progression through acting as a sponge of miR5323p [] So we aimed to identify whether miR578 was a molecular mediator of circZFR in BC progression By contrast miR was prominently upregulated in the two BC cell lines transfected with miR578 mimic Fig a04d We then cloned circZFR fragment containing the miR578binding sites into a luciferase plasmid and mutated the miR578binding sites Fig a0 4e The elevated miR578 expression significantly reduced the activity of circZFR wildtype reporter circZFRWT Fig a04f However the mutation of the target sites circZFRMUT completely abrogated the effect of miR578 on reporter gene expression Fig a04f indicating the validity of the target sequence for interaction Additionally RNA pulldown assays revealed that the enrichment level of circZFR was remarkably elevated by BiomiR578 in both cell lines Fig a04gOverexpression of a0miR‘ restrained BC cell viability colony formation migration invasion and a0glycolysis and a0promoted apoptosis in a0vitroIn BC tissues miR578 expression was significantly decreased compared to the normal control Fig a05a and it was inversely correlated with circZFR level Fig a0 5b Moreover miR578 level was lower in BC cell lines than that of control Fig a05c Subsequently we analyzed the biological effect of miR578 on BC progression In contrast to the control group the increased expression of miR578 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR silencing suppressed BC progression in vitro and in vivo a b qRTPCR for circZFR expression in MCF7 and BT549 cell lines transduced with shNC shcircZFR1 shcircZFR2 or shcircZFR3 CCK8 assay for cell viability c colony formation assay for cell colony formation d e flow cytometry for cell apoptosis f g in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines h i shNCinfected or shcircZFRtransduced BT549 cell line was subcutaneously injected into the nude mice n per group followed by the measurement of tumor volume and weight and the capture of representative pictures P 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR silencing suppressed BC cell migration invasion and glycolysis Transwell assay for cell migration and invasion a b corresponding assay kits for glucose uptake lactate product and ATP level c“e in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines P induced a distinct repression in cell viability Fig a05d and colony formation Fig a0 5e and a strong promotion in cell apoptosis Fig a0 5f as well as a striking reduction in cell migration Fig a05G invasion Fig a05H and glycolysis Fig a05i“kSilencing of a0circZFR regulated BC cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitro by a0up‘regulating miR‘A crucial question was whether circZFR regulated BC progression by miR578 To address this we reduced miR578 expression in shcircZFRtransduced MCF7 and BT549 cell lines As expected in comparison to the negative control the reduced level of miR578 significantly reversed circZFR knockdowninduced antiviability Fig a0 6a anticolony formation Fig a0 6b proapoptosis Fig a0 6c antimigration Fig a0 6d antiinvasion Fig a0 6e and antiglycolysis Fig a06f“hACA AGA A was CircZFR modulated HIF1A expression via a0acting as a0a a0sponge of a0miR‘Using the software TargetScan a predicted miR578binding sequence identified within the ²UTR of HIF1A Fig a0 6a When we cloned the ²UTR fragment containing the putative miR578binding sites downstream of a luciferase coding sequence the cotransfection of the luciferase reporter HIF1A3²UTRWT and miR578 mimic into the two BC cell lines produced lower luciferase activity than in cell lines cotransfected with the miRNC control Fig a07b c However the mutation of the target sequence HIF1A²UTRMUT prominently abolished the suppression of miR578 Fig a07b c By contrast HIF1A mRNA and protein levels were significantly reduced by miR578 overexpression in the two BC cell lines Fig a07d e These data together pointed that HIF1A in BC cell lines was directly targeted and inhibited by miR578 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR directly interacted with miR578 by binding to miR578 a Venn diagrams representing the putative miRNAs that bind to circZFR identified by CircInteractome and CircBANK online algorithms b c The levels of miR578 miR944 and miR5323p by qRTPCR in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines d qRTPCR for miR578 expression in the two BC cell lines transfected with miRNC mimic or miR578 mimic e Schematic of the miR578binding sites within circZFR and the mutation of the seed sequence f Relative luciferase activity in MCF7 and BT549 cell lines cotransfected with circZFRWT or circZFRMUT and miRNC mimic or miR578 mimic g qRTPCR for circZFR level in cell lysates incubated with BioNC or BiomiR578 and streptavidin beads P We then examine whether circZFR influenced HIF1A expression in BC cell lines As expected in comparison to their counterparts HIF1A expression was remarkably downregulated by circZFR silencing at both mRNA and protein levels in the two BC cell lines and this effect was significantly abrogated by antimiR578 introduction Fig a0 7f g Additionally qRTPCR data showed a striking upregulation of HIF1A mRNA level in BC tissues Fig a0 7h Furthermore in BC tissues HIF1A mRNA expression was positively correlated with circZFR expression and negatively correlated with miR578 level Fig a07iHIF1A was a0a a0functional target of a0miR‘ in a0regulating BC cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitroTo provide further insight into the link between miR and HIF1A in BC progression we elevated HIF1A expression using a recombinant overexpressing plasmid in miR578 mimictransfected BC cell lines As a result HIF1A protein level was prominently increased by the overexpressing plasmid in the two cell lines Fig a0 8a Functional experiments results revealed that the upregulation of HIF1A dramatically abolished miR578 overexpressionmediated antiviability Fig a0 8b proapoptosis 0cChen a0et a0al Cancer Cell Int Page of Fig MiR578 overexpression hindered BC progression in vitro a qRTPCR for miR578 expression in pairs of tumor tissues and adjacent normal tissues b Correlation between miR578 expression and circZFR level in BC tissues using the Spearman test c MiR578 expression by qRTPCR in MCF10A MCF7 BT549 MDAMB231 and MDAMB453 cell lines MCF7 and BT549 cell lines were transfected with miRNC mimic or miR578 mimic followed by the determination of cell viability by CCK8 assay d colony formation using a standard colony formation assay e cell apoptosis by flow cytometry f cell migration g and invasion h by transwell assay glucose uptake lactate product and ATP level using assay kits i“k P Fig a08c antimigration Fig a08d antiinvasion Fig a08e and antiglycolysis Fig a08f“hDiscussionTo date the emerging links between circRNAs and BC progression have opened up a novel field for cancer diagnosis and treatment [ ] In the meantime understanding the molecular basis underlying the actions of circRNAs has been still challenging In this study we identified the biological role of circZFR in BC progression in a0 vitro and in a0 vivo and investigated the mechanisms governing itHere we firstly demonstrated that circZFR was overexpressed in BC and the elevated expression of circZFR was associated with the poor prognosis of these patients By using lossoffunction in a0vitro and in a0vivo analyses we were first to uncover that the silencing of circZFR performed a suppressive effect in BC progression Previous reports had highlighted the potential oncogenic role of circZFR in several other malignancies such as hepatocellular carcinoma papillary thyroid cancer and nonsmall cell lung cancer [ ] Conversely circZFR was reported as a tumor suppressor in colorectal cancer and gastric cancer [ ] These contradictory findings may attribute to different type tumor or complex tumor microenvironmentUsing the online algorithms we first identified that circZFR sequestered miR578 through sponging miR in BC cell lines Ji et a0al showed that the increased miR578 level weakened osteosarcoma progression via directly interacting with circ_001621 [] Farhana et a0al unraveled that in pancreatic cancer miR578 was 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR knockdown repressed BC malignant progression by miR578 in vitro shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines were transiently transfected with antiNC or antimiR578 a CCK8 assay for cell viability b A standard colony formation assay for cell colony formation c Flow cytometry for cell apoptosis d e Transwell assay for cell migration and invasion f“h Glucose uptake lactate product and ATP level using assay kits P associated with the tumor cell apoptosis [] As it has been reported [] our data validated the downregulation of miR578 expression in BC tissues and cells Moreover we first identified that miR578 overexpression restrained BC cell malignant behaviors in a0 vitro More importantly for the first time we substantiated that circZFR knockdown hampered BC progression in a0vitro by miR578Using TargetScan software we identified that miR in BC cell lines directly targeted and inhibited HIF1A HIF1A a transcriptional regulator in response to intratumoral hypoxia [ ] contributes to BC metastasis and malignant progression [“] We also uncovered that the upregulation of miR578 hampered BC malignant progression via downregulating HIF1A in a0 vitro Previous studies had reported that several 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR modulated HIF1A expression via sponging miR578 a Schematic of the putative miR578binding sequence and mutated the target sequence b c Relative luciferase activity in MCF7 and BT549 cell lines cotransfected with HIF1A3²UTRWT or HIF1A3²UTRMUT and miR578 mimic or miRNC mimic HIF1A mRNA and protein levels by qRTPCR and western blot in MCF7 and BT549 cell lines transfected with miR578 mimic or miRNC mimic d e shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines transfected with antiNC or antimiR578 f g h Relative HIF1A mRNA expression by qRTPCR in pairs of tumor tissues and adjacent normal tissues i Correlation between HIF1A expression with circZFR or miR578 level in BC tissues using Spearman test P other miRNAs such as miR18a and miR497 exerted an antitumor activity in BC through targeting HIF1A [ ] Furthermore our data first illuminated the role of circZFR as a sponge of miR578 to mediate HIF1A expression in BC cell lines The findings by Liang et a0al underscored that circRNA circDENND4C contributed to BC cell proliferation under hypoxia via regulating HIF1A []In conclusion our present study demonstrated that circZFR was overexpressed in BC and the silencing of 0cChen a0et a0al Cancer Cell Int Page of Fig The repression of miR578 upregulation on BC progression in vitro was mediated by HIF1A a HIF1A protein level by western blot in MCF7 and BT549 cell lines transfected with vector or HIF1A MCF7 and BT549 cell lines were transfected with miRNC mimic vector miR578 mimic vector or miR578 mimic HIF1A followed by the determination of cell viability by CCK8 assay b cell apoptosis by flow cytometry c cell migration and invasion by transwell assay d e glucose uptake lactate product and ATP level using assay kits f“h vector negative control plasmid HIF1A recombinant HIF1A overexpressing plasmid P circZFR suppressed BC malignant progression via the regulation of the miR578HIF1A axis This is the first report of circZFR in BC pathogenesis providing evidence for the crucial involvement of circZFR in BC progressionSupplementary informationSupplementary information accompanies this paper at https doi101186s1293 Additional file a0 Supplement material The STR authentication of MCF7 A and BT549 B cellsAdditional file a0 Supplement material The detailed quantification of the western blot analysisAbbreviati

Thyroid_Cancer

"pharmacological therapies and treatments targeting pancreatic neuroendocrine tumorsPNETs have proven ineffective far too often Therefore there is an urgent need for alternative therapeuticapproaches Zyflamend a combination of antiinflammatory herbal extracts that has proven to be effective invarious in vitro and in vivo cancer platforms shows promise However its effects on pancreatic cancer in particularremain largely unexploredMethods In the current study we investigated the effects of Zyflamend on the survival of betaTC6 pancreaticinsulinoma cells TC6 and conducted a detailed analysis of the underlying molecular mechanismsResults Herein we demonstrate that Zyflamend treatment decreased cell proliferation in a dosedependent mannerconcomitant with increased apoptotic cell death and cell cycle arrest at the G2M phase At the molecular level treatmentwith Zyflamend led to the induction of ER stress autophagy and the activation of cJun Nterminal kinase JNK pathwayNotably pharmacological inhibition of JNK abrogated the proapoptotic effects of Zyflamend Furthermore Zyflamendexacerbated the effects of streptozotocin and adriamycininduced ER stress autophagy and apoptosisConclusion The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer viamodulation of the JNK pathwayKeywords Pancreatic neuroendocrine tumor cells Zyflamend JNK Apoptosis Autophagy ER stressPlain English summaryThrough investigating the effects of treating an experimental model of pancreatic neuroendocrine tumor cells withZyflamend we discovered a novel therapeutic potential ofthis polyherbal blend Findings from this study could helppioneer future advancements in our understanding of howphytochemicals and natural compounds could synergistically prove effective against pancreatic cancer by alteringcancer cell survival and proliferation Furthermore the evidence presented within promotes Zyflamend as an adjuvantprospect where it could enhance the effectiveness of standard cancer therapies In addition we believe that thesenovel findings will be of major interest to a broad spectrumof scientists and may pave the way towards more effectiveand translatable therapies Correspondence abettaieutkedu1Department of Nutrition University of Tennessee Knoxville CumberlandAvenue Jessie Harris Building Knoxville TN USA3Graduate School of Genome Science and Technology University ofTennessee Knoxville TN USAFull list of author information is available at the end of the BackgroundPancreatic cancer remains one of the deadliest types ofcancer in the United States with over new casesand deaths in accounting for of allcancer deaths [] Recent epidemiological studies predict 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 0cPuckett Cell Communication and Signaling Page of that in pancreatic cancer will be the third leadingcause of cancerrelated death [] Although advancements in science and health care have led to decreasedmortality from numerous forms of cancer pancreaticcancer survival rates have not improved significantlyover the past several decades leaving a desperate needfor more effective treatment options The risk of developing pancreatic cancer has been associated with numerous biological environmental pathological andgenetic factors These factors include variables such asfamilial history chronic pancreatitis smoking obesityand diabetes reviewed in [ ] In addition hereditaryfamilial factors and germline mutations could contributeto increased risk of cancer onset However the survivaloutcomehighlydependent upon the time of diagnosis While pancreaticductal adenocarcinoma PDAC is the most commonlycontracted and investigated subtype the pathological nature ofthe rarer pancreatic neuroendocrine tumorsPNETSs remains elusive []effectivenesstreatmentandarePNETs account for less than of all pancreatic cancers and are often diagnosed at a late stage in patientswith advanced metastasis making surgery a nonviabletreatment option [ ] Additionally because of theirheterogeneous clinical presentation and responses tochemotherapeutic agents current pharmacological therapies and treatment options targeting PNETs have toooften proven ineffective [] PNETs treatment optionsoften include the use of chemotherapeutic compoundssuch as streptozocin 5fluorouracil doxorubicin andcisplatin both alone or in combination reviewed in [] The effectiveness of these compounds often increases at higher doses but this directly exacerbates therisk for cytotoxicity and collateral side effects [] Inaddition adjunct therapy involving the combination ofvarious treatment approaches such as surgery and radiotherapy is often implemented [] In pursuit of survivaland improved quality of life patients often seek to enhancetherapiesthrough dietary and supplemental means [ ]effectiveness ofconventionaltheNew Chapter Brattleboro VT first launched Zyflamend based on the idea of combining extracts of ten different herbs to effectively reduce inflammation throughcyclooxygenase COX inhibition [] A large volume ofresearch has emerged over the last two decades that supports the antiinflammatory properties of Zyflamend andits ability to inhibit COX in various types of cancer including prostate [] melanoma [] and oral cancer[] Individually many of the extracted components ofZyflamend have proven to exhibit anticancer activity[ ] However the high doses required to optimizeeffectiveness against cancer could prove infeasible forthe majority In theory the combined effects generatedthrough integrating these unique and powerful herbscould grant superior benefit over their isolated form[] Additionally Zyflamend has shown the capabilityto interact with a variety of integral cellular signalingpathways beyond COX These signaling pathways andmechanisms of interaction include AMPactivated protein kinase AMPK [ ] nuclear factor kappalightchainenhancer of activated B cells NFκB [ ]mammalian target of rapamycin mTORC1 [] apoptosis cell growth [ ] endoplasmic reticulum ERstress [ ] and finally autophagy [] Whilethese studies show that Zyflamend could exhibit profound potential in the therapeutic application more research is required to elucidate the molecular basisunderlying its anticancer effects In the current studywe investigated the effects of Zyflamend on the survivalof betaTC6 pancreatic insulinoma cells TC6 anddeciphered the underlying molecular mechanismsMethodsChemicals and reagentsMedia sera and trypsin for cell culture were purchasedfrom Gibco Thermo Fisher Scientific Waltham MAPrimary antibodies and secondary antibodies were acquired from varying sources Supplementary Table General caspases inhibitor ZVADfmk was obtainedfrom Calbiochem La Jolla CA Zyflamend„¢whole bodywas purchased from New Chapter New Chapter IncBrattleboro VT Zyflamend composition is indicated inSupplementary Table Quality assurance is in full compliance with Good Manufacturing Practicing Standardsas mandated by CRF Part Additionally full description and characterization of Zyflamend and itspreparation have been previously described in detail[] Chemical reagents such as dithiothreitol DTTpercoll digitonin phenylmethylsulfonyl fluoride PMSFprotease inhibitors cocktail sodium deoxycholate Tritonglycolbis2aminoethylNNN€²NX100€²tetraacetic acid EGTA sodium fluoride NaF sodium phenylbutyrate 4PBA Hoechst propidium iodide streptozotocin STZ adriamycin ADRautophagy inhibitor 3methyladenine 3MA and JNKinhibitor SP600125 were acquired from MilliporeSigma Burlington MA Finally AMPK inhibitor BML aka compound C was purchased from Santa CruzBiotechnology Santa Cruz CAethyleneCell cultureMouse betaTC6 pancreatic insulinoma TC6 ATCC®CRL11506„¢ and rat pancreatic insulinoma RIN5FATCC® CRL2058„¢ cells were cultured as monolayersin Eagle€™s modified Dulbecco medium plus Lglutamine mM sodium pyruvate mM and fetal bovineserum FBS GibcoThermo Fisher Scientific WalthamMA Cells were maintained in tissue culture plates 0cPuckett Cell Communication and Signaling Page of Thermo Fisher Scientific Waltham MA at °C in ahumidified atmosphere of CO2 Medium was replaced with fresh medium h before experimentsZyflamend treatmentZyflamend was dissolved in dimethyl sulfoxide DMSOat a concentration of mgml Cells were treated withZyflamend at the indicated concentrations and for theindicated durations Treatments were terminated by twowashes with icecold phosphate buffer saline PBSPlates were then flashfrozen in liquid nitrogen andstored at ˆ’ °C until further analysesProliferation assayCell proliferation assay was performed using the sulforhodamine B SRB MilliporeSigma method as previously described [] with modification Briefly an equalnumber of TC6 cells X cells were seeded in well plates Six h later cells were treated with the indicated concentrations of Zyflamend and incubated at °C in an atmosphere of CO2 for the indicatedtime Treatment was stopped by two washes with icecold PBS and cells were fixed with trichloroaceticacid in PBS Intracellular proteins were stained for min at room temperature using SRB dissolved in acetic acid Excess SRB stain was removed by rinsingthe plates thoroughly with running tap water Plateswere airdried for at least h prior to dissolving the stainin mM Tris pH Intracellular proteins werequantified using the Synergy„¢ HTX MultiMode microplate reader BioTek Instruments Inc Winooski VT ata wavelength of nm The relative survival rates ofcells were determined by dividing the absorbance observed for a given treatment by the absorbance detectedin control cells treated with DMSO and expressed as afold changeCytotoxicity assayThe MTT [45dimethylthiazol2yl]25diphenyltetrazolium bromide cytotoxicity assay was performed aspreviously described with modification [] Briefly cells were plated in a 96well plate for h Then afreshly prepared solution of Zyflamend alone or in combination with ZVADfmk 10uM 4PBA 250uMSP600125 10uM STZ mM or adriamycin μMfor an additional h The experiment was terminatedby adding μl of the MTT solution mgml to eachwell for h then the cell culture medium was removedand the dye was dissolved in μl SDS solution overnight at °C Relative cytotoxicity was determinedby measuring the absorbance at nm using the Synergy„¢ HTX MultiMode microplate readerColognic testColonie formation assay was performed as previously described [ ] with modification Cells were seeded inthe presence of DMSO control Zyflamend alone or incombination with ZVADfmk uM 4PBA uM SP600125 uM STZ mM or adriamycin μM After h media was replaced with a freshlyprepared new cell culture media and plates were incubated for days at °C in an atmosphere of CO2After incubation the colonies were washed with icecoldPBS fixed and stained with a mixture of glutaraldehyde and crystal violet for min The plates werewashed with water dried and colonies with morethan cellscolony were counted The relative number of colonies in each condition was determined bydividing the number of colonies for a given treatmentby the total number of colonies in DMSO treatedcells control and expressed as a percentage relativeto DMSOtreated cells CtrlWestern blotting analysisCells were lysed in radioimmunoprecipitation assayRIPA buffer as previously described [] Lysates wereclarified by centrifugation at g for min andprotein concentrations was determined using bicinchoninic acid assay kit Pierce Chemical Dallas TX Proteins €“ μg were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresisSDSPAGE andtransferred to polyvinylidene fluoride PVDF membranes Immunoblotting of lysates was performed withprimary antibodies Supplementary Table and afterincubation with secondary antibodies proteins were visualized using Luminata„¢ Forte Western Chemiluminescent HRP Substrate MilliporeSigma Pixel intensitiesofusingFluorChem Q Imaging software Alpha Innotech CorpSan Leandro CA Data for phosphorylated proteins arepresented as the intensity of phosphorylation normalizedto total protein expression while total protein expression was normalized to the loading control actinimmunoreactivebands werequantifiedMorphological analysis of apoptosisTC6 cells were exposed to Zyflamend for the indicated duration then washed with PBS and labeledwith Hoechst μgmlbluegreenfluorescence Hoechst binds to condensed nuclearchromatin [] and was used to visualize apoptoticcells green fluorescence by fluorescence microscopyLeica DMI8 Leica Microsystems Inc Buffalo GroveIL For each condition atleast cells werecounted Percentages of apoptotic cells were calculated relative to total cellsin PBS 0cPuckett Cell Communication and Signaling Page of MilliporeSigmaAnnexin V stainingQuantification of externalized phosphatidylserine anearly event in the apoptotic cascade was performedusing flow cytometry as previously described [] withmodification Briefly €“ confluent TC6 cellswere exposed to Zyflamend for h then washed withPBS and resuspended in μl of PBS containing FBS Immediately after an equal volume ofthe 2XGuava Nexin reagentcontainingAnnexin V Fluorescein isothiocyanate FITC and aminoactinomycin D 7AAD was added to each treatment and incubated for min at room temperatureunder lightprotected conditions Intensities of fluorescence emitted by Annexin V FITC and 7AAD weremeasured using the Guava® easyCyte Flow CytometerMilliporeSigma on PM1 and PM2 channels respectively Viable negative for both Annexin V and 7AADstaining and apoptotic cells both at early Annexin Vpositive 7AAD negative and late positive for bothAnnexin V and 7AAD stages were quantified using theInCyte„¢ and GuavaSuite Software package LuminexCorp Austin TXCell cycle analysisCell cycle analysis was conducted through assessing theDNA content of cells stained with propidium iodide aspreviously described [] with modification Briefly €“ confluent TC6 cells were starved in serummedia for h then complete growth media was addedto the cells along with various freshly prepared concentrations of Zyflamend h later cells were harvestedwashed twice with icecold with PBS and fixed overnight in ethanol at °C Next cells were washedtwice with icecold PBS and incubated in a freshly prepared RNase solution [ mM TrisHCl pH containing Uml of DNasefree RNase A AppliedBiosystems Austin TX] for min at °C Cells werewashed twice with icecold PBS and incubated in a solution of propidium iodide PI μgml in PBS overnightat °C under light protected conditions Fluorescenceintensity of PI was measured using the Guava® easyCyteflow cytometer on PM2 channel DNA histogram analysis was performed on cells using the InCyte„¢ andGuavaSuite Software package and the proportions ofcells with one or two copies of their chromosomal DNAwere calculatedStatistical analysisData were analyzed using JMP Pro program SASInstitute NC and presented as means standard errorof the mean SEM Unpaired heteroscedastic twotailStudent€™s t test was used for all statistical analyses anddifferences were considered significant at p Singlesymbol such as or €  was used to indicate a p valuethat is less than while double symbol such as or€ €  corresponds to a p value that is less than ResultsZyflamend decreases cell proliferation causes G2M cellcycle arrest and induces apoptotic cell death inpancreatic cancer cellsWe first examined the effects of varying doses of Zyflamend on the proliferation of pancreatic insulinoma TC6 cells Zyflamend caused a significant dose andtimedependent decrease in cell growth Fig 1a Additionally a Zyflamend dose of μgml was sufficient toinhibit cell proliferation by after h of treatmentwhile a dose of μgml completely abolished cell proliferation Fig 1a In line with these findings cell cycleanalysis demonstrated that Zyflamend alters cell cycledistribution in a dosedependent manner Indeed Zyflamend treatment resulted in the enrichment of the G2Mfraction with N DNA content which was accompaniedby a reduction in cell cycle progression through the G0G1 and S phases Fig 1bc These results suggest thatZyflamendinduced inhibition of cell proliferation is mediated at least in part through cell cycle arrest in theG2M phaseIn order to determine whether Zyflamendinduced inhibition of cell proliferation was associated with apoptotic cell death we determined changes in apoptosis inTC6 cells treated with increasing doses of Zyflamend and μgml for h usingtwo approaches the Guava Nexin Annexin V assay andHoechst stain Using the Annexin V assay thepercentages of both Annexin V positive7AAD negativecells reflective of early apoptotic cells and Annexin Vpositive7AAD positive cells reflective oflate apoptosis exhibited a dosedependent and significant increase in response to Zyflamend treatment Fig 1deConsistent with this observationthe number ofHoechstpositive cells was also higher in Zyflamendtreated cells compared to control cells Fig 1fgHoechst is a nucleic acid dye that binds to condensed chromatin in the nucleus of apoptotic cells thusgiving an assessment of overall apoptotic cell death []At a dose of and μgml the percentages ofapoptotic cells were ± ± and ± respectively further emphasizing the proapoptotic effects of Zyflamend on these cells Similarfindings were obtained using the MTT assay Fig 1hA human equivalent dose of Zyflamend induces apoptoticcell death in TC6 cellsTo further characterize the proapoptotic properties ofZyflamend we conducted a time course analysis using aphysiological relevant fixed dose of Zyflamend μgml [] This dose is representative of the maximum 0cPuckett Cell Communication and Signaling Page of Fig Zyflamend Reduces Cell Survival and Induces Cell Death of Pancreatic Cancer Cells in a Dose Dependent Manner a Effects of Zyflamendon cell survival and proliferation cells were treated with increasing doses of Zyflamend for h Line graphs represent the intensity of SRBstaining reflective of the cell number and presented as means SEM bc Cell cycle analysis and assessment of DNA content in TC6 cellstreated with DMSO control or the indicated concentration of Zyflamend for h Representative histogram distributions for each treatment areshown c Bar graphs represent the percentages of cells in each phase of the cell cycle which were estimated using the GuavaSuite Softwarepackage and are presented as means SEM from three independent experiments p p indicate significant difference betweenthe indicated concentration and control cells treated with the vehicle DMSO de Zyflamend treatment induces apoptosis in TC6 Cells confluent cells were treated with increasing concentrations of Zyflamend and then labeled with Annexin VFITC and 7AAD Representative dotplots are shown Annexin V positive and 7AAD negative cells lower right quadrants represent early stages of apoptosis whereas cells that arepositive for both Annexin V and 7AAD upper right quadrants are in late stages of apoptosis e Bar graphs represent live early and lateapoptotic cells are presented as means SEM of at least three independent experiments p p indicate significant differencebetween the indicated concentration of Zyflamend and control cells treated with the vehicle DMSO fg Chromatin condensation in cells treatedwith increasing doses of Zyflamend for h Representative images are shown Scale bar μm g Bar graphs represent the number of apoptoticcells Hoechst positive as means SEM of at least three independent experiments h Cell toxicity assay using the MTT method Bar graphsrepresent the intensity of formazan produced from MTT by viable cells staining reflective of the cell number and presented as means SEM ofat least three independent experiments In g and h p p indicate a significant difference between cells treated with Zyflamendand nontreated cellstreatmentplasma concentration of a primary ingredient of Zyflamend curcumin that was reported in humans after oraladministration [] At this dose a marked increase inchromatin condensation and apoptotic cell number wasobserved after h ofFig 2ab Subsequently markers of apoptosis and cell survival were investigated using Western blotting Zyflamend inducedcleavage of caspase3 and its downstream target polyADPribose polymerase PARP Fig 2cd In additionwe examined changes in the mitogenactivated proteinkinases MAP kinases pathways in response to Zyflamend Our data revealed that TC6 cells treated withZyflamend exhibited a marked decrease in the phosphorylation of protein kinase B AKT and extracellularsignalregulated kinases ERK particularly after h oftreatmentTo determine whether Zyflamendinduced cell deathwas associated with the caspase dependent pathwaysof apoptosis we tested whether blocking caspases€™activity usingcarbobenzoxyvalylalanylaspartyl[Omethyl]fluoromethylketone ZVADfmk could inhibit Zyflamendinduced chromatin condensation andapoptosis ZVADfmk is a potent cell permeable pancaspase inhibitor which acts by irreversibly bindingto the catalytic site of the caspase proteases and thusinhibiting their activities Our study shows that pretreatment with ZVADfmk caused a significant decreased in the levels of chromatin condensation inZyflamendtreated cells Fig 2ef Additionally ZVADfmk treatment alleviated Zyflamendinduced celltoxicity as judged by the MTT Fig 2g and the colony formation Fig 2hi assays Taken together ourfindings indicate that Zyflamend treatment reducescell viability and induces cell death through the induction of the apoptotic machinery in TC6 cellsZyflamend induces ER stress apoptosis and autophagyresponses in TC6 cellsA plethora of intrinsic and extrinsic pathways can leadto apoptosis in response to stressors including ER stressand autophagy among many more Therefore in orderto dissect the precise molecular mechanism mediatingthe proapoptotic effects of Zyflamend we examined theactivation of key signaling molecules related to thesepathways Zyflamend μgml significantly inducedER stressactivation of ERjudged byasthe 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Apoptotic Cell Death in TC6 Cells ab Effects of Zyflamend on chromatin condensation Cells were treated withZyflamend μgml for the indicated time and chromatin condensation was evaluated by fluorescence microscopy using Hoechst Representative images are shown Scale bar μm b Bar graphs represent the number of apoptotic cells Hoechst positive as means SEMp indicates a significant difference between cells treated with Zyflamend and nontreated cells cd Immunoblots of key proteins in cellsurvival and apoptosis markers in cells treated with μgml of Zyflamend for the indicated time d Bar graphs represent cleaved caspase3 CCasp 3actin cleaved PARP CPARPactin pAKTAKT and pERKERK as means SEM p p indicates a significant differencebetween cells treated with Zyflamend and nontreated cells ef Chromatin condensation in TC6 cells treated with μgml Zyflamend withand without the pancaspase inhibitor ZVADfmk Representative images are shown Scale bar μm f Bar graphs represent the number ofapoptotic cells Hoechst positive as means SEM g Cell toxicity assay using the MTT method Bar graphs represent the intensity of formazanstaining reflective of the cell number and presented as means SEM hi Colony formation assay i Bar graphs represent the relative number ofcolonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSOtreated cells Ctrl and expressed as a percentage In g and i p p indicate a significant difference between cells treated withZyflamend and nontreated cells € p € € p indicate a significant difference between cells treated with ZVADfmk and nontreatedproteinCHOPby Westerntransmembrane sensors protein kinase RNAlike endoplasmic reticulum kinase PERK and inositolrequiringtransmembrane kinaseendoribonuclease 1α IRE1αalong with downstream targets such as eukaryotic translation initiation factor alpha EIF2α and CEBP homologousblottingZyflamend induced ER stress as evidenced by increasedPERK Thr980 EIF2α Ser51 and IRE1α Ser724 phosphorylation Fig 3a Furthermore the level of CHOPexpression was elevated a direct downstream target ofboth the PERK and IRE1 pathways The activation ofCHOP a potent inducer of apoptotic cell death [ ]in response to ER stress Fig 3ab strengthens our conclusions of Zyflamendinduced apoptosis in these cellsMoreover Zyflamend has been shown to activateAMPK and our results recapitulate these previous findings []The AMPK signaling pathway has been shown toinregulate autophagy and cell death [] Thereforeorder to assess whether Zyflamend induces autophagy inTC6 cells we immunoblotted for autophagyrelatedproteins We observed a time dependent increase inbeclin microtubuleassociated proteins 1A1B lightchain LC3I II and autophagyrelated proteins and ATG57 Fig 3cd The increase in the expressionof these proteins is indicative of elevated autophagy inthese cells Because ER stress inflammation and autophagy can all lead to apoptosis we used the pancaspaseinhibitor ZVADfmk to determine which pathway mightbe responsible for the proapoptotic effects of Zyflamend Our data shows that while there was a significantattenuation of Zyflamendinduced cleavage of caspase3and its downstream target PARP treatment with ZVADfmk had no effects on Zyflamendinduced activation of the AMPK autophagy and ER stress signalingcascades Fig 3ef These findings suggestthat ERstress autophagy and MAP kinases pathways are upstream of the apoptotic signaling cascade that might bemediating the proapoptotic effects of Zyflamend in TC6 cellsZyflamendinduced cell death is mediated through the ERstressJNKautophagy pathwayThe exact molecular mechanisms leading to apoptosisby Zyflamend in cancer cells hashave not been revealedyet although recent studies have supported the role ofAMPK in the regulation of cancer cell growth bioenergetics autophagy and cell death To investigate the potential role of AMPK in Zyflamendinduced apoptosiswe pretreated cells with the AMPK inhibitor compoundC CC μM for h prior to Zyflamend treatment foran additional h The dose and duration of exposurewere determined based on the ability of compound C toreverse AMPKdependent inhibitory phosphorylation ofacetylCoA carboxylase ACC data not shown Cellswere then examined for AMPK activation as well as activation of inflammation ER stress autophagy and celldeath Fig 4a While the level of phosphorylated AMPKwas reduced in cotreated cells pretreatment with compound C had no effects on Zyflamendinduced JNKphosphorylation ER stress autophagy or cell death Fig4ab These data suggest that Zyflamendinduced apoptosis in TC6 cells is independent of AMPK activationNext we sought to examine whether blocking autophagyusing 3MA could protect cells against Zyflamendinduced apoptosis 3MA inhibits autophagy by blockingautophagosome formation via the inhibition of class Iand class III phosphatidylinositol 3kinases PI3K []Cells were preincubated with 3MA nM for hprior to Zyflamend treatment Cotreatment with Zyflamend and 3MA significantly decreased the expressionof beclin LC3 and cleaved caspase3 but had no effects on ER stress markers nor on AMPK phosphorylation Fig 4cd These data suggest that autophagymediates Zyflamendinduced apoptosis and that bothAMPK and ER stress activation by Zyflamend occur upstream of autophagy and apoptosisThe relationship between these two fundamental processes ER stress and autophagy is complex and poorlyunderstood Recent literature demonstrates that bothpathways display dual roles in cell survival in multiple 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Inflammatory ER Stress and Autophagy Responses in TC6 Cells ab Total cell lysates from control and Zyflamendtreated cells for and h were immunoblotted for ER stress markers pPERK pEIF2α pIRE1α their respective unphosphorylatedproteins sXBP1 CHOP and actin as a loading control Representative immunoblots are shown b Bar graphs represent pPERKPERK pEIF2αEIF2α pIRE1IRE1 sXBP1actin and CHOPactin as means SEM p p indicate a significant difference between cells treatedwith Zyflamend and nontreated cells cd Markers of autophagy were examined in the same lysates using antibodies against Beclin LC3 IIIATG5 ATG7 and actin as a loading control d Bar graphs represent Beclin 1actin LC3actin ATG5actin and ATG7actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells ef Immunoblots of keyproteins in autophagy AMPK ER stress and apoptosis signaling in TC6 cells treated with μgml Zyflamend with and without the pancaspase inhibitor ZVADfmk Representative immunoblots are shown f Bar graphs represent pAMPKAMPK pPERKPERK pEIF2αEIF2α pIRE1αIRE1α sXBP1actin CHOPactin pJNKJNK Beclin 1actin LC3IIIactin and cleaved caspase3actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells € p € € p indicate a significant differencebetween cells treated with ZVADfmk and nontreated cellscancer celllines Similar to ER stress autophagy hasbeen shown to promote cell survival by clearing unwanted components from the cells Nonetheless a considerable body of evidence also indicates that bothautophagy and ER stress can lead to apoptosis in tumorcells In addition to this a growing body of literaturesupports existing crosstalk between the two pathways[ ] However which pathway is upstream of theother is yet to be determined Our data suggest thatZyflamendinduced autophagy is likely to be downstream of ER stress To test this hypothesis we pretreated TC6 cells with an ER stress inhibitor phenylbutyrate 4PBA mM for h prior to Zyflamend treatment and we examined changes in inflammation ER stress autophagy and cell death Fig 4ef PBA is a cell permeant chemical chaperone that hasbeen shown to inhibit ER stress and ER stressinducedapoptosis in many cancer cell types including pancreaticcancer cells [ ] Our findings show a profound decrease in ER stress autophagy and cell death markers inresponse to Zyflamend Fig 4ef when cells were pretreated with 4PBA Additionally 4PBA treatment alleviated the decrease in cell proliferation Fig 4g and colonycaused by ZyflamendFurthermore pretreatment of TC6 cells with 4PBAreduced Zyflamendinduced chromatin condensationFig 4jk Conversely 4PBA did not alter the activationof AMPK by Zyflamend Fig 4ef suggesting that ERstress occurs upstream of autophagy and apoptotic celldeathformation Fig 4hiPrevious studies have shown that the ER stress sensorIRE1 may promote autophagy through the TRAF2ASK1JNK pathway [ ] To test this hypothesis wetreated TC6 cells with SP600125 a selective JNK inhibitor and investigated changes in inflammation ERstress and proliferation in response to Zyflamend treatmentFig As expected cells pretreated withSP600125 exhibited a significant reduction in the phosphorylation of JNK and reduced expression of autophagyand cell death markers in response to Zyflamend Fig5ab Likewise JNK inhibition protected TC6 cellsfrom Zyflamendinduced reduction in cell survival Fig5c colo

Thyroid_Cancer

"Postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in COVID19 patientsMethods We evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans Complete macroscopic and microscopicautopsies were performed on different ans in COVID19 nonsurvivors Presence of SARSCoV2 was evaluatedwith immunohistochemistry IHC in lung samples and with realtime reversetranscription polymerase chainreaction RTPCR test in the lung and other ansResults Pulmonary findings revealed earlystage diffuse alveolar damage DAD in out of patients andmicrothrombi in small lung arteries in patients Latestage DAD atypical pneumocytes andor acute pneumoniawere also observed Four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedThere was no evidence of myocarditis hepatitis or encephalitis Kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients Spongiosis and vascular congestion were the mostfrequently encountered brain lesions No specific SARSCoV2 lesions were observed in any an IHC revealedpositive cells with a heterogeneous distribution in the lungs of of the patients RTPCR yielded a widedistribution of SARSCoV2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brainConclusions In autopsies revealed a great heterogeneity of COVID19associated an injury and theremarkable absence of any specific viral lesions even when RTPCR identified the presence of the virus in many ansKeywords COVID19 SARSCoV2 Autopsy RTPCR Immunohistochemistry Correspondence IsabelleSalmonerasmeulbacbe1Department of Pathology Erasme Hospital Universit Libre de BruxellesULB Route de Lennik Brussels Belgium2Centre Universitaire inter Rgional d™expertise en Anatomie PathologiqueHospitali¨re CurePath CHIREC CHU Tivoli ULB Rue de Borfilet 12A Jumet BelgiumFull 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 0cRemmelink Critical Care Page of severeacuteincludingBackgroundCoronavirusesrespiratorysyndrome coronavirus SARSCoV and Middle Eastrespiratory syndrome coronavirus MERSCoV causesevere acute respiratory failure which is associated withhigh mortality rates [] The novel SARSCoV2 strainexhibits phylogenetic similaritiesto SARSCoV andcauses coronavirus disease COVID19 which hasresulted in more than deaths worldwide so farAs the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that SARSCoV2 can directly alter cellfunction by a link to the angiotensin converting enzyme ACE2 receptor which is almost ubiquitous in thehuman body []Nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with COVID19remain poorly understood Controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease []In this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of COVID19 Most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] The aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from COVID19MethodsStudy designIn this postmortem study we included the first adultpatients years who died in our hospital either in aCOVID19 unit or an intensive care unit from March with confirmed SARSCoV2 infection ie positiveRTPCR assay on nasopharyngeal swab andor bronchoalveolar lavage specimen Exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination The study protocol wasapproved by the local ethics committee P2020218Data collectionrelevantWe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport Acute respiratory distress syndrome ARDSand acute kidney injury AKI were defined accordingto standard definitions [ ]Postmortem procedureThe Belgian Public Health Institute Sciensano guidelines were integrated into our postmortem procedure[] The cadavers were kept in the refrigerator at °Cand autopsies were performed to h after death toensure the safety of the autopsy team Personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves FFP3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots In the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationAll analyses were performed at normal pressurespleen bone marrow kidney bladderUsing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking The material was biobanked by BiobanqueH´pital ErasmeULB BE_BERA1 CUB H´pital ErasmeBBMRIERIC The banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain For the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken For safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe Additional file Additional Material Formalinfixed paraffinembedded FFPEtissues underwentstandard processing to provide hematoxylin and eosinHEstained sections Special stains and immunohistochemistry IHC were used for lung Masson™s trichromeperiodic acidSchiff [PAS] GomoriGrocott antiCMVIHC antiHSV IHC antiPneumocystis J IHC and kidneyPAS Masson™s trichromeJones methenamine silversamplesMorphological analysisMorphological analysis was performed on HE stainedglass slides using the SecundOs digital platform TribVnHealth Care Chatillon Francefor digital diagnosisafter the acquisition of whole slide digital scans — magnification using a Nanozoomer HT slide scanner Hamamatsu Hamamatsu City JapanSARSCoV2 detection by immunohistochemistrySince no antibody against SARSCoV2 has been validatedfor IHC on FFPE tissues we selected an antiSARSnucleocapsid protein antibody Standard IHC was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display SARSnucleocapsid protein Invitrogen PA141098dilution on Dako Omnis Agilent Technologies 0cRemmelink Critical Care Page of Santa Clara CA USA using the Envision Flexdetection system according to the manufacturer™sprotocol [] The sections were counterstained withhematoxylin Negative tissue controls were obtainedfrom patients who had an autopsy before the COVID pandemic Semiquantitative IHC evaluation wasperformed by two senior pathologists ND MR as follows negative ˆ’ between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one — field SARSCoV2 detection by rRTPCRTotal nucleic acid was extracted from FFPE tissues usingthe Maxwell RSC DNA FFPE Kit reference AS1450Promega Corporation Madison WI USA and thePromega Maxwell extractor following the protocol described by the manufacturer Onestep RTPCR assaysspecific for the amplification of SARSCoV2 E envelopeprotein gene were adapted from a published protocol[] Briefly μL of RNA ng was amplified in μL reaction mixture containing μL of TaqMan FastVirus 1step master mix Life Technologies μM ofeach forward ACAGGTACGTTAATAGTTAATAGCGT and reverse ATATTGCAGCAGTACGCACACAprimers and μM of probe FAMACACTAGCCATCCTTACTGCGCTTCGBBQ The amplification condition was °C for min for reverse transcriptionfollowed by °C for s and then cycles of °C for s and °C for s A clinical sample highly positivefor SARSCoV2 was diluted and used as a positive control in each analysis A clinical sample obtainedfrom a patient who was autopsied before the COVID19pandemic was used as a negative control The quality ofthe RNA from the samples showing negative results wasassessed by amplification of the human MET RNA according to a validated ISO15189 accredited methodused as a routine diagnostic method in our laboratoryStatistical analysisData are reported as counts percentage or medians[interquartile ranges IQRs] All data were analyzedusing GraphPad Prism Version GraphPad Software San Diego CA USAResultsStudy cohortThe main characteristics of the study cohort malesout of median age [“] years are given inTable The time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days All except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n None ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza A and B virusesEleven of the patients were treated with mechanicalventilation Eleven patients died in the ICU and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n Laboratory data are reported in Additional file Table S1Macroscopic findingsOne patient had had a left pneumonectomy and onepatient a right bilobectomy The lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage Thrombi were found in the large pulmonary arteries in patients and lung infarction in patientsPleural adhesions associated with pleural effusions were observed in cases We observed cardiomegaly in and hepatomegaly in patients The kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct The gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis In the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhageMicroscopic findingsfile As shown in Figs and and AdditionalTable S2the main pulmonary findings includedearlystage diffuse alveolar damage DAD which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryII pneumocytehyperplasia Microthrombi were noted in the smallpulmonary arteries in patients Ten of the patientsalso had advanced DAD lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells We observed no viralinclusions or squamous metaplasiaand typeinfiltrateAll the patients who survived more than weeksn had late DAD lesions There was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct In of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi The other patients had longer 0cRemmelink Critical Care Page of Table Characteristics of the study populationIDCT scanComorbiditiesAgeSexrRTPCRPOSTime todeathAntemorteman failureARDSAKIPOSNEGNEGMFMFMCADCVDDiabetesHypertensionCADCRFLiver cirrhosisCOPDCancerHypertensionCancerCVDCOPDCancerGGOPOSMAGGOPOSPOSTreatmentsCause of deathMechanicalventilationAntibioticsHydroxychloroquineAntibioticsCorticosteroidsMechanical ventilationHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMOHydroxychloroquineRemdesivirCorticosteroidsAntiobioticsHydroxychloroquineAntibioticsCardiogenicshockMOFRespiratory failureRespiratory failureRespiratory failureMesentericischemiaMOFRespiratory failureRespiratory failureAntibioticsSeptic shockMOFMechanical ventilationRRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMORRTHydroxychloroquineOseltamivirAntibioticsHydroxychloroquineAntibioticsRespiratory failureRespiratory failureSudden deathMechanical ventilationHydroxychloroquineAntibioticsMOFHydroxychloroquineRespiratory failureHydroxychloroquineAntibioticsRespiratory failureARDSAKIHypoxichepatitisARDSAKIARDSARDSAKIHypoxichepatitisARDSAKIARDSAKIHypoxichepatitisAKIARDSAKIARDSAKIARDSARDSAKIHypoxichepatitisARDSARDSAKIHypoxichepatitisMHypertensionCRFBCPOSMNoneBCPOSMFHypertensionCADCVDCRFDiabetesHypertensionDiabetesEmphysemaPOSGGOPOSMHypertensionDiabetesGGOBCPOSMMMFDiabetesLiver cirrhosisCancerDiabetesHypertensionCADDiabetesDiabetesHypertensionDiabetesBCPOSGGOPOSGGOBCGGOBCPOSPOS 0cRemmelink Critical Care Page of Table Characteristics of the study population ContinuedIDComorbiditiesCT scanAgeSexMGGOLPPOSrRTPCRTime todeathFMHypertensionDiabetesLiver transplantHypertensionCVDGGOBCPOSGGOBCLPPOSAntemorteman failureARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismTreatmentsCause of deathMechanical ventilationRRTHydroxychloroquineRemdesivirAntibioticsMechanical ventilationRRTHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineAntibioticsSeptic shockMOFSeptic shockMOFSeptic shockMOFTime to death time from admission to death days Cause of death was reported by the attending physician M male F female rRTPCR reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test NEG negative POS positive CAD coronary artery disease CVD cerebrovascular disease LP lobarpneumonia GGO groundglass opacity MA minor abnormalities BC bilateral consolidation COPD chronic obstructive pulmonary disease CRF chronic renal failureARDS acute respiratory distress syndrome AKI acute kidney injury ECMO extracorporeal membrane oxygenation RRT renal replacement therapy MOF multiplean failuredelays between hospitalization and death daysthey had no microthrombiFifteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis Histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in In the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients Chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisFig Main histological findings Green finding present gray finding absent black unavailable 0cRemmelink Critical Care Page of Fig Pulmonary histological findings a Earlystage diffuse alveolar damage DAD hyaline membrane HE — magnification with a zoom ona giant cell — magnification b Fibrin thrombi in a pulmonary artery HE — magnification c Latestage DAD fibroblastic proliferationHE — magnification d Latestage DAD fibroblastic proliferation Trichrome staining — magnification e Acute pneumonia HE — magnification f AntiSARSCoV immunohistochemistry IHCpositive cells — magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryLiver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate The histological changes in theabdominal ans including the esophagus stomachand colon are reported in Additional file Table S2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisBrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n We found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalSARSCoV2 detection in the lungs by IHCSARSCoV2 was identified by IHC in the lungs of ofthe patients Fig Howeverthere was largevariability in the distribution of SARSCoV2positivecells in the lung parenchymaSARSCoV2 detection by RTPCRSARSCoV2 RNA was detected in at least one anfrom every patient Fig In the lung RTPCR waspositive in patients with threshold cycle Ct valuesvarying from to Viral RNA was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples Ct valuesfor nonpulmonary ans ranged from to Eight patients had positive RTPCR in all tested ansabnormalitiesDiscussionThis postmortem study showed several histopathologicalin COVID19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though SARSCoV2 was detected in all examined ans using RTPCR We decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe This approach enabled us to 0cRemmelink Critical Care Page of Fig Detection of SARSCoV2 by immunohistochemistry IHC in FFPE post mortem lung samples of patients Semiquantitative evaluationœˆ’ negative result œ scattered positive cells between and positive cellswhole slide œ positive isolated cells cellswhole slide butno foci œ foci of positive cells more than positive cells in one — field NA not availabledocument the considerable heterogeneity of histologicallesions and of SARSCoV2 spread through the bodyThe diagnosis of SARSCoV2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases In a previous autopsy study in COVID19patients [] the authors reported that DAD associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia No obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report [] Desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ARDS of many different causes especially inearlyphase ARDS [] The presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice As in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of SARSCoV2 infection [] Finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to SARSCoV2In the absence of typical postmortem viral featuresour results show that RTPCR is feasible on FFPE blocksand could be used in postmortem analyses to identifythe presence of SARSCoV2 in multiple ans and tounderstand the spread of the virus within the humanbody The discordant RTPCR and IHC results fordetection of SARSCoV2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the RTPCR whereas lowlevel viral replication might not be detected by IHC Moreover IHC wasbased on the only available antibodies which aretargeted against SARSCoV New antibodies againstSARSCoV2 need to be developed to improve theaccuracy of IHC in the analysis of tissue samples fromsuspected or confirmed COVID19 patientsMost of the previous postmortem studies in COVID19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofSARSCoV2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations As for previous coronavirusdiseases [ ] the lungs are the most affected ans inCOVID19 However DAD findings werehighly 0cRemmelink Critical Care Page of Fig Molecular detection of SARSCov2 RNA in postmortem samples Detection of SARSCoV2 by reverse transcription realtime polymerasechain reaction RTPCR in FFPE postmortem tissues of patients œ positive result œˆ’ negative result œNA tissue not available NC noninformative test result due to lowquality RNAheterogeneous including both earlyonset and additionallate lesions This finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase [] As some patients died outsidethe ICU without receiving mechanical ventilation we couldnot estimate lung compliance before death The heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the DADreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections In a recent report needle postmortem biopsies suggested that COVID19 is not associated withDAD but rather with an acute fibrinous and anizingpneumonia AFOP consequently requiring corticoid treatment [] A diagnosis of AFOP is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with DAD and complete lunganalysis not just biopsies are necessary to exclude theirpresence Moreover AFOP may be a fibrinous variant ofDAD [] The limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for SARSCoV2 using RTPCR [] when compared to almost in our series In addition we did notfind specific œendothelitis as previously reported in a smallcase series [] Considering the heterogeneity of postmortem COVID19 associated lesions molecular and IHCassessments are mandatory in the histological analysis ofCOVID19 tissue samplesPatients with COVID19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism PE [] In ourstudy three patients had PE already diagnosed beforedeath Four patients had pulmonary infarction In a previous study acute PE was considered as the main causeof death in four patients [] however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of PEwhen compared to our study Although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofARDS regardless of etiology [ ] As such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in COVID19 0cRemmelink Critical Care Page of patients remains to be further studied In a systematicreview of pathologicalfindings in COVID19 Polak [] identified a timeline in the histopathologicalfindings in the lung with epithelial DAD denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms Few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease Our resultsare consistent with those of Polak [] except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients We did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis The cases ofœacute cardiac injury reported in COVID19 clinicalstudies [] do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia [] However using RTPCR we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theACE2 receptor which is ubiquitous but may notdirectly cause an injury As extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatSARSCoV2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in COVID19associated an dysfunction AsRTPCR mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance []just detect residual viral genomeThis study has several limitations i we only includedpatients who had had a positive RTPCR on nasopharyngeal swab andor bronchoalveolar lavage To ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic CTscan findings suggestive of COVID19 but had negativeRTPCR results This limitation in our study reflects thedifficulty of diagnosing COVID19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death This delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow SARSCoV2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related toCOVID19 Finally we did not evaluate specific mechanisms involved in the pathogenesis of an injuryConclusionThese results underline the heterogeneity of an injuriesduring COVID19 disease and the absence of specificSARSCoV2 lesions Using RTPCR SARSCoV2 couldbe detected in all ans even those without evidentmicroscopic lesionsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s13054020032185Additional file Critical careautopsyCovid Additional materialProcedure to obtain brain samplesAdditional file Critical careautopsyCovid Additional Table S1Laboratory findings on the day of admissionAdditional file Critical careautopsyCovid Additional Table S2Detailed histological findings in all patientsAbbreviationsACE2 Angiotensin converting enzyme AFOP Acute fibrinous andanizing pneumonia AKI Acute kidney injury ARDS Acute respiratorydistress syndrome COVID19 Coronavirus disease Ct Threshold cycleDAD Diffuse alveolar damage FFPE Formalinfixed paraffinembeddedHE Hematoxylin and eosin IHC Immunohistochemistry IQRs Interquartileranges MERSCoV Middle East respiratory syndrome coronavirusPAS Periodic acidSchiff PE Pulmonary embolism RTPCR Realtime reversetranscription polymerase chain reaction SARSCoV Severe acute respiratorysyndrome coronavirusAcknowledgmentsThe authors thank Nathalie Lijsen Christophe Valleys Gees LacroixBarbara Alexiou Dominique Penninck Nicole Haye and Audrey Verrellen fortechnical and logistic supports Prof Frdric Schuind and Dr DjamelEddineYahiaCherif for neurosurgical procedure Egor Zindy DIAPath ULB forproofreading the paper and Dr MariePaule Van Craynest for trainees™supervisionAuthors™ contributionsIS had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis IS FT JLV and CD drafted the paper MRCV LL PL MLR CM ALT JCG LP RDM SD SR ND LP and OD collected thedata MR ND and RDM did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published All authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedFundingThis study received financial support from Fonds Y Bo«l Brussels BelgiumFonds Erasme pour la Recherche Mdicale Brussels Belgium and œAppel  projet Spcial COVID19 ULB Brussels Belgium The CMMI is supported bythe European Regional Development Fund and the Walloon Region ofBelgium Walloniabiomed grant no project œCMMIULBsupport the Center for Microscopy and Molecular Imaging and its DIAPathdepartment CD is a Senior Research Associate with the FNRS BelgianNational Fund for Scientific Research 0cRemmelink Critical Care Page of Availability of data and materialsThe data that support the findings of this study are available from thecorresponding author on reasonable request Participant data without namesand identifiers will be made available after approval from the correspondingauthor and local Ethics Committee The research team will provide an emailaddress for communication once the data are approved to be shared withothers The proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data Additional materials may also be required during theprocess D'Haene N Melndez B Blanchard O De N¨ve N Lebrun L VanCampenhout C Design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas CancersBasel Corman VM Landt O Kaiser M Molenkamp R Meijer A Chu DK et alDetection of novel coronavirus 2019nCoV by realtime RTPCR EuroSurveill de Hemptinne Q Remmelink M Brimioulle S Salmon I Vincent JL ARDS aclinicopathological confrontation Chest “ Menter T Haslbauer JD Nienhold R Savic S Hopfer H Deigendesch N et alEthics approval and consent to participateThe study protocol was approved by the local ethics committee ErasmeHospital P2020218 The ethical committee has waived the need for writteninformed consentPostmortem examination of COVID19 patients reveals diffuse alveolardamage with severe capillary congestion and var

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

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

Thyroid_Cancer

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

Thyroid_Cancer

"Vitamin D is a fatsoluble vitamin vitamin D is essential to sustain health and it protects againstosteoporosis It is crucial to the human body™s physiology in terms of muscular movement and neurological signaltransmission and to the immune system in defense against invading pathogensCase presentation This was a case of a 26yearold Sudanese woman who presented with a 2year history ofanosmia recurrent nasal polyps back pain and chronic fatigue She was diagnosed as having a case of vitamin Ddeficiency and responded well to treatmentConclusion There is an association between vitamin D deficiency and recurrent allergic nasal conditionsKeywords Vitamin D deficiency Allergy Nasal polyps Backache Chronic fatigabilityBackgroundVitamin D is a fatsoluble vitamin it is naturally presentin some foods and as dietary supplements It is also produced endogenously through exposure to ultraviolet raysfrom sunlight Vitamin D obtained from sun exposurefood and supplements is biologically inert and mustundergo two hydroxylations in the body for activationTheand produces hydroxyvitamin D 25OHD also known as calcidiolThe second occurs in the kidney and forms the physiologically active 125dihydroxy vitamin D 125OH2D alsoknown as calcitriol []first occursin theliverVitamin D is found in cells throughout the body vitamin D is essential to sustain health and it protectsagainst osteoporosis It is crucial to the human body™sphysiology in terms of muscular movement and neurological signal transmission and to the immune system indefense against invading pathogens []Although there are different methods and criteria fordefining vitamin D levels the criteria Holick proposed Correspondence drmuhanadkamalhotmailcom1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum SudanFull list of author information is available at the end of the have been widely accepted In this proposal vitamin Ddeficiency is defined as blood level of less than ngmlinsufficiency of vitamin D is defined as blood levels ranging between and ngml and sufficiency if greaterthan or equal to ngml [] About one billion peopleglobally have vitamin D deficiency and of the population has vitamin D insufficiency The majority ofaffected people with vitamin D deficiency are the elderlyobese patients nursing home residents and hospitalizedpatients Vitamin D deficiency arises from multiplecauses including inadequate dietary intake and inadequate exposure to sunlight Certain malabsorption syndromes such as celiac disease short bowel syndromegastric bypass some medications and cystic fibrosis mayalso lead to vitamin D deficiency []Vitamin D deficiency is now more prevalent than everand should be screened in highrisk populations Manyconflicting studies now show an association betweenvitamin D deficiency and cancer cardiovascular diseasediabetes autoimmune diseases and neuropsychiatricdisorders [ ] 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 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of two functional endoscopic sinusCase presentationThis was a case of a 26yearold Sudanese woman married who has a 3yearold boy This woman presentedto our ear nose and throat ENT department complaining of anosmia for the past years She had a history ofsurgeriesFESSs for nasal polyps the first one was years agoand the second one was years prior to presentationShe complained of being highly sensitive to different irritants including dust weather change perfumes andpetsShe also stated that she attended more than three different physicians due to generalized fatigue and gettingtired easily after simple daily activity in addition to sleeping for more than hours a dayShe attended an orthopedic clinic for unspecified lower back pain that was notrelated to any type of trauma or physical activity a lumbosacral magnetic resonance imaging MRI was done andrevealed no abnormal findingsShe mentioned that she isknown to be anxious most of the time and aggressive toward simple reactions from her family members She hadno psychiatric history and was not using any medicationsShe was not known to be diabetic or hypertensive orto have any chronic illnesses she was not on any regularmedication She is a housewife of high socioeconomicstatus she is well educated graduated from dentalschool with a bachelor™s degree but currently notemployed She has never consumed tobacco or alcoholshe practiced regular cardio exercisesOn examinationshe looked healthy well not pale or jaundiced Herpulse rate was 74minute and her blood pressure was Her body mass index BMI was All systems examinations were normal except for bilateral nasalpolyps Complete blood count CBC renal function testREF electrolyte liver function test LFT thyroid function test TFT urine analysis general urine test antinuclear antibody ANA and rheumatoid factor RFwere all normal An imaging profile included lumbosacral MRI a computed tomography CT scan of her sinuses and electrocardiogram ECG which were normalexcept for bilateral nasal polyps and severe sinusitis thatlooked allergic to fungi in natureShe underwent FESSsurgery to remove the polyps and clean out her sinusesup to weeks after surgery she used nasal steroidsmometasone furoate two times a day but hersymptoms regarding anosmia were not improved MRIof her brain and a CT scan of her sinuses were done andboth revealed normal features A vitamin D deficiencywas suggested and the laboratory results revealed a lowvitamin D level of ngml Treatment with vitamin Dsupplement was prescribed at international unitsIU weekly for weeks and then IU maintenancedose daily she was advised to take food rich in vitaminD and get exposed to sunlight for minutes three timesa week after the loading dose of supplement She was atregular followup for months at rates of weekly for thefirst month every weeks for the second month andmonthly for the rest of the followup period At eachvisit she was assessed with clinical history and examination It was noticed that the symptoms of tirednesssleeping anosmia and back pain were dramatically improving during that period At the months followupher blood level of vitamin D was normal she describedher condition as free from all symptoms and shereturned back to normal physical activityDiscussion and sThis was a nonclassical case of vitamin D deficiency ofa 26yearold woman who presented with chronic anosmia and recurrent nasal polyps She was diagnosed ashaving a case of vitamin D deficiency and respondedwell to vitamin D replacement therapy This case correlated an association between decreased levels of vitaminD and recurrent nasal polyps that led in time to chronicanosmia as a result of chronic high sensitivity reactionstriggered by our patient™s autoimmune system The literature links chronic rhinosinusitis with nasal polypsCRSwNP with asthma and allergic rhinitis but the cellular and molecular mechanisms that contribute to theclinical symptoms are not fully understood Sinonasalepithelial cell barrier defectsincreased exposure topathogenic and colonized bacteria and dysregulation ofthe host immune system are all thought to play prominent roles in disease pathogenesis []Despite all the previous surgical and medical interventions over the past years our patient™s condition did notimprove and she still complained of anosmia A study revealed that this patient was experiencing excessive allergicreactions that led to recurrent nasal polyps It is wellknown that classical clinical effects of vitamin D deficiencyare bones and musculoskeletalrelated disorders severallines of evidence demonstrate the effects of vitamin D onproinflammatory cytokines regulatory T cells and immune responses with a conflicting interpretation of theeffects of vitamin D on allergic diseases []The working diagnosis was suggested in relation tosome musculoskeletal symptoms and chronic fatigue especially when the imaging profile for her lower back andall routine investigations were normal It has been suggested that clinicians should routinely test for hypovitaminosis D in patients with musculoskeletal symptomssuch as bone pain myalgias and generalized weaknesswhich might be misdiagnosed asfibromyalgia andchronic fatigue [] The most common causes of anosmia were assessed as well and they were negative theseincluded sinonasal diseases post infectious disorder andposttraumatic disorder and congenital defects and disorders caused by neurodegenerative disease [] 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of Authors™ contributionsMI analyzed and interpreted the findings of the case report and was themajor contributor in writing the manuscript ME reviewed the report andadded valuable comments All authors read and approved the finalmanuscriptFundingNoneAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from Albasar Institutional Review BoardConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum Sudan 2Preventive Medicine and EpidemiologyAlneelain University Khartoum SudanReceived March Accepted July Thus blood level for vitamin D was requested and theresults were of low D levelIn the past history of the previous nasal polyps surgeries our patient noted that there was no anosmia and hermain complaints were classic complaints of sinusitis including sneezing nasal blockage and headache Soonafter surgery her symptoms improved except for theallergyrelated symptoms despite usage of inhaled steroids spray She stated that at the last time the presentation was different since it was only anosmia indicatingthat there was significant inflammation that affected thesmell receptors around the olfactory epithelium Afterthe last nasal polyps and sinuses drainage surgery thesymptoms related to allergic reactions including chronicsneezing did not improve for up to weeks and she wasstill suffering from hyposmia although that was a fairpostoperative period for recoveryThe symptoms of anosmia and sneezing and othersystematic symptoms gradually started to improve aftervitamin D supplements indicating that the main reasonbehind her symptoms was vitamin D deficiency She wasfollowed up for up to months after establishment ofvitamin D supplements and at the last followup she hada normal sense of smell and she was free from backpain fatigue and allergyrelated symptomsThis was a nonclassical presentation as our patientwas young and she did not have alkaline phosphatasecalcium and phosphorus abnormalities [] that are expected in cases of vitamin D deficiencyThis case revealed an association between decreasedlevels of vitamin D and recurrent nasal polyps that ledto anosmia as a result of hypersensitive reactions produced by the body™s systemsAlthough vitamin D deficiency is prevalent measurement of serum 25OHD level is expensive and universalscreening is not supported However vitamin D testingmay benefit those at risk for severe deficiencyIt is highly recommended to consider vitamin D deficiency among all patients with unspecified symptoms orin cases of nondiagnosed disorder regardless of the presenting complaintIn there is an association between vitaminD deficiency and recurrent allergic nasal conditionsAbbreviationsCRSwNP Chronic rhinosinusitis with nasal polyps CT Computedtomography ECG Electrocardiogram ENT Ear nose and throatFESS Functional endoscopic sinus surgery BMI Body mass indexCBC Complete blood count RFT Renal function test LFT Liver function testTFT Thyroid function test ANA Antinuclear antibody RF Rheumatoid factorIU International unitReferencesNational Institutes of Health Vitamin D fact sheet for health professionals httpsodsodnihgovfactsheetsVitaminDHealthProfessionalen2Accessed Apr National Institutes for Health NIH Vitamin D fact sheet for consumers httpsodsodnihgovfactsheetsVitaminDConsumer Accessed Dec Kuriacose R Olive KE Vitamin D insufficiencydeficiency managementSouth Med J “ httpsdoi101097SMJSizar O Khare S Givler A Vitamin D deficiency Treasure Island Stat PearlsPublishing httpswwwncbinlmnihgovbooksNBK532266 PMID Accessed Dec Wlliam B Fatme A Meis M Targeted 25hydroxyvitamin D concentrationmeasurements and vitamin D3 supplementation can have important patientand public health benefits Eur J Clin Nutr “ httpsdoi101038s4143002005640Hanmin W Weiwen C Dongqing L Xiaoe Y Xiaode Z Nancy O et alVitamin D and chronic diseases Aging Dis “ httpsdoi1014336AD20161021 Whitney W Ropert P Robert C Chronic rhinosinusitis with nasal polyps JAllergy Clin Immunol Pract “ httpsdoi101016jjaipThacher TD Clarke BL Vitamin D insufficiency Mayo Clin Proc “ httpsdoi104065mcp20100567Kennel KA Drake MT Hurley DL Vitamin D deficiency in adults when totest and how to treat Mayo Clin Proc “ httpsdoi104065mcp20100138Sanne B Elbrich M Duncan B Antje W Veronika S Joel D et al Anosmia aclinical review Chem Senses “ httpsdoi101093chemsebjx025Shikino K Ikusaka M Yamashita T Vitamin Ddeficient osteomalacia due toexcessive selfrestrictions for atopic dermatitis BMJ Case Rep httpsdoi101136bcr2014204558AcknowledgementsNot applicablePublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Health related quality of life functional impairment and comorbidity in people with mild to moderate chronic kidney disease a cross sectional studySimon DS Fraser Jenny Barker1 Paul J Roderick1 Ho Ming Yuen1 Adam Shardlow2 James E Morris1 Natasha J McIntyre2 Richard J Fluck2 Chris W McIntyre3 Maarten W Taal To cite Fraser a0SDS Barker a0J Roderick a0PJ et a0al Health related quality of life functional impairment and comorbidity in people with mild to moderate chronic kidney disease a cross sectional study BMJ 202010e040286 101136bmj 2020040286 –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 May Revised June Accepted July Authors or their employers Re use permitted under CC BY Published by BMJ1School of Primary Care Population Sciences and Medical Education Faculty of Medicine University of Southampton Southampton UK2The Department of Renal Medicine Royal Derby Hospital NHS Foundation Trust Derby UK3Department of Medical Biophysics University of Western Ontario London Ontario Canada4Division of Medical Sciences and Graduate Entry Medicine University of Nottingham Derby UKCorrespondence toDr Simon DS Fraser S Fraser soton ac ukObjectives To determine the associations between comorbidities health related quality of life HRQoL and functional impairment in people with mild to moderate chronic kidney disease CKD in primary careDesign Cross sectional analysis at year follow up in a prospective cohort studySetting Thirty two general practitioner surgeries in EnglandParticipants participants with CKD stage of people recruited at baseline in the Renal Risk in Derby study who survived to years and had complete follow up data for HRQoL and functional status FSPrimary and secondary outcome measures HRQoL assessed using the level EQ 5D version EQ 5D 5L with domains of mobility self care usual activities paindiscomfort and anxietydepression and index value using utility scores calculated from the English general population and FS using the Karnofsky Performance Status scale functional impairment defined as Karnofksy score ‰ Comorbidity was defined by self reported or doctor diagnosed condition disease specific medication or blood resultResults Mean age was years The numbers reporting some problems in EQ 5D 5L domains were for mobility for self care for usual activities for paindiscomfort and for anxietydepression Only reported no problems in any domain HRQoL index values showed greater variation among those with lower FS eg for those with Karnofsky score of the median IQR EQ 5D index value was to compared with to for those with Karnofsky score of Overall had functional impairmentIn multivariable logistic regression models functional impairment was independently associated with experiencing problems for all EQ 5D 5L domains mobility OR CI to p0001 self care OR CI to p0001 usual activities OR CI to p0001 paindiscomfort OR CI to p0001 anxietydepression CI to p0001 Higher comorbidity count and obesity were independently associated with problems in mobility self care usual activities and paindiscomfort for three or Strengths and limitations of this study –º This study involved a large cohort of people with chronic kidney disease CKD recruited from primary care a setting in which patients with mild to moderate CKD are typically managed in the UK –º A broad range of comorbidities were included but they were identified at baseline only not at follow up by which time the number of comorbidities may have changed –º Health related quality of life and functional status were measured in the same patient group and the use of the EQ 5D 5L index measure and data from the Health Survey for England enabled comparison with a general population –º Health related quality of life and functional status measures were taken at year follow up but not at baseline and we were therefore unable to identify change over time –º This was a cross sectional study of survivors and we are therefore not able to draw causative linksmore comorbidities versus none mobility OR CI to p for trend self care OR CI to p for trend usual activities OR CI to p for trend paindiscomfort OR CI to p for trend and for obese body mass index BMI ‰¥ kgm2 versus BMI kgm2 mobility OR CI to p for trend self care OR CI to p for trend usual activities OR CI to p for trend paindiscomfort OR CI to p for trend Female sex lower FS and lower educational attainment were independently associated with anxietydepression ORs CI to p CI to p0001 and CI to p respectively Older age higher comorbidity count albuminuria ‰¥ mgmmol vs mgmmol lower educational attainment no formal qualifications vs degree level and obesity were independently associated with functional impairment ORs CI to p0001 CI to p for trend Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access CI to p for trend CI to p for trend and CI to respectivelyConclusions The majority of persons with mild to moderate CKD reported reductions in at least one HRQoL domain which were independently associated with comorbidities obesity and functional impairmentTrial registration number National Institute for Health Research Clinical Research Portfolio Study Number INTRODUCTIONChronic kidney disease CKD is common globally affecting about of the general adult population with CKD stage the most prevalent category1 Current treatment guidelines for CKD are disease specific and focus on reducing progression and preventing complications such as cardiovascular disease3 However in the UK most people with CKD stage are managed in primary care and in this context only a minority evidence progression over years4 The risk of end stage kidney disease ESKD is extremely low Conversely comorbidities additional chronic diseases are common in individuals with CKD and can worsen clinical outcomes and health related quality of life HRQoL5 Ninety six per cent of people with stage disease have at least one comorbidity around have a comorbidity count of two or more6A significant body of research has explored HRQoL and the functional status FS of people with ESKD or following kidney transplant but these factors are not well explored in those with less severe CKD Among people with high risk CKD in the Renal Impairment In Secondary Care study reported problems in one or more of the EQ5D domains7 This is a clinically important knowledge gap because mild to moderate reductions in glomerular filtration rate GFR are usually asymptomatic so improved understanding of the comorbidities and symptoms that affect HRQoL and FS in this group of people is important to facilitate a holistic approach to management The objective of this study was therefore to evaluate HRQoL and determine the associations between comorbidities HRQoL and functional impairment in people with mild to moderate CKD in primary careMATERIALS AND METHODSA detailed description of the Renal Risk in Derby RRID study methodology has been published elsewhere9 In summary approximately people with CKD stage were identified from renal registers at primary care clinics in Derbyshire UK between and and invited to participate in the study Of these people attended initial baseline visits and met eligibility criteria age ‰¥ years two estimated GFR eGFR results derived from the Modification of Diet in Renal Disease study MDRD equation of “ mLmin173 m2 at least days apart9 People with a life expectancy of less than year who were unable to attend study visits or who had a solid an transplant were excludedHealthrelated quality of lifeHRQoL was assessed at year follow up using the EQ 5D 5L a widely used validated measure of health status that can be standardised to different populations EQ 5D 5L consists of two aspects a descriptive system in which participants are asked to rate their health state from to against five domains mobility self care usual activities paindiscomfort anxietydepression and the EQ visual analogue scale EQ VAS in which participants rate their health on a scale ranging from ˜the best health you can imagine™ to ˜the worst health you can imagine™ An EQ 5D 5L value set has previously been published for England11 However concerns have been raised about the quality and reliability of the data collected in the valuation study such that the English National Institute for Health and Care Excellence NICE recommend ˜If data were gathered using the EQ 5D 5L descriptive system utility values in reference case analyses should be calculated by mapping the L descriptive system data onto the L value set™ For these analyses individual health states were therefore converted using the EuroQol EQ 5D 5L Crosswalk Index Value Calculator into a single 3L index value a preference based score that typically ranges from states worse than dead to full health with dead at using utility scores calculated from the English general population13 The index value and the EQ VAS score were used to graphically display the relationship between HRQoL and FSFunctional statusFS defined in this paper as the physical ability to perform normal activities and independently self care was assessed at year follow up using the Karnofsky Performance Status KPS scale The KPS is a clinician assessed score originally developed in oncology and was used for assessing prognosis and management in patients with cancer15 The scale ranges from ˜normalno complaints™ to ˜dead™ Theoretically the scale can take any whole number value within the range but in practice results are commonly recorded as multiples of therefore KPS was treated as an ordinal variable in this study The original continuous KPS score is defined as being ˜able to carry on normal activity and to work with no special care needed™ a score of between and inclusive is defined as ˜unable to work able to live at home and care for most personal needs varying amount of assistance needed™ and a score of less than or equal to is defined as ˜unable to care for self requiring the equivalent of institutional or hospital care™ Functional impairment was analysed as a binary outcome due to the small number of patients with low KPS score A KPS score of ‰ versus was chosen to compare those able to carry on normal life with those experiencing some functional impairment as has been used in evaluation of FS in patients with lung cancer16Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cComorbidities identified at baselineThe methods for defining comorbidities in participants have been described in detail elsewhere6 In brief eleven comorbidities were pragmatically identified at baseline using information from a combination of sources and agreed by consensus between three clinicians SF MWT and PJR patient questionnaires in which patients were asked to list chronic medications followed by verbal confirmation with verification of repeat prescriptions where possible blood pressure measurement at the time of baseline study visit and self reported clinical diagnoses Self reported comorbidities included heart failure ischaemic heart disease peripheral vascular disease defined as peripheral arterial revascularisation or amputation and cerebrovascular disease stroke or transient ischaemic attack Diagnoses of chronic respiratory disorder depression painful condition hypertension diabetes and thyroid disorders were made according to medication history or patient report Anaemia was defined according to Kidney Disease Improving Global Outcomes KDIGO guidelines as haemoglobin gdL gL in men and gdL gL in women at baseline17 Hypertension was defined either by medication history or by a systolic blood pressure mm Hg or diastolic mm Hg at baselineKidney functionKidney function was assessed at year follow up eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation and was treated as a continuous variable The urine albumin to creatinine ratio uACR from three consecutive early morning specimens was used for analysis uACR was categorised into three levels according to KDIGO guidelines and fitted as a discrete variable in regression analysesMethods for defining CKD progression have also been detailed elsewhere4 In summary progression of CKD was defined as a decline in GFR coupled with a worsening of GFR category or an increase in albuminuria category CKD remission was defined as the presence of both eGFR mLmin173 m2 and uACR mgmmol at any study visit in an individual who had previously met KDIGO diagnostic criteria for CKDOther baseline measuresBody mass index BMI was calculated from weight in kilograms divided by square of height in metres and was treated as a categorical variable18 Smoking status was categorised as never smoked ex smoker and current smoker Socioeconomic status was assessed using self reported educational attainment categorised into no formal qualifications school or equivalent qualifications and degree or equivalent qualifications as well as the Index of Multiple Deprivation IMD score categorised in quintiles19 The IMD is a measure of relative deprivation for small areas of residence in England and combines information from seven domains income employment education skills and training health and disability crime accessbarriers to housing and services and living environment Self reported ethnicity status was also collectedStatistical analysesDescriptive statistics were used to show the characteristics of the study participants at year follow up Descriptive statistics were also used to show the distribution of functional impairment KPS ‰ among those reporting problems in the five EQ 5D 5L domains Associations between the patient reported EQ 5D 5L domains and FS was assessed using the χ2 test Ratings from the five participant reported EQ 5D 5L domains were also compared between the RRID cohort and those reported by people aged years and over in the Health Survey for England HSE”which is representative of the England population20 A comparison of basic characteristics was also made between those with and without complete year follow up dataUnivariable logistic regression models were used to assess the relationships between having ˜some problems™ in each EQ 5D 5L domain and each predictor variable including comorbidity count and year five eGFR Variables considered to be clinically relevant and where p01 on univariable analysis were subsequently included in multivariable logistic regression models This process was then repeated for the relationship with the outcome variable functional impairment Due to the small number of non white participants ethnicity was not included in the modelsIn the regression models interactions between the individual and area measures of socioeconomic status were also tested because of the potential for the relationship between individual socioeconomic status indicated by educational attainment and HRQoL to vary by area deprivation particularly for older people21 The level of significance was set at All analyses were performed using StataIC V150Patient and public involvementThe RRID study design was discussed with a patient and two feedback meetings for participants and their families were anised after the year visits which were well attended In addition a web page provides updates and information for participants httpswww uhdb nhs uk renal risk in derby rrid studyRESULTSOf participants recruited survived to years and of these participants of survivors had complete year follow up data for HRQoL and FS figure The mean age of the cohort was years SD and the majority n621 were female table Approximately half n506 reported having had no formal education just under half n497 lived in areas of lower deprivation IMD quintiles four or five and the majority n994 were white The Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Figure Flow chart of study participants CKD chronic kidney disease KDIGO Kidney Disease Improving Global Outcomes GP general practitioner QoL quality of lifemean eGFR at follow up was mLmin173 m2 SD and almost half n460 had had stable CKD over the preceding year period Only had no comorbidities and about a third n344 had three or more comorbidities For comparison of basic characteristics of those with and without complete year follow up data see online supplementary table S1 A slightly higher proportion of those with incomplete follow up data had three or more comorbidities and only a very small proportion had functional impairment online supplementary table S1The majority reported some impairment in HRQoL overall with a median score of out of IQR “ on the EQ VAS A minority n378 had an EQ 5D 3L index score higher than the agesex matched median and only of people n191 reported no problems across any of the individual HRQoL domains Furthermore a majority of participants reported some problems with mobility n582 and paindiscomfort n712 table When comparing the self reported HRQoL domains with HSE data the proportion of people in the RRID population reporting problems with mobility or paindiscomfort was higher vs and vs respectively than in the HSE population table For clinician assessed FS only two participants had performance status assessed as KPS ‰ ˜unable to care for self requiring the equivalent of institutional or hospice care™ and were assessed as KPS “ ˜unable to work able to live at home and care for most personal needs varying amount of assistance needed™The association between clinician assessed FS and patient reported HRQoL was complex either when based on the index score figure 2A or the VAS scale figure 2B HRQoL was generally higher among those with better FS However the spread of HRQoL scores using either of the HRQoL metrics was broader among those with lower FS suggesting a greater degree of variation in HRQoL among those with lower FS than among those with higher FS figure A higher proportion of people with clinician assessed functional impairment KPS ‰ reported having some degree of problems in each of the five EQ 5D 5L domains than people without functional impairment online supplementary table S2Using the mobility domain as an example table on univariable analysis older age greater area deprivation level higher number of comorbidities poorer FS lower eGFR higher level of albuminuria lower educational attainment and higher BMI were associated with having some problemsIn the fully adjusted multivariable model these associations remained for older age higher number of comorbidities poorer FS and higher BMI table A summary of the main independent associations identified in the multivariable logistic regression models for usual activities self care paindiscomfort and anxietydepression is shown in table and the full analyses in online supplementary tables S3 to S6Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cTable Characteristics of patients at year follow up in the Renal Risk in Derby study n1008 unless otherwise statedVariableCategoryAge in years mean SDAge group n years Sex  n “ years yearsMaleFemaleEducational attainment  n n1007Index of Multiple Deprivation IMD quintile relative to England  n n1006Ethnicity  n WhiteOther¡No formal qualificationsGCSE A level NVQ “First or higher degree NVQ “Quintile most deprivedQuintile Quintile Quintile Quintile least deprivedNormal or underweight kgm2Overweight “ kgm2Obese ‰¥ kgm2Never smokedEx smokerCurrent smokerBody mass index  n Smoking status  n eGFR in mLmin173 m2 mean SD n1007uACR in mgmmol median IQR n1007KDIGO uACR categories n KDIGO eGFR categories eGFR in mLmin173 m2Progression of kidney disease n Number of comorbidities  n A1A2A3G1 eGFR ‰¥G2 eGFR “G3a eGFR “G3b eGFR “G4 eGFR “G5 eGFR StableProgressionRemissionNone CKD onlyOneTwoThree or moreDescriptive statistics “ ContinuedTable ContinuedVariableCategory accessDescriptive statisticsIndividual comorbidities  n Quality of life domains any problems reported in each EQ 5D 5L domain n Functional status KPS score n HypertensionPainful conditionAnaemiaIschaemic heart diseaseDiabetesThyroid disorderCerebrovascular diseaseChronic respiratory disorderDepressionPeripheral vascular diseaseHeart failureMobility problemsSelf care problemsUsual activity problemsPaindiscomfortAnxietydepressionNo problems in any domainFunctional impairment KPS ‰KPS able to carry on normal activity and to work no special care needed Where variable category percentages sum to less than or more that this is due to roundingVariables assessed at year follow up Variables assessed at baseline¡Includes mixed Asian Cypriot and otherCKD chronic kidney disease eGFR estimated glomerular filtration rate GCSE General Certificate of Secondary Education KDIGO Kidney Disease Improving Global Outcomes KPS Karnofsky Performance Status A level advanced level NVQ National Vocational Qualifications uACR urine albumin to creatinine ratioFactors associated with a lower FS on univariable analysis included older age lower socioeconomic status assessed by both IMD score and educational attainment higher number of comorbidities obesity reduced eGFR and greater degree of albuminuria Other than reduced eGFR all of these factors remained significant after adjustment table No interactions were identified in any analysesDISCUSSIONIn this cross sectional study of people with mild to moderate CKD who survived to year in a UK primary care cohort overall patient reported HRQoL was relatively high Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Table Comparison of the EQ 5D 5L quality of life domains between the Health Survey for England HSE and the Renal Risk in Derby RRID cohortHSE cohort n258nRRID CKD cohort n1008nMobility no problems in walking about “ some problems Self care no problems washing or dressing “ some problemsUsual activities no problems ng usual activities “ some problems Paindiscomfort no pain or discomfort “ some pain or discomfort Anxietydepression not anxious or depressed “ some anxiety or depression All participants were aged years or aboveCKD chronic kidney diseasethough a substantial proportion of participants reported problems in each HRQoL domain A majority reported problems with mobility and paindiscomfort Although most people had a clinician assessed FS suggesting that they were able to carry on normal activity and to work with no special care needed about a quarter were assessed as having functional impairment being unable to work but able to live at home and care for most personal needs with a varying amount of assistance needed HRQoL was generally higher among those with better FS but there was more variation in HRQoL among those with lower FS and low FS was independently strongly associated with low HRQoL in regression analyses Higher number of comorbidities and obesity were independently associated with problems in most EQ 5D 5L domains and with functional impairment Functional impairment was independently associated with experiencing some problems across all EQ 5D 5L domainsFigure Relationship between quality of life and functional status A Functional status by Karnofsky score and quality of life by EQ 5D 3L Index score B Functional status by Karnofsky score and quality of life by EQ 5D self reported visual analogue scale VASThis study had several strengths including the large size of the cohort and recruitment from primary care a setting in which patients with mild to moderate CKD are typically managed The RRID cohort is pragmatic and likely to represent a population of typical patients with mild to moderate CKD in the UK22 We were able to identify a broad range of comorbidities but they were identified at baseline only The number of comorbidities may therefore have changed by the time of follow up assessment meaning that our comorbidity prevalence data were likely underestimates of the true prevalence in some patients Similarly certain other exposures were assessed at baseline and could potentially have changed by the time of follow up We recognise these as important limitations but consider that they are unlikely to significantly alter the main findings of our study with regard to HRQoL and FS A further strength is that we were able to measure HRQoL and FS in the same patient group and the HRQoL and FS data were relatively complete The use of the EQ 5D 5L index measure and data from the HSE enabled comparison with a general population However the index values for HRQoL required conversion to 3L values as reliable 5L index values are not yet available for all standard populations Evidence from a previous RRID analysis on prior renal function change provided depth to our analyses for this cross sectional study However there were also several important limitations”this was a Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0cTable Logistic regression models examining associations between lower quality of life EQ 5D 5L mobility domain categorised as ˜no problems™ vs ˜any problems™ and patient characteristics accessUnivariableOR CIP valueMultivariableOR CIP value“   to “  to to Quintile most deprived Quintile Quintile Quintile Age yearsFemale sex vs maleIndex of Multiple Deprivation IMD quintile relative to England vs quintile least deprived n1006 Number of comorbidities vs no comorbidities Functional status KPS score vs KPS eGFR mLmin173 m2 N1007 to to to to to to to One Two Three or more Functional impairment KPS ‰ to   to to to to to to to to to to to to A2 “ mgmmol A3 ‰¥ mgmmol No formal qualifications GCSE A level or NVQ “uACR KDIGO categories vs category A13 mgmmol n1007 Educational attainment vs first or higher degree or NVQ “ n1007 BMI vs kgm2 Smoking status vs never smoked Overweight BMI “ kgm2 Obese BMI ‰¥ kgm2 to to to to to to Current smoker Ex smoker     to to to to    to to  “““n1008 in univariable models unless otherwise stated n1005 for final multivariable logistic regression modelsAdjusted for age deprivation level number of comorbidities functional status estimated glomerular filtration rate eGFR at year follow up urinary albumin to creatinine ratio uACR at year follow up educational attainment and body mass index BMI P value for trendGCSE General Certificate of Secondary Education KDIGO Kidney Disease Improving Global Outcomes KPS Karnofsky Performance Status A level advanced level NVQ National Vocational Qualificationscross sectional study of survivors and we were therefore not able to draw causative links HRQoL and FS measures were taken at year follow up but not at baseline and we were therefore unable to identify change over time The RRID cohort predominantly comprises people of white ethnicity limiting generalisability of our findings Comparison with HSE data was undertaken only via univariable analyses such that potential confounding factors may have influenced the differences observed between the two groups We also did not have sufficient numbers to allow for reliable exploration of associations between specific comorbidities and HRQoL or FS We also recognise the need for caution in the interpretation of the associations between functional impairment and problems in individual domains due to small numbers in some individual categories leading to wide CIs A further limitation is that one inclusion criterion was the ability to attend study visits which would have resulted in some selection bias by excluding the very frailPeople with CKD are likely to have multiple comorbidities due both to the nature of the disease process and the relationship between CKD and older age We have identified that comorbidity count was an independent determinant of both HRQoL and FS highlighting the importance of a holistic approach that includes attention to comorbidities in the management of people with Fraser a0SDS et a0al BMJ 202010e040286 101136bmj 2020040286 0c access Table Summary matrix of the independent associations with ˜some problems™ in each domain of the EQ 5D 5L from multivariable logistic regression analysesMobility Self care Usual activitiesPaindiscomfortAnxietydepressionIncreasing ageFemale sexGreater area deprivation levelHigher number of comorbiditiesFunctional impairmentLower eGFRHigher level of albuminuriaLower educational attainmentHigher BMISmokingΟ ΟΟ Ο ΟΟ ΟΟBMI body mass index eGFR estimated glomerular filtration rate ΟΟ Ο ΟΟ Ο Ο Ο Ο mild to moderate CKD As reported previously of people in this cohort with stage disease had at least two comorbidities6 There are clearly shared risk factors for several of the included comorbidities It is therefore perhaps unsurprising that in a large cohort of over half a million Canadian patients with CKD comorbidities such as hypertension and diabetes were common and respectively However a substantial number of patients also had ˜discordant™ comorbidities such as chronic pulmonary disease and of patients had chronic pain5 Comorbidities were all associated with an increased risk of hospitalisation5 It is striking that the majority of people in our cohort reported problems with mobility and chronic paindiscomfort and that both were more prevalent than in a nationally representative sample of the English general population of similar age About of our cohort were taking analgesic medication but about reported pain or discomfort in the EQ 5D 5L This likely reflects the association of CKD with comorbidities since mild to moderate reductions in GFR are unlikely to cause poor mobility or pain Nevertheless this observation further highlights the need to pay attention to mobility issues and pain management in order to improve quality of life in people with stage CKDThe prevalence of diabetes in this population of people with CKD stage was This is lower than the prevalence of noted in analyses of CKD prevalence in the HSE2 It is possible that this relates to this study population comprising survivors at years in a cohort study and those with diabetes may have been more likely to die prior to these analyses than those without The study population was also predominantly white and those ethnic groups with greater diabetes prevalence were therefore under representedMental health problems are common with of adults in England reporting a diagnosis at some point in time24 In our study about of people were classified as having a depressive condition defined pragmatically based on current antidepressant use or patient self report although about reported some anxiety or depression in the EQ 5D 5L so this was probably an underestimate In a large meta analysis approximately of adults with CKD stage “ had symptoms suggestive of depression25 This appears to persist even in milder forms of the disease and a large study from showed the prevalence of depression in people with CKD whose eGFR was ‰¥ mLmin173 m2 was These data imply that careful attention to mental health problems including screening for depression may also be key interventions to improve HRQoL in

Thyroid_Cancer

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so 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 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Intervertebral disc degeneration IDD is the most common degenerative disease all over the word Our previous studyconfirmed that the downregulated circGRB10 directly interacts with miR3285p which modulate ERBB2 and leads tothe degeneration of intervertebral disc however the underpinning mechanism of circGRB10 dysregulation remainsunclear We identified that FUS and demonstrated that circGBR10 biosynthesis in nucleus pulposus NP cells waspromoted by FUS whose expression was controlled by miR1413p In addition ERBB2 downregulation led todecreased Erk12 phosphorylation which enhanced miR1413p production in NP cells In vivo data indicated that circGRB10 inhibited IDD in rat model The present study revealed that miR1413p and FUS are key factors that regulatecircGRB10 synthesis in NP cells In addition circGBR10 participates in the molecular circuitry that controls human IDDdevelopment These findings provide a basis for further functional diagnostic and therapeutic studies of circGRB10in IDDIntroductionThe Global Burden of Disease Study stated that lowback pain LBP represents an important cause of disability worldwide1 LBP is tightly associated with intervertebral disc degeneration IDD which involves ofall LBP cases causing significant economic and socialburdens worldwide23 According to previous reports of the world population have low back pain during theirlifetime with being chronically disabled4Currently IDD pathogenesis is largely unclear howeverit could be due to microenvironmental alterations in theintervertebral discs caused by various factors such asgenetic features aging sex a predisposing injury and theCorrespondence Wei Guo guow0319163com1Department of Orthopaedics Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R China2Department of Breast Surgery Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R ChinaFull list of author information is available at the end of the Edited by G Calinenvironment56 The main feature of IDD pathology iselevated biosynthesis of catabolic enzymes combined withreduced extracellular matrix ECM accumulation causedby imbalanced anabolism and catabolism7 Intervertebraldiscs comprise a central nucleus pulposus NP a peripheral annulus fibrosus AF and cartilaginous endplates which connect overlying capillary beds craniallyand caudally The NP maintains homeostasis by producing an ECM mostly comprising type II collagen andproteoglycans the main functional components of intervertebral discs which are indispensable to maintain thedisc height and absorb various mechanical loads8 It iswell known that loss of collagenII and aggrecan is anearly critical event in the degenerative cascade in Intervertebral disc tissue9 MMP13 is the most importantenzymes that hydrolyze collagens10 ADAMTS5 is classified as the major aggrecanases due to their high efficiency in cleaving aggrecan11 A large body of evidencesupporting the involvement of MMP13 and ADAMTS5in IDD pathogenesis12 During IDD the main histologicalalteration involves the centrally located NP cells which 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 0cGuo Cell Death and Disease Page of after a phenotypic transformation are substituted bysmaller fibrochondrocytelike cells with reduced proteoglycan production and a global shift towards synthesizing fibrotic materials and compromising the structuralintegrity of discs1314 Therefore unveiling the mechanisms underpinning such imbalance is urgently needed forthe development of new therapeutic targets in IDDMounting evidence supports roles for circular RNAscircRNAs in IDD15“ Previous research demonstratedthat circRNAs are closed RNAs produced by backsplicingof single premRNAs18 It is not completely known howcircRNAs are biosynthesized although complementaritybetween inverted sequences in flanking introns and theactivity of RNAbinding proteins RBPs increase thecontiguity of splice sites contributing to backsplicing inmammalian cells19“The RBP FUS affects splicing regulation23 with manysplicing factors termed FUS interactors24“ FUS mutations could lead to protein mislocalization to the cytosolwith decreased nuclear FUS amounts and occurrence ofabnormal cytosolic aggregates2728 The FUS protein isinvolved in regulating intracellular RNA transport mRNAsynthesis alternative splicing and polyadenylation siteselection29 He demonstrated that FUS combinedwith circ_002136 and promoted the generation ofcirc_002136 in Glioma30 It was recently shown that FUScontrols the expression of circRNAs by binding tointrons that flank the splicing junction31 Moreover FUSwas reported to be regulated by many miRNAs includingmiR1413p3233 Studies revealed miR1413p is upregulated in NP tissue specimens from IDD cases anddemonstrated that miR1413p is associated with discdegeneration34 However the function and mechanism ofFUS as well as the interaction between FUS and miR1413p in IDD have not been reportedOur previous research confirmed that circGRB10amounts are markedly reduced in NP cells from IDDpatients which accelerates IDD development by enhancing miR3285p mediated ERBB2 suppression in NPcells15 However the mechanism of circGRB10 downregulation in degenerative NP cells remains unclear Inthis study we demonstrated that the miR1413p whichis significantly increased in degenerative NP cells34 regulate expression of the FUS which is responsible for thegeneration of circGRB10 in NP cells Furthermore weshowed that ERBB2 downregulation led to decreasedErk12 phosphorylation and the decreased levels of Erk1 phosphorylation enhanced miR1413p biogenesis indegenerative NP cells promoting IDD developmentTaken togetherthese findings suggested circGBR10contributes to the molecular circuitry controlling IDDdevelopment in humansOfficial journal of the Cell Death Differentiation AssociationResultsCircGRB10 regulates NP cell functions through the ERBB2Erk signaling pathwayOur previous study revealed circGRB10 promotes NPcell survival by increasing ERBB2 amounts via suppression of miR3285p However the effect of circGRB10expression on NP cell anabolism or catabolism remainsobscure To further assess circGBR10™s functions in IDDpathogenesis circGRB10 or circGRB10 small interferingRNA siRNA was transiently transfected into culturedprimary human NP cells As shown in Supplementary FigS1 overexpression and knockdown of circGRB10 haveno effect on linear GRB10 but only affect circular GRB10The immunofluorescence results demonstrated that afteroverexpressing circGRB10 in NP cellssignificantlyupregulation of collagen II and aggrecan and decreasedamounts of MMP13 and ADAMTS5 were found Conversely circGRB10 knockdown resulted in oppositeeffects Fig 1a b These findings were confirmed by qRTPCR Fig 1csignificantlyincreased while MMP13Our previous research demonstrated circGRB10 inhibits IDD development by regulating ERBB2 expression inNP cells Increasing evidence supports an important rolefor the ERBB2 gene and Erk signaling pathways in theprogression of many human diseases35“ Meanwhile theErk pathway is altered during IDD38 and plays a significant role in extracellular metabolism39 These resultsprompted us to assess the plausible association of circGRB10 with ERBB2 Erk signaling In this study primaryhuman NP cells underwent transfection with circGBR10circGRB10 siRNA and respective negative controlsrespectively As shown in Fig 1d e western blot assayshowed that pErk12 collagen II and aggrecan amountswereandAMADT5 levels were reduced in NP cells overexpressingandcircGRB10 Conversely pErk12aggrecan were downregulatedandAMADT5 amounts were increased in NP cells transfected with circGRB10 siRNA Fig 1d e FurthermoreERBB2 affected pErk12 in a similar way as circGRB10Fig 1d e suggesting cricGRB10 modulates IDD progression via ERBB2Erk signaling Therefore in order tofurther validated whether ERBB2 was the downstreammediator of circGRB10 in the NP cells We cotransfectedcircGRB10 and ERBB2 siRNA into NP cells andobserved that the positive effects of circGRB10 on NPcells functions were attenuated in the absence of ERBB2Fig 1f g Moreover upregulation of ERBB2 counteracted the inhibitory effect of circGRB10 knockdown onNP cells function Fig 1hi Collectively the abovefindings indicated that circGRB10 associated protectionin IDD may involve ERBB2Erk signalingcollagen IIand MMP13 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig circGRB10 regulates NP cell functions through ERBB2Erk signal pathway a CollagenII aggrecan MMP13 ADAMT5 expression wereanalyzed in circGRB10 or circGRB10 siRNA transfected cultured primary human NP cells using Immunofluorescence staining analysis b Thecorresponding bar graphs show quantitative analysis of the relative fluorescent value of each group n replicates per group p p Scale bar μm c qRTPCR showing the expression levels of collagen II aggrecan MMP13 ADAMT in human NP cells after circGRB10overexpression or knockdown Three independent experiments are presented as mean ± SEM error bars P P d The expressionlevels of CollagenII aggrecan MMP13 ADAMT5 and pErk12 were detected by western blot Quantitative analysis was shown in e and threeindependent repeats were performed in each experiment p f NP cell were cotransfected with circGRB10 and ERBB2 siRNA Western blotassay showed that ERBB2 siRNA blocked the effect of circGRB10 on CollagenII aggrecan MMP13 and ADAMT5 expression Quantitative analysis wasshown in g and three independent repeats were performed in each experiment p h NP cell were cotransfected with circGRB10 siRNAand ERBB2 Western blot assay showed that ERBB2 attenuated the effect of circGRB10 siRNA on CollagenII aggrecan MMP13 and ADAMT5expression Quantitative analysis was shown in i and three independent repeats were performed in each experiment p Key factors regulating circGRB10 formationIn this study we found a highly reverse complementarysequence nt upstream the ² splice site in intron andone nt downstream the ² splice site in intron which were named 2RC reversecomplementarysequence in intron and 6RC reverse complementarysequence in intron respectively Then wildtypesequence spanning from intron to intron of theGRB10 gene and multiple deletion constructs “for circGRB10 were introduced into pcDNA31respectively Fig 2a Upon transfection the wildtypevector unlike the 2RC andor 6RC deletion constructs “ overexpressed circGRB10 indicating 2RCand 6RC may contain the binding sites that regulate circGRB10 biogenesis Fig 2bcould beregulated by RBPs postAs circRNAs are derived from premRNAs and circRNAstranscriptionally18212240 we hypothesized that circGBR10 ismodulated by RBPs posttranscriptionally in IDD development To identify the RBPs which might regulateGRB10 premRNA splicing to generate circGRB10 weincubated biotin labeled sequences cloned from circGRB10 back splicing site nt upstream P1 or ntdownstream P2 with nuclear protein extracts fromnormal human NP cells Fig 2c Nuclear proteins boundto RNA underwent separation by SDSPAGE and silverstaining Fig 2dfollowed by mass spectrometry foridentification A total of proteins SupplementaryTable S1 were retrieved and mapped to the STRINGdatabase screening significant interactions with scoresabove Fig 2e Enrichment analysis demonstratedthat these proteins were mainly involved in thepathways of gene expression processing of capped introncontaining premRNA mRNA splicing mRNA splicingmajor pathway mRNA processing and formation andmaturation of mRNA transcript related signaling pathways Fig 2f Among these proteins were involved inthe mRNA splicing and mRNA splicingmajor pathwaySupplementary Table S2 In addition the web tool CircInteractome predicted RBPs which can potentiallybind circGRB10 premRNA Fig 2g Notably FUS wasOfficial journal of the Cell Death Differentiation Associationthe only RBP that was involved in mRNA splicing andcould potentially bind to circGRB10 premRNA suggesting circGRB10 generation may be associated withFUS expression in NP cellsFUS promotes the generation of circGRB10 in NP cellsRecently FUS was reported to have a role in regulatingcircRNA biosynthesis via binding of introns surroundingthe backsplicing junctions31 As shown in Fig 3A FUSamounts in IDD NP tissues were remarkably lower thanthose of controls In addition Western blot further confirmed this result Figs 3b and S2 To assess whetherFUS contributes to circGRB10 production in NP cells weoverexpressed or suppressed FUS and determined circGRB10 amounts qRTPCR demonstrated that FUSoverexpression led to significantly increased circGRB10amounts in NP cells while FUS knockdown reduced theexpression of circGRB10 Fig 3c Moreover FUS had noeffects on linear GRB10 expression Fig 3c Overexpression of FUS resulted in increased collagenII andaggrecan amounts and decreased MMP13 and ADAMT levels in NP cells while the circGRB10 siRNA attenuated these changes Fig 3d FUS knockdown resultedin downregulated collagenII and aggrecan and upregulated MMP13 and ADAMT5 in NP cells while circGRB10 markedly counteracted the effects of FUS knockdown indicating that FUS exerted its functions throughcircGRB10 Fig 3eTo assess whether FUSbinding sequences are important in circGRB10 biosynthesis FUSbinding sequenceswere searched in circGRB10 and surrounding intronsand two putative FUSbinding sites were detected Fig3f Next two short circGRB10 minigenes were engineeredincluding circGRB10s and circGRB10sEmPrecisely circGRB10s comprises presumed FUSbingingsites on both flanking introns preserved with the inverselyinserted ²intron in circGRB10 removed to preventcomplementary sequences from reacting Fig 3f circGRB10sEm resembles circGRB10s but with FUS sitesdeleted from the surrounding introns Fig 3f RIPrevealed an overt interaction of FUS with circGRB10s 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig RBPs interact with GRB10 premRNA a A schematic drawing of four types of circGRB10 overexpressing vectors to The genomicregion for circGBR10 green bars with its wildtype flanking introns black lines was inserted into the pcDNA31 expression vector 2RC and 6RCare indicated by red bars A series of deletions are indicated by black crosses to b qRTPCR showed the expression of circGBR10 aftertransfection with the four types of circGRB10 overexpressing vectors to Three independent repeats were performed in each experimentp c Schematic diagram of RNAs corresponding to different fragments of GRB10 premRNA P1 P2 produced by in vitro transcription inthe presence of biotin for RNA pulldown experiments d Silver stain acrylamide gel of total nuclear proteins before Input and after pulldown withbiotinlabeled RNA probe P1 P2 M molecular weight marker kDa e Proteins identified from mass spectrometry were integrated to STRINGdatabase and constructed Proteinprotein interaction PPI network A densely connected module which contains proteins including FUS wasscreened from the PPI network and these proteins were participate in biological process of mRNA splicing via spliceosome f Pathways enrichmentanalysis of proteins in PPI network g RBPs which can potentially bind circGRB10 premRNAunlike circBsEm Fig 3g indicating FUS required theputative sites in surrounding introns for binding We nextexpressed circGBR10sin FUS overexpressing orknocked down NP cells and circGRB10s yielded elevated amounts of circGRB10 transcript after FUS overexpression and reduced amounts upon FUS knockdownconfirming FUS is important in circGRB10 biosynthesisin NP cellsFig 3h Next circGRB10sEm wasexpressed in NP cells and it yielded markedly reducedcircGRB10 amounts in comparison with circGRB10sFig 3h This indicated that the putative FUSbindingsequences in the surrounding introns were important incircGRB10 biosynthesis Taken togetherthe abovefindings demonstrated that FUS had a critical regulatoryfunction in circGRB10 biosynthesis in NP cells viabinding to recognition sites in the introns surrounding thecircGRB10forming exonsFUS in NP cells is regulated by miR1413pThe mechanism of FUS downregulation in NP cells ofIDD patients remains unclear Previous studies havedemonstrated that FUS is regulated by miRNAs in manydeseases3233 Therefore we hypothesized that FUS maybe regulated by miRNAs in NP cells Using the Targetscan Microt4 miRanda PITA and RNAhybird databasesall predicted miRNAs were retrieved and submitted toVenn analysis Fig 4a The results showed that FUS waspredicted to be regulated by miRNAs SupplementaryTable S3 including miR1413p Svetoni confirmedthat miR1413p regulates FUS expression during neuraldifferentiation and Ji revealed miR1413p is associated with disc degeneration3334 Furthermore qRTPCRshowed that miR1413p was markedly upregulated in NPtissue samples from IDD cases in comparison with controlvalues Fig 4b Therefore we supposed that FUSexpression was regulated by miR1413p in NP cells Tofurther assess miR1413p interaction with FUS luciferasereporter assays were carried out Cotransfection of FUSWT wild type and miR1413p mimic in primary humanNP cells resulted in markedly decreased luciferase activityin comparison with the FUSmut mutantmiR1413pmimic cotransfection group Fig 4c d These findingsOfficial journal of the Cell Death Differentiation Associationwere further confirmed at the gene and protein levels inhuman NP cells in vitro Fig 4e f pointing to FUS as amiR1413p target Then primary human NP cellsunderwent transfection with miR1413p mimic and miR1413p inhibitor and the corresponding negative controls respectively The results showed that circGBR10was significantly downregulated in cells overexpressingmiR1413p Fig 4g Conversely circGRB10 was upregulated in the miR1413p inhibitor group Fig 4gMoreover upregulation of FUS alleviated the suppressiveeffects of miR1413p on circGRB10 expression Fig 4hwhile FUS knockdown attenuated the effects of miR1413p inhibitor on circGRB10 upregulation Fig 4i Theabove results indicated that miR1413p regulates circGRB10 expression in NP cells primarily through targetingof FUSERBB2 regulates miR1413p in NP cells byphosphorylating Erk12Induced Erk12 signaling causes widespread miRNArepression via suppression of the main steps of miRNAbiogenesis4142 In this study we found decreased levels ofErk12 phosphorylation in circGRB10 or ERBB2 knockeddown NP cells Fig 1d e Previous studies demonstratedthat phosphorylated Erk12 can cause widespreadmiRNA repression through suppressing the major stepsof miRNA biogenesis41“ As ERBB2 amounts werereduced in degenerative NP cells we hypothesized thatmiR1413p may be regulated by Erk12 phosphorylationin NP cells To explore this possibility we overexpressedor knocked down ERBB2 in NP cells qRTPCR resultsdemonstrated that overexpression of ERBB2 significantlydownregulated miR1413p while ERBB2 knockdownincreased miR1413p amounts Fig 5a In addition pretreatment of NP cells with the Erk12 phosphorylationinhibitor U0126 downregulated ERBB2 and attenuatedERBB2 induced phosphorylation of Erk12 decreasing theexpression of FUS Fig 5b Moreover blocking Erk12phosphorylation in NP cellssignificantly attenuatedERBB2™s effects on miR1413p biogenesis Fig 5c anddecrease the expression of circGRB10 Fig 5d Collectivelythe above findings demonstrated that ERBB2 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig FUS regulates the generation of circGRB10 in NP cells a qRTPCR showing FUS mRNA levels in normal and IDD NP tissues Threeindependent repeats were performed in each experiment p b Western blot showing FUS protein amounts were decreased in IDD NPtissues c qRTPCR analysis of circGRB10 expression level after FUS overexpression or knockdown in NP cells FUS overexpression led to significantlyincreased circGRB10 amounts in NP cells while its knockdown reduced circGRB10 levels Moreover FUS had no linear effects on GRB10 expressionThree independent repeats were performed in each experiment p d e qRTPCR analysis of the expression of CollagenII aggrecan MMP and ADAMT5 in NP cells f Schematic illustrating the putative FUSbinding sites on the flanking introns in the circGRB10s minigene The ²terminus of the circular exons of circGRB10 was defined as position Putative FUSbinding sites A and B are located in the intron at the ² terminusof the circGRB10 exon position ˆ’ to ˆ’ and on the intron at the ² terminus of the circGRB10 exon position “ g RIP analysis ofFUSbinding to circGRB10s and circGRB10sEm minigenes in NP cells Bound complexes were pulleddown using an antibody against FUS qRTPCRwas then used to measure circGRB10s binding to FUS Values were normalized to the level of background RIP as detected by an IgG isotype controlh qRTPCR analysis of the expression of circGRB10 relative to GAPDH in NP cells Cells were cotransfected with FUS or FUS siRNA and a circGRB10minigene circGRB10s or circGRB10 minigene containing deleted FUSbinding sites circGRB10Em Quantitative data from three independentexperiments is presented as mean ± SEM error bars P P regulated miR1413p expression in NP cells by phosphorylating Erk12Next we found that decreased ERBB2 amounts indegenerative NP cells could promote miR1413p generation which suppressed the expression of FUS resultingin circGRB10 downregulation our previousstudydemonstrated that circGRB10 downregulation leads toERBB2 reduction by enhancing miR3285p mediatedsuppression of ERBB2 in NP cells15 These findings suggested circGBR10 contributed to the molecular circuitrycontrolling IDD development in humans Fig 5ephosphorylation collagenII and aggrecan upregulationand inhibited the expression of MMP13 ADAMT5 inthe rat model of IDD Fig 6g Moreover Immunofluorescence staining also confirmed that the increasedexpression of collagenII aggrecan and the downregulation of MMP13 ADAMT5 expression in the circGRB10 group compared with noninjection group at weeks Fig 6h Jointly the above findings suggested atherapeutic role for circGRB10 in protecting the discsrevealing circGRB10 as a candidate therapeutic target inIDDIntradiscal injection of circGRB10 alleviates IDD in a ratmodelNeedle puncture has been one of the most commonmethods to establish animal models of IDD4445 We havesuccessfully established a rat model of IDD by needlepuncture according to the above methods in this studySupplementary Fig S3 At and days upon modelingadenoviral human circGRB10 was injected into thepunctured intervertebral discs with 33G needles In vivoRNA FISH indicated circGRB10 in the NP region at weeks after surgery Fig 6b CT scan at and weeks revealed progressive disc space narrowing in allpunctured animals and a significant increase in DHI wasnoted at and weeks post surgery in rats treated bycircGRB10 Fig 6c CT scan revealed that overexpression of circGRB10 markedly preserved the progressive disc space narrowing in rat IDD modelFig 6dAnd safranin O staining results demonstrated that overexpression of circGRB10 can inhibit the degradation ofextracellular matrix of NP cells Fig 6e These resultssuggesting circGRB10 exerted protective effects in surgically induced IDD After injection of adenoviral circGBR10 FUS and ERBB2 amounts in degenerative NPtissues were remarkably elevated Fig 6f while miR1413p amounts were decreased Fig 6f In addition injectionof adenoviral circGBR10 alleviated the degenerativealterations ofincluding enhanced Erk12the NPDiscussionNumerous circRNAs are found in the human transcriptome playing critical roles in the regulation of cellfunctions174647 Our previous study showed that circGRB10 downregulation is associated with human NP cellapoptosis15 Howeverthe mechanism of circGRB10dysregulation in IDD has not been previously describedHere we found that FUS regulated and promoted circGRB10 biosynthesis by interacting with its flankingintrons In addition FUS expression in NP cell wasregulated by miR1413p Our findings suggest a regulatory network in NP cells FUS bound to GRB10 premRNA to regulate circGRB10 synthesis while circGRB10 acted as a molecular sponge for miR3285p withsuppressive effects on ERBB2 production and modulatedIDD development downregulation of ERBB2 decreasedErk12 phosphorylation and promoted the generation ofmiR1413p which bound to the ²UTR region of FUS toinhibit its expression constituting a positive feedbackloop promoting intervertebral disc degenerationThe differential expression of circGBR10 betweennormal and degenerative NP tissues indicates that circGRB10 biosynthesis is controlled differently in variouscells with NP cells possessing specific factors required forcircRNA biogenesis Indeed introns and of the GRB10premRNA had binding sites to regulate circGRB10biogenesis Furthermore multiple RBPs were highlyOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig miR1413p inhibits FUS expression in NP cells a The Venn diagram indicates the predicted miRNAs regulate FUS expression miR1413p was intersected predicted by different databases b Expression of miR1413p in IDD NP tissues showing that miR1413p expression wassignificantly higher than that of controls Quantitative data from three independent experiments is presented as mean ± SEM error bars P c Sequence alignment of a putative miR1413pbinding site within the ²UTR of FUS mRNA Bottom mutations in the ²UTR of FUS mRNAsequence to create the mutant luciferase reporter constructs d Luciferase reporter assay in NP cells after transfected with scramble oligo or miR1413p mimics Renilla luciferase vector and the reporter constructs Both firefly and Renilla luciferase activities are measured in the same sample Fireflyluciferase signals were normalized with Renilla luciferase signals Quantitative data from three independent experiments is presented as mean ± SEMerror bars P e f FUS expression level was detected by qRTPCR western blot in primary human NP cells Three independent experimentsis presented as mean ± SEM error bars P g NP cells from control tissues were transfected with miR1413p mimic or miR1413p inhibitorqRTPCR was used to detect the relative expression level of circGRB10 compared with controls Three independent experiments is presented asmean ± SEM error bars P h NP cells from control tissues were transfected with miR1413p with or without FUS overexpress plasmidqRTPCR was used to detect the relative expression level of circGRB10 compared with controls i miR1413p inhibitor with or without FUS siRNA wastransfected into NP cells from control tissues and the expression level of FUS Three independent experiments is presented as mean ± SEM errorbars P Fig ERBB2 regulate miR1413p expression in NP cells a miR1413p expression level in NP cells with ERBB2 overexpression or ERBB2knockdown Three independent experiments is presented as mean ± SEM error bars P b NP cells overexpressing ERBB2 were treated withU0126 or not for one hour Western blot was used to detect the phosphorylated level of Erk12 c d NP cells overexpressing ERBB2 were treated withU0126 or not qRTPCR was used to detect the expression level of miR1413p and circGRB10 Three independent experiments are presented asmean ± SEM error bars P e Schematic representation of mechanisms by which circGRB10 mediates IDD development On the basis offindings described in the manuscript miR1413p downregulates FUS level in NP cells leading to circGRB10 decreased This downregulated circGRB10 in turn enhanced miR3285p mediated suppression of ERBB2 in NP cells leads to decreased Erk12 phosphorylation level And the decreasedErk12 phosphorylation level enhances the generation of miR1413p in NP cellsOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig cricGRB10 attenuates IDD development in vivo a Overview of the experimental setup with injections of circGRB10 or their negativecontrol at and days after surgery b Six weeks after surgery in vivo RNA FISH found circGRB10 located in the NP region Blue fluorescence diamidino2phenylindole DAPI indicating cell nucleus Red fluorescence indicating circGRB10 Scale bar μm c Changes in disc height indexDHI of the indicated groups after needle puncture The DHI was measured at and weeks A significant decrease of the DHI was observedin all puncture groups at week after surgery P A significant increase in DHI was noted at and weeks post surgery in rat treated withcircGBR10 P d CT scan of the indicated groups were obtained weeks after needle puncture e The intervertebral disc degenerationevaluated by Safranin O staining Scale bar µm f qRTPCR showed that the increased levels of FUS ERBB2 and the decreased level of miR1413p in the punctured discs treated with circGRB10 Three independent experiments are presented as mean ± SEM error bars P g Westernblot showing the expression levels of collagen II aggrecan pErk12 MMP13 ADAMT5 in rat NP tissues h Immunostaining for collagenII andaggrecan in IDD model treated by circGRB10 at and weeks Scale bar µmOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of enriched in circGRB10™s flanking introns and FUS contributed to circGRB10 biogenesis as shown aboveAlthough FUS induced circGRB10 biosynthesisitssilencing only decreased circGRB10 levels by asshown above It is known that two or more RBPs couldthe synthesis of circRNAs synergistically4048controlwhich might explain the above incomplete suppressionTherefore circGRB10 modulation in NP cells deservesfurther examinationAs shown above altered FUS expression might profoundly affect circGRB10 biogenesis Further deletion ofFUSbinding sequences in the introns flanking of circGBR10 dramatically reduced circGRB10 amounts Takentogether the above findings indicate FUS may directlycontrol backsplicing to upregulate circGRB10 in NPcells by interacting with putative binding sequences onboth flanking introns of circGRB10Recently miR141 has been detected in NP tissuesamples from IDD cases and its levels correlate with discdegeneration Therefore miR141 NPs have been used inIDD treatment with commendable efficacy34 As shownabove miR1413p which is a key regulator of IDDdirectly regulated FUS further revealing the FUScircGRB10 axis as an IDDrelated regulatory pathwayAccumulating evidence indicates that Erk signaling hasan important role in IDD394950 In the present study wefound that circGRB10 significantly upregulated collagenII and aggrecan in NP cells and mediated the protectiveeffects in IDD likely via ERBB2Erk signaling There mightalso be cellular pathways that compensate for ERBB2expression after its knockdown For example the longintergenic noncoding RNA lincRNA BCLIN25 upregulates ERBB2 by inducing promoter CpG methylation ofmiR125b resulting in miR125b repression44 A previousstudy indicated the Erk pathway regulates the miRNAgenerating complex43 In additi

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

"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adults™population of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [“] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and 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 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [“] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients™ intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at ˆ’ °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients™ intakeUsual Macronutrients™ intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants™ physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrients™intake and physical activity between different FTO genotypes The post hoc Tukey™s test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrients™protein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [“] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ‰¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableˆ’Calorieˆ’ˆ’ˆ’ˆ’ATˆ’ˆ’ˆ’ˆ’P valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele ß ˆ’ kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrients™intakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [“] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients™ intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3“ and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors™ contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet “Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States “ JAMA “Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol “Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab “Fredriksson R Hagglund M Olszewski PK Stephansson O Jacobsson JAOlszewska AM Levine AS Lindblom J Schiöth HB The obesity gene FTO isof ancient origin upregulated during food deprivation and expressed inneurons of feedingrelated nuclei of the brain Endocrinology May “Doaei S Hajiesmaeil M Aminifard A MosaviJarrahi SA Akbari MEGholamalizadeh M Effects of gene polymorphisms of metabolic enzymeson the association between red and processed meat consumption and thedevelopment of colon cancer a literature review J Nutritional Sci Doaei S Kalantari N Mohammadi NK Izadi P Gholamalizadeh M EiniZinabH Salonurmi T Jarrahi AM Rafieifar S Janipoor R Sadeghypor M Upregulation of FTO gene expression was associated with increase in skeletalmuscle mass in overweight male adolescents Arch Med Sci Sep155Hakanen M Raitakari OT Lehtimäki T Peltonen N Pahkala K Sillanmäki LLagstrom H Viikari J Simell O Ronnemaa T FTO genotype is associatedwith body mass index after the age of seven years but not with energyintake or leisuretime physical activity J Clin Endocrinol Metab Apr “Thorisson GA Smith AV Krishnan L Stein LD The international HapMapproject web site Genome Res Nov “Smemo S Tena JJ Kim KH Gamazon ER Sakabe NJ GómezMarín C AneasI Credidio FL Sobreira DR Wasserman NF Lee JH Obesityassociatedvariants within FTO form longrange functional connections with IRX3Nature Mar5077492371“ Antonio J Knafo S Kenyon M Ali A Carson C Ellerbroek A Weaver CRoberts J Peacock CA Tartar JL Assessment of the FTO gene 0cMehrdad Lipids in Health and Disease Page of Ronkainen J Huusko TJ Soininen R Mondini E Cinti F Mäkelä KAKovalainen M Herzig KH Järvelin MR Sebert S Savolainen MJ Fat massandobesityassociated gene Fto affects the dietary response in mouse whiteadipose tissue Sci Rep Mar Poritsanos NJ Lew PS Fischer J Mobbs CV Nagy JI Wong D Rüther UMizuno TM Impaired hypothalamic Fto expression in response to fastingand glucose in obese mice Nutr Diab 2011110e19 Doaei S Kalantari N Izadi P Salonurmi T Jarrahi AM Rafieifar S Azizi Tabesh GRahimzadeh G Gholamalizadeh M Goodarzi MO Interactions between macronutrients™ intake FTO and IRX3 gene expression and FTO genotype in obeseand overweight male adolescents Adipocyte Jan “ Olszewski PK Fredriksson R Olszewska AM Stephansson O Alsiö JRadomska KJ Levine AS Schiöth HB Hypothalamic FTO is associated withthe regulation of energy intake not feeding reward BMC Neurosci Dec1011“ Razquin C Martinez JA MartinezGonzalez MA A 3year intervention with aMediterranean diet modified the association between the rs9939609 genevariant in FTO and body weight changes Int J of Obesity “ McTaggart JS Lee S Iberl M Church C Cox RD Ashcroft FM FTO isexpressed in neurones throughout the brain and its expression is unalteredby fasting PLoS One 2011611e27968 httpsdoi101371journalpone0027968Stratigopoulos G Padilla SL LeDuc C A Watson E Hattersley AT McCarthyMI Zeltser LM Chung WK Leibel RL Regulation of FtoFtm gene expressionin mice and humans Am J Physiol Integr Comp Physiol 2008294R1185“ Wang P Yang FJ Du H Guan YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May ““ Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3“ N Engl J Med Sep “ Wardle J Carnell S Haworth CM Farooqi IS O™Rahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab “ httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E O™Daly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspolymorphisms rs1421085 rs17817449 and rs9939609 in exercisetrainedmen and women the effects of a 4week hypocaloric diet J Int Soc SportsNutr Dec Blundell JE Lawton CL Cotton JR Macdiarmid JI Control of humanappetite implications for the intake of dietary fat Annu Rev Nutr “Ludwig DS The glycemic index physiological mechanisms relating toobesity diabetes and cardiovascular disease Jama “Sonestedt E Roos C Gullberg B Ericson U Wirfält E OrhoMelander M Fatand carbohydrate intake modify the association between genetic variationin the FTO genotype and obesity Am J Clin Nutr Sep “Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relativevalidity of food group intake in a food frequency questionnaire developedfor the Tehran lipid and glucose study J Epidemiol “ Azar M Sarkisian E Food composition table of Iran National Nutrition andfood research institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes “ Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455“ Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May “ Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov “ Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul “ Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov “Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript levels but does not altertheir DNA methylation profiles in rat white adipose tissues Gene “ 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

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

"Sphingosine1phosphate receptor S1PR1 is involved in vascular development a key process intumorigenesis This study aimed to evaluate its roles in tumor development and prognosisMethods S1PR1 expression levels were analyzed using TIMER and Oncomine database and the prognosticsignificance of S1PR1 was assessed using PrognoScan and KaplanMeier plotter databases The relationship betweenS1PR1 and tumorinfiltrated immune cells was analyzed using TIMERResults S1PR1 expression was remarkably lower in breast and lung cancer tissues than in the correspondingnormal tissues Lower expression was related to poor overall survival and diseasefree survival in breast invasivecarcinoma BRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC A functional networkanalysis confirmed the function of S1PR1 in regulating vasculogenesis In addition S1PR1 levels were significantlynegative with regard to the tumor purity of BRCA r ˆ’ P 176e66 LUAD r ˆ’ P 605e16 andLUSC r ˆ’ P ˆ’ 520e20 Furthermore S1PR1 levels were significantly positive with regard to infiltratingCD8 r P 591e35 and CD4 T cells r P 103e26 macrophages r P 367e12neutrophils r P 203e7 and dendritic cells DCs r P 914e11 in BRCA S1PR1 levels weresignificantly positive with regard to CD8 T cells r P 361e12 macrophages r P 101e17neutrophils r P 415e8 and DCs r P 416e6 in LUAD and positive with regard to B cells r P 157e15 CD8 r P 383e26 and CD4 T cells r P 398e14 macrophages r P 261e45 neutrophils r P 179e25 and DCs r P 212e40 in LUSCConclusions S1PR1 levels are positively correlated with multiple immune markers in breast and lung cancer Theseobserved correlations between S1PR1 and the prognosis and immune cell infiltration provide a foundation forfurther research on its immunomodulatory role in cancerKeywords S1PR1 Breast cancer Lung cancer Tumorinfiltrating Prognosis biomarker Correspondence caodl126com wenrenlyf2008163com1The First Affiliated Hospital Guangzhou University of Chinese Medicine No Airport Road Baiyun District Guangzhou China2Department of Laboratory Medicine Guangdong Second Provincial GeneralHospital No Xingang Middle Road Haizhu District Guangzhou Guangdong Province 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 0cZhong BMC Cancer Page of BackgroundSphingosine1phosphate S1P produced by sphingosine kinase Sphkis a biologically active signalinglipid [] S1P regulates vascular development andfunction including vascular maturation [ ] S1P receptor S1PR1is a biologically active sphingolipidmetabolite that mediates S1P activity and promotescell proliferation and survival [ ] S1PR1 is widelyexpressed in vascular endothelial cells and is requiredfor embryonic vascular development and maturation[] Estrogen the growthstimulating hormone inbreast cancer cells was shown to stimulate endothelial cell growth via S1PR1 [ ] In the tumor microenvironment S1P exhibits multiple functionsitincreases the survival of macrophages b it serves asthe œcomeandgetme signal of dead cells attractingand enhancing macrophage migration by combiningwith S1PR1the polarization ofTAMM2 macrophages by activating S1PR124 [“] Accumulating evidence demonstrated that tumorprogression requires new blood vessel growth whichis achieved by producing angiogenic factors that canactivate vascular endothelial cells [] Tumor cellsreleasevascularendothelial growth factor VEGFa which leads toangiogenesis and tumor growth [] Studies haveshown that S1PR1 inhibits VEGF signaling by prointeraction between VEcadherin andmoting theVEGFR2thereby inhibiting VEGFinduced vascularsprouting [ ]angiogenicstimulatesstimulicitasuchasHowever the role of S1PR1 in tumorigenesis andits prognostic value are unclear A preclinical studyon human breast cancer cells found that S1PR1 antibody can enhance the cytotoxic and antiproliferativeeffect of carboplatin on MDAMB231 and SKBR3HER2 subtype cells respectively [] Lei et alfound that S1PR1 signaling has tumorsuppressiveeffects and survival benefits in breast cancer []Therefore it is necessary to clarify the role of S1PR1in tumor development and progression Transcriptome analysis can be used to predict important issues such as the intrinsic subtype of the primarytumor tumor grade drug reactivity and recurrencerisk [“]Herein we used Oncomine KaplanMeier plotterPrognoScan UALCAN and GEPIA datasetstoanalyze S1PR1 expression and its relationship withthe prognosis of cancer patients Furthermore westudied the correlation between S1PR1 and tumorinfiltrated immune cells in the tumor microenvironment using TIMER Our results shed light on theimportant role of S1PR1 in breast and lung cancerand determined that it is closely related to tumorimmunityMethodsOncomine database analysisThe Oncomine database wwwoncomineresourceloginhtml was used to evaluate the expressionlevel of S1PR1 in various types of cancers [] Thethresholds were a Pvalue of fold change of and data type was mRNAPrognoScan database analysisThe PrognoScan database wwwprognoscan wasused to test S1PR1 expression and survival in varioustypes of cancers [] The threshold was an adjustedCox Pvalue of CBioPortal database analysiscBioPortal httpcbioportal contains multidimensional cancer genomics data sets [] S1PR1 mutationsand copy number variation CNV in breast and lungcancers were analyzed using cBioPortal The OncoPrinttab was used to obtain an overview of the genetic alterations for each samplethe prognosticKaplanMeier plotterKaplanMeier Plotter kmplotcom was applied to assessvalue of S1PR1Grouped according to the median expression ofS1PR1 high vs low expression all patients were analyzed for overall survivalOS and progressionfreesurvival PFS and KaplanMeier was used to draw asurvival chart []Immune infiltrates analysis using the TIMERTIMER cistromeshinyappsiotimer wasused to analyze immune infiltrates across different typesof cancer [] Especially the expression of S1PR1 in different cancer types and the correlation between the expression of S1PR1 and the abundance ofimmuneinvasion was determined In addition the correlation between S1PR1 expression and tumor infiltrating immunecell gene markers was also explored through relatedmodulesGene correlation analysis using GEPIAGEPIA httpgepiacancerpkucnindexhtml wasused to confirm the genes with significantly correlatedexpression levelsin TIMER [] The Spearmanmethod was used to determine the correlation coefficients The tumortissue datasets were used foranalysisLinkedOmics database analysisThe LinkedOmics database httpwwwlinkedomicsloginphp was used to analyze S1PR1 coexpressioncorrelationPearson™sbasedon 0cZhong BMC Cancer Page of coefficients The results were visually evaluated usingvolcano plots and heat maps The function moduleof LinkedOmics was used to analyze gene ontologyGOKyotoEncyclopedia of Genes and Genomes KEGG pathways by a gene set enrichment analysis GSEA Therank criterion was FDR and simulationswere performed []biologicalprocessesBPandUALCAN database analysisUALCAN httpualcanpathuabeduincluded theCancer Genome Atlas TCGA level RNA sequencesClinical data from cancer types were used toanalyze the relative expression of genes in tumorand normalstagetumor grade or other clinicopathological characteristics []according to tumorsamplesS1PR1 mRNA expression level analysisGene expression data of breastinvasive carcinomaBRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloadedin UCSC Xena xenabrowsernet S1PR1 mRNAexpression level was compared between cancerous andnormal tissue using MannWhitney test with P setting as a cutoffStatistical analysisGene expression data in the Oncomine database wasanalyzed using pvalue fold change and mRNA datatype The survival curves were generated via KaplanMeier plots and PrognoScan database are displayedwith HR and P or Cox Pvalues from a logrank testSpearman correlation analysis was used to evaluatethe correlation of gene expression in TIMER and LinkedOmics databases P was considered statistically significantResultsS1PR1 mRNA expression levels in different types ofhuman cancersThe Oncomine database was used to analyze S1PR1mRNA levels in tumor tissues and normal tissues ofvarious cancer types S1PR1 expression was lower inmost tumor tissues including sarcoma bladder brainleukemiacentral nervous system breast colorectallung myeloma and ovarian cancer tissuesthan innormal tissues Fig 1a The mRNAseq data fromTCGA were analyzed using TIMER to verify thesefindings Data from TCGA shown that the differentialexpression of S1PR1 between the tumor and adjacentnormal tissues is shown in Fig 1b Compared withadjacent normal tissues S1PR1 expression was significantlycarcinomaBLCA BRCA cholangiocarcinoma CHOL colonadenocarcinomacarcinomaESCA head and neck squamous cell carcinomaHNSC kidney chromophobe KICH kidney renalpapillary cell carcinoma KIRPliver hepatocellularcarcinoma LIHC LUAD LUSC prostate adenocarREADcinomaskinstomachrectum adenocarcinomacutaneous melanomain bladderesophagealurothelialSKCMreducedCOADPRADFig S1PR1 expression levels in different types of human cancers a Differences in S1PR1 between cancer tissues and normal tissues based ondata in the Oncomine database P 1E04 Fold change Data type mRNA b Human S1PR1 expression levels in different tumor types fromTCGA database were determined using TIMER P P P 0cZhong BMC Cancer Page of adenocarcinoma STAD and uterine corpus endometrial carcinoma UCEC However S1PR1 expression was significantly higher in kidney renal clear cellcarcinoma KIRC and thyroid carcinoma THCAthan in adjacent normal tissues Fig 1b These datashowed that alterations in S1PR1 expression dependon the tumor type suggesting that this gene exertsdiverse functions in various tumorscancerand GSE12276in three breastPrognostic evaluation of S1PR1 in cancersWe investigated whether S1PR1 expression is relatedto prognosis The effect of S1PR1 expression on survival wasevaluated by PrognoScan Two probes204642_at and 239401_at matching S1PR1 were detected NotablycohortsGSE1456GPL96 GSE7378lowS1PR1 expression was significantly associated with apoorer prognosis breast cancer Fig 2a“f We usedthe KaplanMeier plotter database to further examinethe prognostic value of S1PR1 in breast cancer Poorprognosis based on recurrencefree survival RFS inbreast cancer was significantly correlated with lowS1PR1 expression HR P 71e13 but a significant correlation was not observed for overall survival OS HR P and postprogressionsurvival PPS HR P Fig 2g“i Its determined that the low expression of S1PR1 is an inderisk factorpendentfor poor prognosis of breastcancer In additionlow S1PR1 expression was alsorelated to poor prognosis in two cohorts of patientswith lung cancer GSE31210 and GSE8894 as determined using two probes 204642_at and 239401_atFig 2j“l KaplanMeier plotter database also showedthat low expression of S1PR1 was an independent riskfactor for poor prognosis of lung cancer overall survival HR P 69e08 recurrencefree survivalHR P but notrelated to postin lung cancer HR P progression survival Fig 2m“oFurthermore we found that low S1PR1 expressionwas associated with a poor prognosis in patientswith soft tissue blood and brain cancers Fig S1a“c In contrastlow S1PR1 expression was an independent risk factor for a good prognosis in gastricFig S1d“g These results confirmed thecancerprognostic value of S1PR1 in specific types of cancer both high and low S1PR1 expression was associated with prognosis depending on thetype ofcancer Based on the consistent results for the associations between S1PR1 expression and survivalinbreast and lung cancers we focused on the preciseeffects of S1PR1 in these two cancer types as wellas the underlying mechanismsP Correlations between clinical characteristics and S1PR1expression in breast cancer and lung cancerWe used the KaplanMeier plotter to study the relationship between S1PR1 expression and clinical characteristics in patients with breast cancer and lungcancer Low expression of S1PR1 was associated withworse overall survival OS in male and female patients with lung adenocarcinoma P Table In particular low S1PR1 mRNA expression was correlated with worse OS in stage P 920E13 andearlystage AJCC stage Mlung cancerTable Low S1PR1 mRNA expression was relatedto poor OS in patients with P or withoutP a smoking history Table In additionlow S1PR1 mRNA expression was related to worseOS in patients who did not receive chemotherapy orradiotherapy These findings strongly suggest that lowS1PR1 mRNA expression is correlated with poor OSin lung cancer Table In BRCA low S1PR1 mRNAexpression was related to poor OS in ERpositive orHER2negative patients and in the luminal androgenreceptor subtype Table Taken together high expression of S1PR1 could be considered a good prognostic indictor for breast and lung cancers dependingon the clinical characteristicsDecreased expression of S1PR1 in breast cancer and lungcancer patientsWe further analyzed the expression of S1PR1 inbreast and lung cancers Gene expression data ofbreast invasive carcinoma BRCAlung adenocarcinoma LUAD and lung squamous cell carcinomain TCGA were downloaded and S1PR1LUSCmRNA expression level was compared between tumorand normal tissue As shown in Fig 3a the expression of S1PR1 was significantly decreased in tumortissues of BRCA LUAD and LUSC Fig 3a In comparison with normal control tissues breast cancer andlung cancertissues presented lower expression ofS1PR1 which was also observed by GEPIA analysisFig 3b Furthermore we analyzed TCGA data usingthe UALCAN database Compared to normal tissuesS1PR1 mRNA expression was significantly decreasedin primary tumors and tumor stages stage stage stage and stage of BRCA LUAD and LUSCFig 3c“e Taken together these data confirmed thedownregulation of S1PR1 expression in breast cancerand lung cancer patientsRegulators of S1PR1 in breast cancer and lung cancerWe used the LinkedOmics function module to detectthe S1PR1 regulatory network to further understandthe biological role of S1PR1 in breast cancer and lung 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig Prognostic value of S1PR1 in cancers a“f KaplanMeier survival curves comparing high and low expression of S1PR1 in breast cancersusing PrognoScan Survival curves based on OS DSS and DFS in three breast cancer cohorts [GSE1456GPL96 n GSE7378 n andGSE12276 n ] g“i Survival curves for breast cancers based on mRNAseq data from TCGA of Kaplan“Meier plotter databases j“l Kaplan“Meier survival curves comparing high and low expression of S1PR1 in lung cancers using PrognoScan Survival curves based on RFS in two threelung cancer cohorts [GSE31210 n and GSE8894 n ] m“o Survival curves for lung cancers based on mRNAseq data from TCGA ofKaplan“Meier plotter databases OS Overall survival RFS RelapseFree Survival PPS Postprogression survival DSS Diseasespecific survivalDFS Diseasefree survivalcancer Figure 4a“c shows genes with significantlypositive dark red dots and negative dark greendots correlations with S1PR1 false discovery rateFDR The top positively and negatively related genes are shown in a heat map in Fig 4d“f AGene Ontology GObased gene set enrichment analysis GSEA showed that genes that are coexpressedwith S1PR1 are enriched for vasculogenesis and thepurinergic receptor signaling pathway while genes related to mitochondria and RNA transcript processingwere inhibited in breast cancer Fig 4g SimilarlyGO annotation resultscoexpressed with S1PR1 are primarily associated withvasculogenesis the purinergic receptor signaling pathway and the phospholipase Cactivating G proteincoupled receptortRNAmetabolic process RNA modification and RNA transcript processing werecancerFig 4h“i A KEGG pathway analysis showed enrichlineage Staphylococcusmentaureusinboth breast cancer and lung cancer SpliceosomeDNA replicationand proteasome pathways wereinhibited in both tumor types Fig 4jl These resultssuggest that S1PR1 contributes to various processesin tumor development at least partially through regulate vasculogenesisinfection and renin secretion pathwayssignaling pathway whilefor hematopoietic cellshowedthatgenesinhibited in lungamplifications and deletionsGenomic alterations in S1PR1 in breast cancer and lungcancercBioPortal database was used to determine the typesand frequencies of S1PR1 alterations in BRCA LUADand LUSC S1PR1 was altered in of patients withBRCA These alterations included mRNA missensemutationsFig 5aS1PR1 was altered in of patients with LUAD and of patients with LUSCincluding mRNA missense mutations truncating mutations amplificationsand deletions Fig 5a Moreover S1PR1 CNV wasassociated with OS in LUAD but not with OS or DFSin BRCA and LUSC Fig 5b“d These results suggestthat mutations in S1PR1 are associated with prognosisin LUADRelationship between immune and S1PR1 expression inbreast cancer and lung cancerTumor infiltrating lymphocytes TIL are lymphocytes that leave the blood circulation and migrate tothe vicinity of the tumor The amount of TIL in thetumor is an important indicator to predict the prognosis of cancer patients and the response to immunotherapy [ ] Tumor purity is a key factorin analyses ofimmune infiltration by genomic approaches [] Therefore we use TIMER to investigate whetherthe expression of S1PR1 in breastcancer and lung cancer is related to immune infiltration We found a significant negative correlation between the S1PR1 expression level and tumor purityin both breast cancer and lung cancer Fig 6a“fLeft S1PR1 was a determinant of immune infiltration in BRCA tumor purity r ˆ’ P 176e including subtypes of BRCA BRCABasal r ˆ’ P 128e06 BRCAHer2r ˆ’ P 444e06 and BRCALuminal r ˆ’ P 915e S1PR1 was related to immune infiltration inincluding LUAD tumor purity r ˆ’lung cancer P 605e16 and LUSC tumor purity r ˆ’ P 520e20Furthermore the relationship between S1PR1 andspecific immune infiltrates in breast cancer and lungcancer were analyzed The S1PR1 expression levelwas significantly positively correlated with levels ofr P 597e35infiltrating CD8 T cellsCD4 T cells r P 103e26 macrophagesr P 367e12 neutrophilsr P 203e07 and DCs r P 914e11 in BRCAFig 6a In BRCABasal there were slight positivecorrelations between S1PR1 expression levels andinfiltrating CD8 T cells r P levels of176e03 and CD4 T cells r P 852e03Similarly there were positive correlations with infiltrating levels of CD8 T cells r P 113e CD4 T cells r P 200e05 neutror philsP 135e04 in BRCAHer2 S1PR1 expression levelswere positively correlated with levels ofinfiltratingCD8 T cells r P 343e21 CD4 T cellsr P 626e14 macrophages r P r P 857e03 and DCs 0cZhong BMC Cancer Page of Table Correlation between S1PR1 mRNA expression andprognosis in lung cancer with respect to clinicopathologicalfactorsClinicopathologicalcharacteristicsOverall survivalNHazard ratioPvalueTable Correlations between S1PR1 mRNA expression andclinical prognosis in breast cancer with respect toclinicopathological factorsClinicopathologicalcharacteristicsOverallNHazard ratioPvalue “ “490E05ER statusER positiveER negativePR status “ “ “ “590E06PR positivePR negativeHER2 status920E13HER2 positive113E01920E01HER2 negativeIntrinsic subtypeSexFemaleMaleHistologyAdenocarcinomaSquamous cell carcinomaStageGradeIIIIIIAJCC stage TAJCC stage NAJCC stage MSmoking historyExclude those never smokedOnly those never smokedChemotherapyNoYesRadiotherapyNoYes “ “ “NA “ “ “ “ “ “ “ “ “ “NA “NANA “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “NA “NABasalLuminal ALuminal BHER2Lymph node statusLymph node positiveLymph node negativeGradeTP53 statusMutatedWild typePietenpol subtypeBasallike Basallike ImmunomodulatoryMesenchymalMesenchymal stem likeLuminal androgen receptorSystemically untreated patientsIncludeExclude “ “71E13Bold values indicate P NA noneBold values indicate P NA none414e04 neutrophils r P 667e04 andDCs r P 644e07tumors Fig 6a We also found that S1PR1 expressionlevels were positivelycorrelated with levels ofin BRCALuminalr P 361e12infiltrating CD8 T cellsr P 101e17 neutrophilsmacrophagesr P 415e08r P 416e06 in LUAD In addition there were positiveinfiltrating B cells r correlations with levels ofand DCs 0cZhong BMC Cancer Page of Fig Decreased expression of S1PR1 in breast and lung cancer patients a Gene expression data of breast invasive carcinoma BRCA lungadenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloaded in UCSC Xena S1PR1 mRNA expression level wascompared between cancerous and normal tissue using MannWhitney test with P setting as cutoff b The expression of S1PR1 in BRCALUAD and LUSC were analysis using GEPIA T tumor N normal tissue NUM number c“e S1PR1 mRNA expression level was expressed as boxplots using the UALCAN database mRNA expression of S1PR1 in normal control and BRCA LUAD and LUSC tumors Left primary tumors Rightindividual cancer stage P P P 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig S1PR1 coexpression genes in breast and lung cancer a“c The S1PR1 highly correlated genes identified by Pearson test in BRCA a LUADb and LUSC c d“f The heat map shows that in BRCA d LUAD e and LUSC f the first genes are positively red and negatively bluecorrelated with S1PR1 g“i Significantly enriched GO annotations of S1PR1 in BRCA g LUAD h and LUSC i j“l Significantly enriched KEGGpathways of S1PR1 in BRCA j LUAD k and LUSC lin LUSC These results strongly suggest P 127e15 CD8 T cells r P r P 398e14383e26 CD4 T cellsr P 261e45 neutrophilsmacrophagesr P 179e25 and DCs r P 212ethatS1PR1 plays a special role in the immune infiltrationof breast and lung cancers and has a particularlystrong effect on T cells macrophages neutrophilsand DCs These observed correlations betweenS1PR1 and various types of immune cells in breastand lung cancers indicated that S1PR1 may havehigh prognostic valuefurthercorrelationsimmune cellsevaluated theCorrelations between S1PR1 expression and immunemarkersbetweenWeS1PR1 and markers of variousinbreast cancer and lung cancer using TIMER Table and GEPIA databases Table S1 The correlationsbetween S1PR1 expression and immune markergenes for different immune cell populations including CD8 T cells T cells general B cells monocytes TAMs M1 and M2 macrophages neutrophilsNK cells DCs and various functional T cells suchas Th1 cells Th2 cells Tfh cells Th17 cells andTregs as well as exhausted T cells were analyzed byTIMER After adjusting for tumor purity S1PR1 expression levels were significantly positively correlatedwith marker sets for various immune cells exceptfor NK cells Th17 and T cell exhaustion in BRCATable and Fig However S1PR1 expressionlevels were highly positively correlated with most immune marker sets and both T cell populations andexhausted T cells in LUAD and LUSC Table andFig We further analyzed the correlation betweenS1PR1 expression and the markers using the GEPIAdatabaseincluding data for BRCA LUAD andLUSC The results for correlations between S1PR1and markers of immune infiltrating cells were similarto those of the TIMER analysis Table S1 This further confirms that S1PR1 is significantly related toimmune infiltrating cells in lung and breast cancersuggesting that high levels of S1PR1 could induceimmune activity in the lung and breast cancermicroenvironmentin normalDiscussionWe systematically analyzed the expression levels ofS1PR1 and the prognostic value in different types ofcancers Compared with levelstissuesS1PR1 expression was significantly lower in BLCABRCA CHOL COAD ESCA HNSC KICH KIRPLIHC LUAD LUSC PRAD READ SKCM STADand UCEC and was significantly higher in KIRC andTHCA Accordingly S1PR1 expression patterns depend on the type of cancer Prognostic data fromKaplanMeier plotterlow levels ofS1PR1 are significantly related to poor prognosis inbreast cancer and lung cancershowed thatsuch a complex biologicalThe downregulation of S1PR1 was associated withworse prognosis in breast cancer and lung cancer andwassignificantly related to clinical characteristicssuch as gender population smoking status and stageThese results suggested that S1PR1 is a prognosticbiomarker in breast cancer and lung cancer Howeversome literatures have reported the oncogenic role ofS1PR1 in breast cancer Lee H demonstrated thatStat3induced S1PR1 expression as well as S1PS1PR1 pathway is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression [] This maybe one of the molecular mechanisms by which S1PR1mediatesresponse Weconsidered that the main reason for this inconsistencyis that our study analyzed the expression of S1PR1 atthe overalllevel We further verified the significantreduction of S1PR1 expression in breast cancer andlung cancer patients through TCGA analysis Anotherstudy has also claimed a survival function benefit ofS1PS1PR signaling in BRCA patients which mightexplain the obstacle to relative antagonist therapy inclinics [] A recent study determined that attenuated endothelial S1PR1 function led to increasedtumor growth and metastasis whereas S1PR1 overexpression led to smaller tumors and strategies to enhance S1PR1 function in the tumor vasculature maypotentiate the efficacy of cytotoxic and targeted anticancer therapies [] These studies support our findings that high expression of S1PR1 is beneficialfortumor survivalThe tumor microenvironment refers to noncancerimmunecells in and around tumorsinfiltrated of 0cZhong BMC Cancer Page of Fig S1PR1 genomic alterations in breast and lung cancer a OncoPrint of S1PR1 alterations in BRCA LUAD and LUSC Different types of geneticalterations highlighted in different colors b“d The relationship between genetic alterations and S1PR1 OSDFS in BRCA b LUAD c and LUSCd Logrank test was used in analysis of OSDFScells in the tumor microenvironment plays a vitalfunction in the occurrence and development of tumors [ ] Lymphocyte infiltration in the tumormicroenvironment is an independent predictor of cancer patient survival and lymph node metastasis [] Studies have shown that S1PR1 can affect the 0cZhong BMC Cancer Page of Fig Correlations between S1PR1 expression and immune infiltration levels in breast and lung cancer a S1PR1 expression was significantlynegatively related to tumor purity and significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in BRCA n b S1PR1 expression was significantly negatively related to tumor purity and was significantpositively correlated with infiltrating levels of CD8 T cells CD4 T cells and dendritic cells in BRCABasal n c S1PR1 expression wassignificantly negatively related to tumor purity and was significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cellsneutrophils and dendritic cells in BRCAHer2 n d S1PR1 expression was significantly negatively related to tumor purity and wassignificantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophages neutrophils and dendritic cells in BRCALuminal n e S1PR1 expression was significantly negatively related to tumor purity and was significantly positively correlated withinfiltrating levels of CD8 T cells macrophages neutrophils and dendritic cells in LUAD n f S1PR1 expression was significantly negativelyrelated to tumor purity and was significant positively correlated with infiltrating levels of B cells CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in LUSC n Spearman™s correlation coefficients were used for analyses P P P proliferation and differentiation of lymphocytes in thetumor microenvironment [] The evaluation of immune cell infiltration in breast and lung cancers usingthe TIMER database revealed strong negative correlations between S1PR1 and tumor purity in BRCALUAD and LUSC Furthermore the S1PR1 expression level was positively correlated with levels ofCD8 T CD4 T neutrophils macrophages and DCsin BRCA The correlation between S1PR1 expressionand immune cell marker genes suggests that S1PR1regulates lung cancer tumor immunity through multiple immune cell populations These results indicatethat high levels of S1PR1 could increase the cytotoxicity of the immune system and immune activation in 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMERLUSCPurityLUADPurityBRCAPurityDescriptionGene markersCD8 T cellT cell generalB cellMonocyteTAMvarXCD8ACD8BCD3DCD3ECD2CD19CD79ACD86CD115 CSF1RCCL2CD68IL10M1 MacrophageINOS NOS2M2 MacrophageIRF5COX2 PTGS2CD163VSIG4MS4A4ANeutrophilsCD66b CEACAM8CD11b ITGAMNatural killer cellDendritic cellTh1Th2CCR7KIR2DL1KIR2DL3KIR2DL4KIR3DL1KIR3DL2KIR3DL3KIR2DS4HLADPB1HLADQB1HLADRAHLADPA1BDCA1CD1CBDCA4NRP1CD11c ITGAXTbet TBX21STAT4STAT1IFNg IFNGTNFa TNFGATA3STAT6corˆ’ˆ’p126E17242E08471E12715E19320E10738E07198E08128E01129E10468E04463E01835E02176E16618E01490E28772E02114E02196E13203E01824E01155E24727E01110E01395E01263E03319E02875E01268E01389E14211E02717E07226E11176E53158E59621E03472E13592E19261E04784E01808E 143E02669E13corˆ’ˆ’ˆ’ˆ’ˆ’p219E04166E02128E02885E07400E04537E05121E04297E07310E08386E02586E11110E09793E18355E01358E02436E14975E10539E17195E08816E06257E13130E06996E04506E01104E04879E02581E01834E03413E09479E02869E07453E07100E09107E04269E03481E05366E03310E01913E02930E02301E01220E03corˆ’p651E21122E17760E21282E26799E24378E13329E13127E45267E56589E24118E30149E36864E02431E01237E06152E57477E53928E54299E06266E30170E33136E03263E07845E04485E11168E06343E01105E06386E52884E20129E48187E52869E24669E28158E24517E20473E32103E04185E02134E01300E07625E01 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMER ContinuedTfhTh17TregSTAT5AIL13BCL6IL21STAT3IL17AFOXP3CCR8STAT5BTGFb TGFB1T cell exhaustionPD1 PDCD1CTLA4LAG3TIM3 HAVCR2GZMBBRCAˆ’ˆ’181E07127E01352E08977E01175E01929E02394E01671E01858E20321E25412E04575E01600E04219E01782E02LUADˆ’227E08115E01801E03234E01265E05462E01198E01461E04467E12943E06256E01727E02439E01178E06599E01LUSCˆ’422E21120E05924E01492E06609E04409E01415E19727E27247E03164E01380E16388E20311E06844E46333E09shown in recently reports endothelialBRCA LUAD and LUSC by increasing the infiltrationof CTLs CD4 T cells and DCs On the contrarylow expression of S1PR1 could lead to reduced infiltrated effector cells in the tumor microenvironmentAsloss ofS1PR1 led to a reduction in CD45 cells macrophages and DCs which influences tumor growth andmetastasis [] In addition S1P is involved in enhancing endocytosis and migration of mature dendriticcells through S1PR3 an event that may increase theimmune response to cancer cells Our findings areconsistent with such reports and these discoveriesimply that S1PR1 plays an important role in recruiting and governing immune infiltration in BRCALUAD and LUSCthe purinergicTo further elucidate the molecular mechanismsunderlying the role of S1PR1 in b

Thyroid_Cancer

American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ‰¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was “ mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research “ Fu 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 the work 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 For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ‰¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundary“lower hyoid margin lower boundary“superior sternal fossa upper margin of the unknown artery and external“lateral margin of the carotid sheath and lower boundary“anterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ‰¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients™ complete medical records were available All of the surgeons had years™ experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the œat least  principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using χ2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients™ age range was “ years old with a median of ± years The maximum diameter range of the cancer lesion was “ mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ‰¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients™ gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimoto™s thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimoto™s thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1“ According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about “ On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8“ These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japan™s Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ‰¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ‰¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 œ American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimoto™s thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter‰¤ mm mm x ‰¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMχ2PYes n No n “““Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβˆ’ˆ’SEWaldPOR CI OR““““““““Abbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ‰¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The χ2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions χ2 P nodule morphology χ2 P and extra thyroidal expansion of cancer focus χ2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimoto™s thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol “ doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg “ doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence “ Cancer Causes Control “ doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid “ doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA “ United States “ doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid “ doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery “ discussion “ doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin “ doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery “ doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid “ doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid “ doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary “ doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol “ doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid “ doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res “ doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid “ doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c'

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

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