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

Thyroid_Cancer

Body fat distribution predicts cardiovascular events better than bodymass index BMI Waist circumference WC and neckcircumference NC are inexpensive anthropometric measurements We aimed to present the conditional distribution of WC andNC values according to BMI stratified by age and sex from the Brazilian Longitudinal Study of Adult Health ELSABrasilbaseline data We analyzed ELSABrasil participants with complete data We used spline quantile regression modelsstratified by sex and age to estimate the NC and WC quantiles according to BMI To test a putative association between ageand median NC or WC values we built sexspecific median regression models using both BMI and age as explanatoryvariables We present estimated 25th 50th 75th and 90th percentiles for NC and WC values according to BMI age and sexPredicted interquartile intervals for NC values varied from to cm and for WC values from to cm Median NCwas not associated with age in men P011 nor in women P079 However median WC increased with advancing age inboth sexes Po0001 for both There was significant dispersion in WC and NC values for a given BMI and age strata for bothmen and women WC but not NC values were associated with increasing age The smaller ‚uence of advancing age on therelationship between BMI and NC compared to WC values may be useful in longitudinal studiesKey words Nomograms Adiposity Bodymass index Waist NeckIntroductionThe prevalence of obesity is increasing worldwideand a growing body of evidence shows that body fatdistribution might add important information for predictingcardiovascular events above and beyond bodymass indexBMI itself A seminal work published in the 1950s already reportedthat differences in the localization of adiposity between menand women are linked to their different cardiovascularprofile The study of fat tissue distribution and cardiovascular risk has gained more attention recently and ithas been hypothesized that specific fat depots couldincrease vascular damage through mediators thatcan ‚uence glucose homeostasis and lipid metabolism‚ammation and coagulation Certain locations of fataccumulation have been linked to diverse cardiometabolic profiles suggesting that regionalfat distribution could play an important role in the development ofCorrespondence CP Baena cristinabaenapucprbrReceived March Accepted June Braz J Med Biol Res 1015901414431X20209815the consensus point outcardiovascular diseases in both nonobese and obesepeople Recently the Consensus Statement of theInternational Atherosclerosis Society argued for theinclusion of waist circumference WC as a vital sign giventhat the prevalence of abdominal obesity is increasing anddysfunctional adipose tissue could be estimated moreaccurately by WC than BMI as shown in recent studiesAdditionallythe gap inknowledge with a recommendation for description of WCvalues for a given BMI category across different agesby sex as the limitations of BMI have been increasingly demonstrated in different populations especially indemonstrating changes in adiposity during aging Moreover it is difficult to measure body fat mass directlywhile WC and neck circumference NC are inexpensiveand easily obtainable anthropometric measurementsfor 0cNeck and waist circumference percentilesAnother study looked at one Brazilian population ofthe Baependi Heart Study in a crosssectional analysisand showed that WC in men discriminated the hypertensives better than visceral and body adiposity indexes Body fat distribution patterns vary and the positivecorrelations between BMI and both NC and WC do notfollow perfectlinear associations and despite beingassociated with higher cardiovascular risk in differentpopulations “ factors as sex age physical activitysmoking habits number of pregnancies and geneticpredisposition have also been linked to body fat distribution “Hingorjo studied young universitystudents in Pakistan and found that approximately of NC variance in males and of NC variance infemales was not explained by BMI values In additionthese discrepancies may vary in different populationsand in different time periods Stern showed thatthe predicted WC according to BMI in Chinese men andwomen increased from to In the same countryand time interval Du reported that the prevalence of central obesity in adults with normal o25 kgm2BMI increased from to during yearsThis underlines the importance of studying the WCand NC values according to BMI in large epidemiologicstudies conducted within different populations To datethere is no such study conducted in large samples Therefore our aim was to present the conditional distribution ofWC and NC values according to BMI stratified by ageand sex among midadult and elderly men and womenparticipants at the baseline assessment of the BrazilianLongitudinal Study of Adult Health ELSABrasil studya large multicenter cohort study in BrazilMaterial and MethodsStudy designELSABrasil is a multicenter prospective cohort study that enrolled civil servants aged to years from Brazilian cities Belo Horizonte PortoAlegre Rio de Janeiro Salvador S£o Paulo and Vitria In this crosssectional analysis we used baselinedata “ Approvals were obtained from theinstitutional review boards of all the centers and all thesubjects signed an informed consent formStudy sampleFrom ELSABrasil participants at baseline weexcluded that did not have complete BMI WCor NC data Our sample comprised menand women with complete dataStudy variablesHeight and weight were measured using a standardized scale and a fixed stadiometer and BMI was calculated by dividing body weight by the squared height inBraz J Med Biol Res 1015901414431X20209815meters kgm2 WC was measured using an inelastic tapeof cm MabisGulick USA at the midpoint betweenthe lowest rib margin and the iliac crest NC wasmeasured with an inelastic tape mm right under thethyroid cartilage and perpendicular to the long axis of theneck with the participant in a sitting position All measurements were performed by trained nurses The intraclass correlation coefficient for repeated measurementswas 95CI “ Age is presented as a continuous variable and alsostratified as “ years “ years “ years and“ years Race was selfreported as White BrownBlack Asian and Native Educational level was stratifiedas up to incomplete high school highschool and collegeor above Smoking status was selfreported as neverformer and current smoker Monthly family income atbaseline was converted from Brazilian reals BRL to USdollars USD at a rate USD1 BRL2 and stratifiedas oUSD1245 USD1245“ and XUSD3320Excessive alcohol drinking was defined as gweekfor men and gweek for women Blood pressurewas obtained in the sitting position after a minimum restperiod of min Three consecutive readings were obtainedfor each participant after oneminute interval between eachone The mean of the two last measurements was definedas the casual blood pressureLaboratory measurements were obtained after anovernight fast Fasting glucose was determined enzymatically by the hexokinase method Total cholesterol highdensity cholesterol HDLcholesterol lowdensity cholesterol LDLcholesterol and triglycerides were determinedby the enzymatic colorimetric method Hypertension was defined as the use of medications to treathypertension systolic blood pressure X140 mmHg ordiastolic blood pressure X90 mmHg at baseline Diabeteswas defined by a medical history of diabetes use ofmedications to treat diabetes a fasting glucose X126 mgdL glycated hemoglobin HbA1C levels X65 or a 2horal glucose tolerance test X200 mgdL Dyslipidemiawas defined as use of lipidlowering treatment or a LDLcholesterol level X130 mgdLStatistical analysisCategorical variables are reported as absolute countsand proportions Continuous variables are reported asmeans±SD or median interquartile range We usedspline quantile regression models stratified by sex andage to estimate the conditional distribution of NC and WCaccording to BMI These models were used to estimatethe 25th 50th 75th and 90th percentiles for NC and WCvalues in the BMI range between and kgm2 To testa putative association between age and median NC orWC values in men and women we built sexspecificmedian regression models using both BMI and age asexplanatory variables Analyses were performed using theR software Significance level was set at 0cNeck and waist circumference percentilesResultsTable details the characteristics of the sample according to sex The mean age was years Most of theparticipants selfreported being of White race having a college education and never havingsmoked Table shows the estimated 25th 50th75th and 90th percentiles for NC values in cm accordingto BMI age strata and sex Predicted interquartile intervals75th“25th percentile for NC values varied from to cm to of predicted median values Similarly Table shows the predicted conditional distribution for WC valuesTable Characteristics of the study samplein cm also according to BMI age strata and sexPredicted interquartile intervals for WC values varied from to cm to of predicted median valuesGraphical presentations ofthe results are available inFigures and We built sexspecific median regression models usingboth BMI and age as explanatory variables to testifmedian NC or WC were associated with age in men andwomen We found median NC was not associated withage in men P011 nor women P079 Howevermedian WC increased with advancing age in both sexesPo0001 for both Figure shows predicted median WCAge years mean±SD“ years N “ years N “ years N “ years N RaceWhite N Brown N Black N Other N Educational levelIncomplete high school N High school N College or above N Monthly family incomeoUSD1245 N USD12453319 N XUSD3320 N Hypertension N Diabetes N Dyslipidemia N SmokingNever N Past N Current N Excessive drinking N Systolic blood pressure mmHg mean±SDDiastolic blood pressure mmHg mean±SDBodymass index kgm2 mean±SDNeck circumference cm mean±SDWaist circumference cm mean±SDFasting plasma glucose mgdL mean±SDTotal cholesterol mgdL mean±SDLDLcholesterol mgdL mean±SDHDLcholesterol mgdL mean±SDTriglycerides mgdL median [P25“P75]MenN6879± WomenN8206± AllN15085± ±±±±±±±±± [“] ±±±±±±±±± [“] ±±±±±±±±± [“]LDL lowdensity cholesterol HDL highdensity cholesterolBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesTable Neck circumference predicted quantiles for sex age and body mass index BMIAge“ years“ years“ years“ yearsBMIMenWomenP25P50P75P90P25P50P75P90and NC values for men and women with BMIs of and kgm2DiscussionWe presented the conditional distribution of WC andNC values according to age sex and BMI values in alarge sample of Brazilian adults There was a significantvariance in WC and NC values for a given BMI and agestrata for both men and women In addition we found thatWC but not NC values were associated with increasingage Some mechanisms have been proposed to explaindifferent fat tissue distribution within individuals with thesame BMI such as dysfunctional adipose tissue sedentary lifestyle or both As mentioned above local bodyfat mass and its clinical markers as NC and WC areassociated with multiple phenotypes of higher cardiovascular risk The association between these phenotypes andNC or WC cannot be explained exclusively by higherBMIs Evidence from the Framingham Study shows thatbody fat distribution and fat depots could be betterpredictors of cardiovascular diseases CVD than BMI Braz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesTable Waist circumference predicted quantiles for sex age and body mass index BMIAge“ years“ years“ years“ yearsBMIMenWomenP25P50P75P90P25P50P75P90Population data from the European Prospective Investigation into Cancer and Nutrition of the Norfolk cohort showedthat WC and waisttohip ratio were more consistentpredictors of coronary heart disease than BMI Theresults of the Framingham Heart Study showed that NCwas associated with CVD risk factors after adjustment forBMI In addition for specific scenarios the associationbetween these anthropometric measurements and cardiovascular risk may be heterogeneous or even additive In acomparison of the clinical usefulness of NC and WC inindividuals with severe obesity mean BMI meanage years NC values had stronger associations withtype diabetes insulin resistance metabolic syndromeand hypertension compared to WC values In theELSABrasil NC was significantly associated withcardiometabolic risk factors as insulin resistance hypertriglyceridemia and higher blood pressure after adjustment for WC and BMI The study of body fat distribution patterns in subjectswith similar BMI may be importantfor both identifyingindividuals at a higher cardiovascular risk compared topeers with the same BMI and understanding the factorslead to unfavorable fat distribution profiles Therethatis evidence that WC values are increasing more thanBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Predicted 25th 50th 75th and90th percentiles for waist circumferenceWC values according to age body massindex and sexBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Predicted 25th 50th 75th and90th percentiles for neck circumferenceNC values according to age body massindex and sexBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Median waist WC and neck circumference NC values according to age strata for men and women with body mass index of and kgm2 Data are reported as meansexpected for the increase in BMI values in recent decadesin different populations Stern analyzed datafrom Chinese men and women aged to yearsin and Chinese men and women with thesame age range in They found that for every agestrata and in both sexes predicted WC for individuals witha BMI of or kgm2 were higher in than in Janssen compared data from subjectsaged to years in to individuals also aged to years who were evaluated in “ inCanada They found that for individuals with a BMI of kgm2 the predicted WC values in “ were to cm higher than in In addition each kgm2 increasein BMI value was associated with higher WC increases in“ compared to Walls comparedNHANES data from “ participants and“ participants and found that WC valuesin American adults younger than years of age but notin older individuals increased cm more than expectedfor the rise in BMI values during this period Another studywith aggregated data from three crosssectional surveystaken in “ and “ n8313 and respectively looked at WC change inAustralians and found an independent increase of WCshowing that the proportion of obese people detected byWC increased for women and for men On the other hand Elobeid analyzed adifferent timeframe in the United States “ anddid not find a slope for the relationship between WCand BMI over time significantly different from zero Therelationship between NC and BMI is less studied and toour knowledge there are no large epidemiological studies describing the conditional distribution of NC valuesaccording to BMI and age strata Our results highlight theimportance of such descriptions as we found that therelationship between BMI and NC values was ‚uencedless by age strata than the relationship between BMI andWC values Future longitudinal analysis of ELSABrasildata will provide important information about the clinicalrelevance of this findingA study by Stern shows predicted WCvalues for Chinese adults with a BMI of kgm2 and forthose with a BMI of kgm2 We compared their data which matches the inclusion period for ELSABrasilto our predicted median WC values for men and womenwith the same BMI values We found slightly higher predicted WC values for men and lower predicted WC values for women in ELSABrasil compared to the Chinesepopulation In all cases estimates did not differ by morethan cm Some differences between these two studiesmay be partially accountable for this finding First Stern used linear regression which is a least squareBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesmodel for mean values and in our study we used quantileregression which is a linear mathematical optimizationtechnique for estimating quantile valuesincluding themedian Although we aimed to compare similar agestrata it is possible that heterogeneity in age stratificationcutoffs may also have yielded different estimates as bothstudies point to higher WC values according to BMI withincreasing ageOur study had some limitations As it is a descriptivestudy with crosssectional design causal inferences werenot focused Although inexpensive both WC and NC maybe prone to measurement errors and NC values may alsobe ‚uenced by neck muscular volume Thereforemeasurements in other samples should be studied beforeusing these values as a screening tool As strengths ourstudy described the distribution oftwo anthropometricmeasurements in a very large multicenter epidemiologic study in Brazil The conditional distribution of thesevalues according to BMI may be used as markers ofbody fat distribution in future prospective ELSABrasilanalyses To our knowledge analyses of large samplesfocusing on NC values distributions conditioned to BMIand age were not previously published Although it mustbe confirmed by prospective data the smaller ‚uence ofadvancing age on the association between BMI and NCcompared to WC values may be useful to help understand distribution of body fat in longitudinal studies Webelieve our study contributes to fill the gap of evidencementioned in the recent statement ofthe InternationalAtherosclerosis Society in terms of providing a description oftwo adiposity measures by different BMI ageand sex Moreover our study adds to the previous bodyof evidence on the change of waist and neckcircumferences according to BMI age and sex as aneasy and reproducible tool to identify adverse fat depotsphenotypesIn this study we estimated sex and agespecificquantile values for NC and WC according to BMI Therewas significant dispersion in WC and NC values for agiven BMI and age strata for both men and women WCbut not NC values were associated with increasing ageAcknowledgmentsThis work was supported by the Brazilian Ministry ofHealth and CNPq grant numbers 0106001000RS 021200BA 0106030000ES 0106027800MG 00SP 0106007100RJ The authors would like to thank the participants of the ELSABrasil who made this studypossibleReferences Canoy D Boekholdt SM Wareham N Luben R Welch ABingham S Body fat distribution and risk of coronaryheart disease in men and women in the European prospective investigation into cancer and nutrition in Norfolk cohorta populationbased prospective study Circulation “ 101161CIRCULATIONAHA106673756 Vague J The degree of masculine differentiation ofobesities a factor determining predisposition to diabetesatherosclerosis gout and uric calculous disease Obes Res “ 101002j155085281996tb00536x Lim S Meigs JB Ectopic fat and cardiometabolic and vasInt J Cardiol “ riskcularjijcard201308077 Lim S Meigs JB Links between ectopic fat and vasculardisease in humans Arterioscler Thromb Vasc Biol “ 101161ATVBAHA114303035 Anothaisintawee T Sansanayudh N Thamakaison SLertrattananon D Thakkinstian A Neck circumference asan anthropometric indicator of central obesity in patients withprediabetes a crosssectional study Biomed Res Int Preis SR Massaro JM Hoffmann U D™Agostino Sr RBLevy D Robins SJ Neck circumference as a novelmeasure of cardiometabolic riskthe Framingham heartstudy J Clin Endocrinol Metab “101210jc20091779 Ross R Neeland IJ Yamashita S Shai I Seidell J Magni P Waist circumference as a vital sign in clinical practicea Consensus Statement from the IAS and ICCR WorkingBraz J Med Biol Res 1015901414431X20209815Group on Visceral Obesity Nat Rev Endocrinol “ 101038s4157401903107 Amankwah N Brunetti R Kotha V Mercier C Li L Ding J Abdominal obesity index as an alternative centralobesity measurement during a physical examination OpenNutr J “ Gearon E Tanamas SK Stevenson C Loh VHY Peeters AChanges in waist circumference independent of weightImplications for population level monitoring of obesity PrevMed Baltim “ 101016jypmed2017 Lohman TG Roche AF Martorell R Anthropometric standIL Humanardization reference manual ChampaignKinetics De Oliveira CM Ulbrich AZ Neves FS Dias FAL HorimotoARVR Krieger JE Association between anthropometric indicators of adiposity and hypertension in a Brazilianpopulation Baependi heart study PLoS One e0185225 101371journalpone0185225 Liang J Wang Y Li H Liu X Qiu Q Qi L Neckcircumference and early stage atherosclerosis the cardiometabolic risk in Chinese CRC study Cardiovasc Diabetol 101186s129330140107x Zhou J Ge H Zhu M Wang L Chen L Tan Y Neckcircumference as an independent predictive contributor tocardiometabolic syndrome Cardiovasc Diabetol Baena CP Lotufo PA Santos I de S Goulart AC BittencourtMS Duncan BB Neck circumference is associatedwith carotid intimalmedia thickness but not with coronary 0cNeck and waist circumference percentiles Schmidt MI Duncan BB Mill JG Lotufo PA Chor D BarretoSM Cohort profile longitudinal study of adult healthELSABrasil Int J Epidemiol “ 101093ijedyu027 Mill JG Pinto K Griep RH Goulart A Foppa M Lotufo PA Medical assessments and measurements in ELSABrasil [in Portuguese] Rev Saude Publica “101590S003489102013047003851 Schmidt MI Griep RH Passos VM Luft VC Goulart ACMenezes GM de S Strategies and development ofquality assurance and control in the ELSABrasil [in Portuguese] Rev Saude Publica “ S003489102013047003889 Despr©s JP Lemieux I Abdominal obesity and metabolic syndrome Nature “ nature05488 Britton KA Massaro JM Murabito JM Kreger BE HoffmannU Fox CS Body fat distribution incident cardiovasculardisease cancer and allcause mortality J Am Coll Cardiol “ 101016jjacc201306027 Assyov Y Gateva A Tsakova A Kamenov Z A comparisonof the clinical usefulness of neck circumference and waistcircumference in individuals with severe obesity Endocr Res “ Janssen I Shields M Craig CL Tremblay MS Changes inthe obesity phenotype within Canadian children and adults to “ Obesity “ oby2011122 Walls HL Stevenson CE Mannan HR Abdullah A ReidCM McNeil JJ Comparing Trends in BMI and WaistCircumference Obesity 101038oby Elobeid MA Desmond RA Thomas O Keith SW AllisonDB Waist circumference values are increasing beyondincreases Obesity those expected from BMI“ 101038oby2007282artery calcium Results from The ELSABrasil Nutr MetabCardiovasc Dis “ 101016jnumecd Baena CP Lotufo PA Fonseca MGM Santos IS GoulartAC Bensenor IMJ Neck circumference is independentlyassociated with cardiometabolic risk factors crosssectionalanalysis from ELSABrasil Metab Syndr Relat Disord “ 101089met20150083 Bouchard C Tremblay A Genetic ‚uences on theresponse of body fat and fat distribution to positive andnegative energy balances in human identical twins J Nutr 943S“947S 101093jn1275943S Chantler S Dickie K Micklesfield LK Goedecke JHGoedecke JH Micklesfield LK Determinants of change inbody weight and body fat distribution over years in asample of freeliving black South African women Cardiovasc J Afr “ 105830CVJA2016038 Suder A Socioeconomic and lifestyle determinants of bodyfat distribution in young working males from Cracow PolandAm J Hum Biol “ 101002ajhb20687 Hingorjo MR Qureshi MA Mehdi A Neck circumference asa useful marker of obesity a comparison with body massindex and waist circumference J Pak Med Assoc “ Stern D Smith LP Zhang B GordonLarsen P Popkin BMChanges in waist circumference relative to body mass indexin Chinese adults “ Int J Obes “ 101038ijo201474 Du T Sun X Yin P Huo R Ni C Yu X Increasing trends incentral obesity among Chinese adults with normal bodymass index “ BMC Public Health Aquino EML Barreto SM Bensenor IM Carvalho MS ChorD Duncan BB Brazilian Longitudinal Study of AdultHealth ELSABrasil objectives and design Am J Epidemiol “ 101093ajekwr294Braz J Med Biol Res 1015901414431X20209815 0c'

Thyroid_Cancer

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

Thyroid_Cancer

"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 following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer “ httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet “ httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir Scand“Dargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta “ Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA “Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA “ httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid “ httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk 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

"The principal function of iodine acts on thyroid function but in recent years the role of iodinedeficiency in metabolism has also been gradually revealed We aimed to investigate the current status of iodizedsalt consumption and urinary iodine concentration UIC in an urban Chinese population with type diabetes andto further explore whether UIC was associated with diabetic microvascular complicationsMethods Four thousand five hundred fiftynine subjects with diabetes from communities in downtownShanghai were enrolled in the crosssectional Metal Study in UIC was detected using an inductively coupledplasmamass spectrometer Diabetic kidney disease DKD was defined as urinary albumintocreatinine ratioUACR mgg or estimated glomerular filtration rate mLmin173 m2 Diabetic retinopathy DR wasevaluated by highquality fundus photographs and was remotely read by ophthalmologistResults The median UIC of subjects with diabetes was μgL “ in downtown Shanghai Among allthe subjects consumed noniodized salt and were iodine deficient Iodine deficiency UIC μgLwas associated with an increased odds of DKD OR 95CI “ after adjustment for age sex educationcurrent smokers BMI HbA1c duration of diabetes dyslipidemia thyroidstimulating hormone and free thyroxineNo association was observed between UIC and DR after multivariable adjustmentConclusions A concerning number of subjects with diabetes consumed noniodized salt and suffered from iodinedeficiency in coastal regions of China Low UIC might be a risk factor for DKD which should be further confirmedby longitudinal prospective studiesKeywords Iodized salt Type diabetes Diabetic kidney disease Urinary iodine concentration Epidemiology Correspondence wnj486126com luyingli2008126com Chi Chen and Yi Chen contributed equally to this workInstitute and Department of Endocrinology and Metabolism Shanghai NinthPeople™s Hospital Shanghai JiaoTong University School of MedicineShanghai China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen Nutrition Metabolism Page of IntroductionType Diabetes Mellitus T2DM has become a seriousglobal health care burden causing microvascular complications which are associated with increased disabilityreduced quality of life and life expectancy [ ] It wasestimated that there were million cases of adult diabetes worldwide in and the number was projectedto increase to million by [] leading to highincidence and prevalence of microvascular complications Approximately one third with T2DM will developdiabetic retinopathy DR and one quarter will developdiabetic kidney disease DKD [] Thus it is critical toidentify and control some novel modifiable risk factorscontributing to microvascular complications in patientswith T2DMIodine is an indispensible micronutrient for the synthesis of thyroid hormones Iodine deficiency ID in early lifeimpairs neurodevelopment and also has many adverse effects throughout various life stages [] Over the past years substantial progress has been achieved in the worldwide effort to eliminate iodine deficiency disorders IDDby salt iodization program However this program is vulnerable and requires a longterm commitment from governments In several countries where ID had been onceeliminated salt iodization programs were discontinuedand ID has now reappeared []The principal function of iodine acts on thyroid function but in recent years the role of ID in metabolismhas also been gradually revealed Analysis of data fromthe National Health and Nutrition Survey NHANES“ found that in US adults low urinary iodineconcentration UIC was associated with dyslipidemia[] and coronary artery disease [] Moreover O S AlAttas [] reported that UIC is markedly decreasedin T2DM and urinary iodine was negatively associatedwith insulin resistance in patients with T2DM Most recently Mingyue Jin [] also found that at a lowerUIC μgL the prevalence of diabetes significantlyincreased relative to an UIC of “ μgL Animalstudies also showed that iodine supplementation couldreduce the blood glucose levels and improve the insulinsensitivity in goats [] However the role of iodine nutrition on diabetic microvascular complications has notbeen studiedThe aim of this study was to investigate the currentstatus of iodized salt consumption and UICs in an urbanChinese population with T2DMand additionallywhether UIC is associated with diabetic microvascularcomplications including DKD and DRMethodsStudy populationThe crosssectional METAL study Environmental Pollutant Exposure and Metabolic Diseases in Shanghaiwwwchictrcn ChiCTR1800017573 was launchedto investigate the association between iodine nutritionand microvascular complications in Chinese adults withdiabetes We recruited study participants from sevencommunities in Huangpu and Pudong new districtShanghai China Huangpu district located in downtownShanghai is the administrative economic and culturalcenter of the metropolitan coastal city [] Pudong newdistrict is the symbol of China™s reform and ingup[] We randomly selected half of patients with diabetesfrom the registration platform in each communityhealthcare center Chinese citizens ‰¥ years old whohad lived in their current area for ‰¥ months were included In August a total of subjects withT2DM who were “ years of age received an examination Participants with missing UIC values n were excluded Finally participants were involvedin the present analysisThe study received ethical approval from the EthicsCommittee of Shanghai Ninth People™s Hospital Shanghai Jiao Tong University School of Medicine All procedures followed were abided by the ethical guidelines ofthe Declaration of Helsinki as reflected in a prioriapproval by the appropriate institutional review committee Informed consent was received from all participantsincluded in the study prior to the data collectionMeasurementsinThe same welltrained and experienced personnelSPECTChina study [“] used a questionnaire to collect information on sociodemographic characteristicseducation medical history family history and lifestylerisk factors Weight and height were measured using abalance beam and a vertical ruler in light clothing andwithout shoes Body mass index BMI was calculated asthe ratio of weight in kilograms divided by height in meters squared Current smoking was defined as havingsmoked at least cigarettes in one™s lifetime and currently smoking cigarettes [] Especially œFor the pastthree years which type of salt was used in your familywas applied to collect information about type of saltThree options for this item were provided only iodized salt only noniodized salt bothBlood samples were drawn between am and am after an overnight fast Blood was refrigerated immediately after phlebotomy and in hours it was centrifugated and the serum was aliquoted and frozen in acentrallaboratory Glycated hemoglobin HbA1c wasmeasured by highperformance liquid chromatographyMQ2000PT Medconn Shanghai China Fastingplasma glucose serum creatininetotalcholesterol high HDL and lowdensity lipoproteinLDL were performed with a Beckman Coulter AU Brea USA Serum thyroidstimulating hormone TSHtriglycerides 0cChen Nutrition Metabolism Page of and free thyroxine FT4 were measured by electrochemiluminescence Roche E601 GermanyMorning fasting spot urine samples collected were refrigerated immediately and frozen at ˆ’ °C in hoursUIC was detected using an inductively coupled plasmamass spectrometer ICPMS No 7700x Agilent Technologies Inc USA The concentrations of urine albuminand creatinine were determined with a Beckman CoulterAU Brea USA using a turbidimetric immunoassayand an enzymatic method respectively Urinary albumintocreatinine ratio UACR was calculated as the urinaryalbumin concentrations divided by the urinary creatinineconcentrations and expressed in mggDR screening was evaluated by mydriatic binocular indirect ophthalmoscopy Topcon TRCNW400 NonMydriatic Retinal Camera Oakland USA Fundus photographs were read by an experienced ophthalmologistspecialized in retinaOutcome definitionDyslipidemia was defined as total cholesterol ‰¥mmolL mgdL triglycerides ‰¥ mmolL mgdL LDL ‰¥ mmolL mgdL HDL mmolL mgdL or selfreported previous diagnosisof hyperlipidemia by physicians according to the modified National Cholesterol Education ProgramAdultTreatment Panel IIIThe estimated glomerular filtration rate eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration CKDEPI equation for œAsian origin []As suggested by American Diabetes Association ADAhigh UACR was defined as UACR ‰¥ mgg reducedeGFR as eGFR mlmin173m2 and DKD as UACR mgg or eGFR mLmin173 m2 []The internationally accepted DR classification by theœGlobal Diabetic Retinopathy Project Group in was applied [] The classification was no retinopathynonproliferative DR intraretinal microaneurysmshemorrhages venous beading prominent microvascularabnormalities and proliferative DR neovascularizationor vitreouspreretinal hemorrhagesStatistical analysisStatistical analysis was run with SPSS IBM Corporation Armonk NY USA General characteristics arepresented as median with the interquartile range IQRfor continuous variables or as proportion for categoricalvariables MannWhitney U test and the KruskalWallistest were used for comparison of two or more groups ofnonnormally distributed data Pearson™s χ2 tests wereperformed to compare categorical variablesThe associations of UIC with elevation of UACR reduction of eGFR DKD and DR were analyzed with logistic regression analyses The results are presented as oddsratio OR and confidence intervals CIs Model was unadjusted Model was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Table General characteristics of study participants by UIC categoriesUrinary iodineAge yrAdequate “ “ “ “ “ “Low “ “ “ “ “ “Women UIC μgLFPG mmolLHbA1c BMI kgm2Duration of diabetes yrCurrent smokers Beyond high school education TSH mIULFT4 pmolLBlood lipidsTotal cholesterol mmolLLDLC mmolLHDLC mmolLTriglycerides mmolLMore than adequate “ “ “ “ “ “ “ “Excessive “ “ “ “ “ “ “ “ “ “ “ “P “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Data are summarized as median interquartile range for continuous variables or as number with proportion for categorical variablesUIC Urinary iodine concentration FPG Fasting plasma glucose HbA1c Glycated hemoglobin BMI Body mass index LDLC Low density lipoprotein cholesterol HDLC High density lipoprotein cholesterol TSH Thyroidstimulating hormone FT4 Free thyroxineUrinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL 0cChen Nutrition Metabolism Page of ResultsGeneral characteristics of the study populationThe general characteristics are presented in Table The mean age of the study population was nearlyone half were women About of the studypopulation were overweight or obese BMI ‰¥ kgm2and were current smokers The percentage of aneducational level beyond high school was and theaverage duration of diabetes was yearsCompared to those with adequate iodine nutritionsubjects with ID were slightly older and were more likelyto be women These subjects also had higher TSH lowerBMI and a lower percentage of current smokers Inaddition subjects with more than adequate and excessiodine nutrition were slightly younger and had higherBMI but comparable TSHUrinary iodine concentration and type of salt intake inthe populationThe distribution of UICs in the study population is presented in Fig The median 25th“75th percentile UICof subjects with diabetes was μgL “ indowntown Shanghai which falls within the range of μgL that WHOUNICEFICCIDD categorize asadequate Urinary iodine measurements indicative of IDUIC μgL were present in of the studypopulation Meanwhile and of the populationshowed more than adequate UIC “ μgL andexcess iodine intake UIC ‰¥ μgL respectivelyThe distribution of type of salt intake is presented inFig As high as of the study population consumed noniodized salt consumed iodized saltand consumed both Logistic regression analysisshowed that compared to those who consumed iodizedsalt subjects consumed noniodized salt were morelikely to be women OR 95CI “ and havea higher educational attainment OR 95CI “ but a comparable age OR 95CI “Association of urinary iodine concentration with elevationof UACR reduction of eGFR and DKDThe association of UIC with elevation of UACR reduction of eGFR and DKD is shown in Table Comparedwith those with adequate iodine nutrition subjects withID had an increased risk of elevation of UACR OR 95CI “ reduction of eGFR OR 95CI“ and DKD OR 95CI “ Adjustment for age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 did not attenuate the association of ID with UACRand DKD However multivariable adjustment weakenedthe association between ID and reduction of eGFR further such that it was no longer significant Meanwhilesubjects with more than adequate and excess iodine nutrition did not have an increased risk of elevation ofUACR reduction of eGFR and DKD after multivariableadjustmentAssociation of urinary iodine concentration with DRTable presents the association of UIC with nonproliferative and proliferative DR Compared with thosewith adequate iodine nutritionthe ORs of nonproliferative and proliferative DR in subjects with morethan adequate iodine nutrition were 95CI “Fig Distribution of UICs in the study population 0cChen Nutrition Metabolism Page of Fig Distribution of type of salt consumed in the study populationthan adequate and 95CI “ respectively Multivariable adjustment weakened the association betweenmoreiodine nutrition and nonproliferative DR further such that the association was nolonger significant There was no significant associationobserved between DR and ID and excessiodinenutritionDiscussionIn this study among over communitydwellingChinese adults with diabetes we found that of thesubjects consumed noniodized salt and had IDIodine deficiency was significantly associated with ahigher prevalence of elevated UACR and DKDindependently of age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 To the best of our knowledge this is the first studyto investigate the current status of iodized salt consumption and iodine nutrition status in a relatively largepopulation with diabetes and further investigate the association between UIC and diabetic microvascularcomplicationsChina was once severely affected by IDD and hence amandatory Universal Salt Iodization USI program wasTable Association of urinary iodine with elevation of UACR and reduction of eGFRUrinary iodineHigh UACRAdequateLowMore than adequateExcessivePrevalenceOdds RatioModel Model Reduced eGFRPrevalence Odds RatioModel Model DKDPrevalence Odds Ratio Ref Ref Ref Ref “ “ “ “ “ “ “ “ “ “ “ “Model Model Ref Ref “ “Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with elevation of UACR reduction of eGFRand DKD P Model was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4High UACR was defined as UACR ‰¥ mgg reduced eGFR as eGFR mlmin173 m2 and DKD as UACR mgg or eGFR mLmin173 m2Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL “ “ “ “ 0cChen Nutrition Metabolism Page of Table Association of urinary iodine with DRUrinary iodineNonproliferative DRAdequatePrevalence Odds RatioModel Model Proliferative DRPrevalence Odds RatioModel Model Ref Ref Ref RefLowMore than adequateExcessive “ “ “ “ “ “ “ “ “ “ “ “P Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with DRModel was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgLintroduced in and was successful in eliminatingIDD However since the prevalence of thyroid diseaseshas markedly increased in recent years some concernsabout the USI have circulated especially among coastalresidents in urban areas [] In the present analysis themedian UIC of residents with diabetes in downtownShanghai has fallen to marginal levels of iodine sufficiency μgL and more surprisingly more thanhalf of the subjects consumed noniodized saltand were iodine deficient Compared with a studyconducted by the Shanghai Municipal Center for DiseaseControl and Prevention CDC in of participants used iodized salt and were iodine deficientat that time [] During “ Zhongyan Shanand her colleagues performed a crosssectional study ineastern and central China and reported that the medianUIC was μgL in schoolaged children and μgLin the total cohort population [] Our previous studyconducted on the general population found that consumed noniodized salt in the urban area of Shanghai in []The present study indicates that an increasing numberof urban residents in downtown Shanghai prefer to usenoniodized salt in recent years and suffer from IDWomen and those with a higher educational level weremore tended to consume noniodized salt WhyChanges in the reported spectrum and growing incidence of thyroid disorders have been linked to the increased iodine intake resulting from USI in the localmedia and international medical literature [] Thosewith high educational attainment were more likely to beconfused by these information and worry about theirthyroid health and call for liberalizing provincial controlof sales of noniodized salt Formerly needed a prescriptionthe sale of noniodized salt has now beenunofficially allowed by some coastal city authorities []In addition T2DM is associated with an increased riskof multiple thyroid disorders such as thyroid nodule[] thyroid cancer [] and autoimmune thyroid diseases [] Since the prevalence of thyroid abnormalitieswere found to be much higher in females than males[] it is reasonable to deduce that these women wouldhave a higher tendency towards consuming noniodizedsalt after diagnosis of thyroid abnormalitiesseafood alone to provide sufficientConsidering that Shanghai is a coastal city local residents believe that they should never suffer from IDDwith high iodineenriched aquatic products consumption However it may not be true to rely on consumption ofiodineActually the environmental levels of iodine in Shanghaiare deficient μgL [] Furthermore based on theresearch initiated by Shanghai Municipal CDC iodizedsalt contributed of the total dietary iodine inShanghai[] Aquatic products which residentsthought to be rich in iodine accounted for only ofthe total dietary iodine []Thus iodized salt is still themain source foriodine supplementation in coastalpopulationsDKD wascommonlydefinedby ADA asœUACR‰¥30mgg or eGFR60 mlmin per m2 []We found that ID was associated with elevated UACRand DKD The mechanism underlying the association ofID with DKD is not yet fully understood Deficiency ofiodine could reduce thyroid hormone production andelevate TSH It has been reported that lower free triiodothyronine and elevated TSH have significant associationwith risk for albuminuria in T2DM [ ] Moreoverinadequate iodine intake is significantly correlated withan increase in oxidative stress [] Recent evidence hasshown thattumorinflammatory cytokinessuch as 0cChen Nutrition Metabolism Page of necrosis factoralpha and interleukin1 play a pivotalrole in the pathogenesis of DKD [] Therefore we suppose the possible mechanism may be via inflammatoryresponseIn view of the evergrowing prevalence of T2DM andDKD all over the world successive intervention in thislarge population can have important impact on publichealth ID unlike most micronutrient deficiencies is notrestricted to people in developing countries with poordiets Since salt iodization is simple effective and inexpensive the best strategy to control ID is addition ofiodine into salt in nearly all countries [] Monitoringiodine situation of people with diabetes is of critical significance and education programs to diabetes especiallywomen with high academic background may also include information of adequate iodine intake in our clinical practice Our study shed light on the possiblebeneficial effect of iodine supplementation in reducingalbuminuria in T2DM which warrants further investigation in welldesigned randomized controlled trialOur study benefited from its welldefined communitybased participants with a relatively large sample sizeSecond regarding the novelty our study is the first toprovide iodine status and linked iodine insufficiency toan increased risk of albuminuria in people with diabetesThird we used ICPMS to detect UIC in the presentanalysis which was considered as the goldstandardmethod [] There were also some limitations weshould acknowledge First no causal relationship couldbe determined due to the crosssectional design of thestudy and thus our findings need to be validated by longitudinal prospective studies Second although UIC isrecommended by the WHOICCIDDUNICEF for evaluation of iodine status at the population level and widelyused in largescale epidemiological studies [ ] thesingle spot urine measurement may not accurately assesslongterm iodine status at the individual level Actuallyinter and intraindividual variability exists in UIC []However the application of a large sample size from to subjects per subgroup may counteract thebias related to the use of only one casual urine sample[] Future followup studies collecting 24h urine specimens twice are needed to replicate the present resultsConclusionA large proportion of diabetic patients in downtownShanghai consumed noniodized salt and had ID IDmay increase the risk of DKD independent of thyroidfunction in diabetic patients Maintaining USI at an appropriate level is indispensable for diabetic patients Cohort and intervention studies as well as basic researchexploring the effect and mechanism of iodine supplementation on renal function are warrantedAbbreviationsT2DM Type Diabetes Mellitus DKD Diabetic kidney disease DR Diabeticretinopathy ID Iodine deficiency UIC Urinary iodine concentrationTSH Thyroidstimulating hormone BMI Body mass index OR Odds ratioCI Confidence interval UACR Urinary albumintocreatinine ratioeGFR Estimated glomerular filtration rateAcknowledgementsThe authors thank Xiaojin Wang and Bingshun Wang from the Departmentof Biostatistics and Shanghai Jiaotong University School of Medicine for dataprocessingAuthors™ contributionsYL and NW designed the study CC YC HZ FX BH WZ YW HWand NW conducted the research CC and YC analyzed the data and wrotethe manuscript CC and YC contributed equally The final manuscript wasread and approved by all authorsFundingThis study was supported by National Natural Science Foundation of China Science and Technology Commission of ShanghaiMunicipality the Fourth Round of ThreeYear Public HealthAction Plan of Shanghai by the Shanghai Municipal Commission of Healthand Family Planning 20164Y0079 Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai 2017YQ053 Fundamental research program funding of NinthPeople™s Hospital affiliated to Shanghai Jiao Tong university School of MedicineJYZZ099 Shanghai Sailing Program 20YF1423500Availability of data and materialsThe datasets during andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Ethics Committee of Shanghai NinthPeople™s Hospital Shanghai Jiao Tong University School of Medicine Writteninformed consent was received from all participantsConsent for publicationNACompeting interestsNo potential conflicts of interest relevant to this were reportedReceived May Accepted August ReferencesKahm K Laxy M Schneider U Rogowski WH Lhachimi SK Holle R Healthcare costs associated with incident complications in patients with type diabetes in Germany Diabetes Care “Chen C Chen Q Nie B Zhang H Zhai H Zhao L Trends in bonemineral density osteoporosis and oste ia among US adults withprediabetes Diabetes Care “Cho NH Shaw JE Karuranga S Huang Y da Rocha Fernandes JD OhlroggeAW IDF diabetes atlas global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract “Valencia WM Florez H How to prevent the microvascular complications oftype diabetes beyond glucose control Bmj 2017356i6505Zimmermann MB Boelaert K Iodine deficiency and thyroid disordersLancet Diabetes Endocrinol “Zimmermann MB Andersson M Assessment of iodine nutrition inpopulations past present and future Nutr Rev “Lee KW Shin D Song WO Low urinary iodine concentrations associatedwith dyslipidemia in US adults Nutrients Tran HV Erskine NA Kiefe CI Barton BA Lapane KL Do VTH Is lowiodine a risk factor for cardiovascular disease in Americans without thyroiddysfunction Findings from NHANES Nutr Metab Cardiovasc Dis “AlAttas OS AlDaghri NM Alkharfy KM Alokail MS AlJohani NJ AbdAlrahman SH Urinary iodine is associated with insulin resistance in 0cChen Nutrition Metabolism Page of Wu J Li X Tao Y Wang Y Peng Y Free triiodothyronine levels areassociated with diabetic nephropathy in Euthyroid patients with type diabetes Int J Endocrinol Vidal ZE Rufino SC Tlaxcalteco EH Trejo CH Campos RM Meza MN et alOxidative stress increased in pregnant women with iodine deficiency BiolTrace Elem Res “Jha JC Banal C Chow BS Cooper ME JandeleitDahm K Diabetes andkidney disease role of oxidative stress Antioxid Redox Signal “ Pearce EN Andersson M Zimmermann MB Global iodine nutrition wheredo we stand in Thyroid “Ittermann T Johner S Below H Leiterer M Thamm M Remer T et alInterlaboratory variability of urinary iodine measurements Clin Chem LabMed “Jeon MJ Kim WG Kwon H Kim M Park S Oh HS Excessive iodineintake and thyrotropin reference interval data from the Korean NationalHealth and nutrition examination survey Thyroid “Inoue K Leung AM Sugiyama T Tsujimoto T Makita N Nangaku M et alUrinary iodine concentration and mortality among US adults Thyroid“ Chen W Li X Guo X Shen J Tan L Lin L Urinary iodine excretion UIEestimated by iodinecreatinine ratio from spot urine in Chinese schoolagechildren Clin Endocrinol Oxf “ Andersen S Karmisholt J Pedersen KM Laurberg P Reliability of studies ofiodine intake and recommendations for number of samples in groups andin individuals Br J Nutr “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationssubjects with diabetes mellitus type Exp Clin Endocrinol Diabetes “Jin M Zhang Z Li Y Teng D Shi X Ba J Ushaped associationsbetween urinary iodine concentration and the prevalence of metabolicdisorders a crosssectional study Thyroid “ Nudda A Battacone G Bomboi G Floris B Decandia M Pulina G Effect ofdietary iodine on thyroid hormones and energy blood metabolites inlactating goats Animal “ Qu Y Zhuo L Li N Hu Y Chen W Zhou Y Prevalence of poststrokecognitive impairment in China a communitybased crosssectional studyPLoS One 201510e0122864Sun X Li Y Liu S Lou J Ding Y Liang H Enhanced performance ofcommunity health service centers during medical reforms in Pudong NewDistrict of Shanghai China A longitudinal survey PLoS One e0125469 Wang N Wang X Li Q Han B Chen Y Zhu C The famine exposure inearly life and metabolic syndrome in adulthood Clin Nutr “ Zhai H Chen C Wang N Chen Y Nie X Han B Blood lead level isassociated with nonalcoholic fatty liver disease in the Yangtze River Deltaregion of China in the context of rapid urbanization Environ Health Chen C Zhao L Ning Z Li Q Han B Cheng J Famine exposure inearly life is associated with visceral adipose dysfunction in adult females EurJ Nutr “ Chen C Zhai H Cheng J Weng P Chen Y Li Q Causal link betweenvitamin D and Total testosterone in men a Mendelian randomizationanalysis J Clin Endocrinol Metab “Stevens LA Claybon MA Schmid CH Chen J Horio M Imai E et alEvaluation of the chronic kidney disease epidemiology collaborationequation for estimating the glomerular filtration rate in multiple ethnicitiesKidney Int “Tuttle KR Bakris GL Bilous RW Chiang JL de Boer IH GoldsteinFuchs J Diabetic kidney disease a report from an ADA consensus conferenceDiabetes Care “ Wilkinson CP Ferris FL 3rd Klein RE Lee PP Agardh CD Davis M et alProposed international clinical diabetic retinopathy and diabetic macularedema disease severity scales Ophthalmology “ Zou Y Lou X Ding G Mo Z Zhu W Mao G Iodine nutritional status afterthe implementation of the new iodized salt concentration standard inZhejiang Province China BMC Public Health Zou S Wu F Guo C Song J Huang C Zhu Z Iodine nutrition and theprevalence of thyroid disease after salt iodization a crosssectional survey inShanghai a coastal area in China PLoS One 20127e40718Shan Z Chen L Lian X Liu C Shi B Shi L Iodine status and prevalenceof thyroid disorders after introduction of mandatory universal salt iodizationfor years in China a crosssectional study in cities Thyroid “ Chen C Xu H Chen Y Chen Y Li Q Hu J Iodized salt intake and itsassociation with urinary iodine thyroid peroxidase antibodies andthyroglobulin antibodies among urban Chinese Thyroid “Teng X Shan Z Chen Y Lai Y Yu J Shan L More than adequateiodine intake may increase subclinical hypothyroidism and autoimmunethyroiditis a crosssectional study based on two Chinese communities withdifferent iodine intake levels Eur J Endocrinol “ Wu Y Li X Chang S Liu L Zou S Hipgrave DB Variable iodine intakepersists in the context of universal salt iodization in China J Nutr “ Chen Y Zhu C Chen Y Wang N The Association of Thyroid Nodules withmetabolic status A crosssectional SPECTChina study Int J Endocrinol Qi J He P Yao H Song R Ma C Cao M Cancer risk among patientswith type diabetes a realworld study in Shanghai China SarfoKantanka O Sarfo FS Ansah EO Yorke E Akpalu J Nkum BC et alFrequency and determinants of thyroid autoimmunity in Ghanaian type diabetes patients a casecontrol study BMC Endocr Disord M B T G M P A P PC W Gender differences in thyroid system functionrelevance to bipolar disorder and its treatment Bipolar Disord “ Microvascular Complications and Foot Care Standards of medical Carein Diabetes2018 Diabetes Care 201841S105“s118 Yasuda T Kaneto H Kuroda A Yamamoto T Takahara M Naka T et alSubclinical hypothyroidism is independently associated with albuminuria inpeople with type diabetes Diabetes Res Clin Pract 201194e75“ 0c"

Thyroid_Cancer

"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagan™s nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming People™s Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expˆ’b·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expˆ’bD f · expˆ’bD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [“] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [“] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Begg™s test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deek™s plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Begg™s testor Deek™s test indicated the potential of publication bias[] Inconsistency index I2 and Cochran™s Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagan™s nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies“ scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The χ2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ““ “ ± “NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA “ ± NA “ ± “Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of ˆ’ mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Begg™s Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of Begg™sTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of ˆ’ P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Begg™s Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Begg™s Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deek™s funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagan™s nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks™ funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagan™s nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors™ contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors™ informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming People™sHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol “ httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med “ httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology “ httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol “ httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis “httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L

Thyroid_Cancer

"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

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

Thyroid_Cancer

"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ‰¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ‰¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ‰¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ “ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinson™s and angina κ “ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworld™s older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ‰¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV “ two algorithms which determine an individual™scurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [“] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ‰¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ‰¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ‰¥ years The first wave ofdata collection began in October through toFebruary N participants aged ‰¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDA™s study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ‰¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ‰¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ‰¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participant™s TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinson™s disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity “ Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ‰¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system [“]Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question eg˜donepezil™ was required to be dispensed on ‰¥ consecutive prescriptions to link this medication withthe RxRiskV condition ˜dementia™ This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ‰¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions “ eg antiwere notthrombotic agents B01AC04 “ B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ‰¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohen™s Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair “ moderate “ good “ and verygood “ []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ‰¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ‰¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ‰¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ‰¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ‰¥ years N Poor delayed recall years N ‰¥ years N years N ‰¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients™ selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ‰¥ years measures of morbidity High blood pressure or hypertension yearsN ‰¥ years N and highcholesterol years N ‰¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ‰¥ years years N “Age‰¥ years N “GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinson™s κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ “ All other conditions had poor agreement κ “ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was ˜very good™ agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe ˜very good™ Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinson™s disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was ˜good™ agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was ˜moderate™ agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was also˜moderate™ agreement for high cholesterol in both agecohorts and for angina and Parkinson™s disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ‰¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPaget™s disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinson™s disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack “ “StrokeTIAHigh Cholesterol “ “ “HeartTrouble “Cancer “EmotionalProblems “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Depressiononly “ “ “ “ “ “Stomachulcers “ “ “ “ “ “Asthma “ “ “ “ “ “Arthritisgeneral “ “ “ “ “ “RheumatoidArthritis only “ “ “ “ “ “ “ “ “Angina “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Congestive HeartFailure “Abnormal HeartRhythm “ “ “ “ ““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““ “ “ “““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Anxiety “ “ “ “ “ “ “ “ “ “LungDisease “ “ “ “ “ “ “ “ “ “ 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis “Psychosisonly “StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinson™s disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinson™s diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only ˜fair™ agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso ˜fair™ agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified ˜taking pain medication™ Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was œpoor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also ˜poor™ agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed ˜diapers and pads supplies™ []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo

Thyroid_Cancer

catalytic activity of human Telomerase Reverse Transcriptase TERT compensates for the loss of telomere length eroded during each cell cycle to ensure a correct division of stem and germinal cells In human tumors ectopic TERT reactivation most frequently due to hotspot mutations in the promoter region TERTp ie c1124 C T c1146 C T confers a proliferative advantage to neoplastic cells In gliomas TERTp mutations TERTpmut mainly occur in oligodendroglioma and glioblastoma We screened for TERTp hotspot mutations adult patients with gliomas and identified heterozygous mutations in cases of oligodendroglioma of glioblastoma and of diffuseanaplastic astrocytoma Besides the recurrent c1124 C T and c1146 C T two cases of glioblastoma harbored novel somatic TERTp variants which consisted of a tandem duplications of nucleotides ie a TERTp c1100_179dup and TERTp c1110_189 both located downstream c1124 C T and c1146 C T In silico analysis predicted the formation of and new transcription factor™s recognition sites for TERTp c1100_179dup and TERTp c1110_189 respectively TERTp duplications TERTpdup mainly affected the binding capacity of two transcription factors™ families ie the members of the Etwentysix and the Specificity ProteinKr¼ppelLike Factor groups In fact these new TERTpdup significantly enhanced the Etwentysix transcription factors™ binding capacity which is also typically increased by the two c1124 C Tc1146 C T hotspot TERTpmut On the other hand they were distinguished by enhanced affinity for the Kr¼ppel proteins The luciferase assay confirmed that TERTpdup behaved as gainoffunction mutations causing a fold increase of TERT transcription The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors with the identification of new recurrent somatic gainoffunction mutations occurring in of glioblastoma IDHwildtypeKeywords TERT Gliomas Gainoffunction mutation ETS and Kr¼ppel transcription factorsIntroductionThe abnormal reactivation of human Telomerase Reverse Transcriptase TERT is a common hallmark of human solid tumors Although it may be caused by Correspondence cristinamecucciunipgit robertalastarzaunipgit Cristina Mecucci Roberta La Starza have equally contributed to this work Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia ItalyFull list of author information is available at the end of the several mechanisms ie methylation mutations rearrangementsfusions and DNA copy number amplifications TERT promoter TERTp methylation and gainoffunction mutations are the most frequent [ ] In particular two recurrent hotspot mutations are respectively located at TERTp124 and TERTp146 base pairs bp from the TERT ATG start site [ “ ] Both mutations generated from a cytidine to thymidine dipyrimide transition C T are usually heterozygous mutually exclusive and produce The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPierini a0et a0al acta neuropathol commun Page of an identical a0bp ˜CCC CTT CCGGG™ sequence resulting in the creation of de novo consensus binding motifs for Etwentysix ETS transcription family members These new binding sites recruit a larger number of ETS factors enhancing the transcription of TERT []TERT promoter mutations TERTpmut typically occur in tumors that arise from low selfrenewal tissue such as melanomas thyroid hepatobiliary carcinoma and central nervous system CNS tumors with a variable frequency that range from to of cases in diverse histological subtypes [ ] In CNS tumors TERTpmut are typically associated with glioblastoma GBM “ and oligodendroglioma ODG “ whereas their frequency decreases in other glioma subtypes such as diffuseanaplastic astrocytoma DAAA “ medulloblastoma and meningioma about [ ] Although the clinical value of TERTpmut in refining the diagnostic classification of gliomas is widely accepted [] its role as prognosticpredictive biomarker is still largely debated TERTpmut have been associated with a poor disease outcome in GBM IDHwildtype GBM IDHwt but there is no full agreement on its impact on DAAA [ ] It is worth noting however that DAAA IDHwildtype DAAA IDHwt harboring genomic abnormalities typically associated with GBM ie TERTp mutations or EGFR amplification or gain of whole chromosome in combination with monosomy of chromosome have a clinical outcome similar to or only slightly longer than GBM [] Thus the cIMPACT NOW Update recommended to use one of these molecular criteria to classify this subgroup of astrocytomas as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV and to revise the classification of DAAA IDHwt accordingly []Herein we report two new TERTp mutations that were identified in two patients with GBM IDHwt Both these new variants originated from the duplication of a stretch of nucleotides at TERTp TERTpdup and although slightly different shared an overlapping sequence of nucleotides We demonstrated the somatic nature of one of these TERTpdup and that enhancing the binding affinity for ETS transcription factors TFs they both elicit the TERT transcription thus widening the spectrum of recurrent gainoffunction mutations of TERTp in GBMCase presentationCohortThe study was carried out on a cohort of patients affected by primary CNS tumours and referred to our laboratory during the last a0years Table a0 There were males and females ratio with a median age of range age According to the WHO the diagnosis was grade II DA IDHwt cases and DA IDHmutant DA IDHmut cases grade III AA IDHwt cases and AA IDHmut grade IV GBM IDHwt and GBM IDHmut grade IIIII ODG Three patients had a diagnosis of uncommon glioma Table a0 The study was approved by Institutional Bioethics Committee University of Perugia and Santa Maria della Misericordia Hospital of PerugiaItaly Protocol no284316 all patients gave informed consent for sample collection and molecular analyses in agreement with the Declaration of HelsinkiIndex casesA 71yearold male UPN131 had a left frontal lesion of a0mm diameter partially infiltrating the corpus callosum the second case UPN171 a male of a0years presented with a right frontal lesion Histopathology and immunohistochemistry were consistent with a diagnosis of GBM IDHwt in both patients In case UPN131 neoplastic cells showed marked cytoplasmic and nuclear pleomorphism there was a discrete number of atypical mitotic figures widespread necrosis a diffuse GFAP positivity and few neoplastic elements with strong nuclear TP53 stain Case UPN171 was characterized by striking atypia of neoplastic cells diffuse necrosis vascular proliferation strong and diffuse positivity for GFAP and nuclear TP53 Fig a0 No IDH1IDH2 hotspot mutations were detected while both cases showed MGMT promoter methylation Monosomy of chromosome cooccurred with EGFR amplification UPN131 or with gain of the whole chromosome UPN171Materials and a0methodsTERT promoter mutational analysisGenomic DNA was extracted from FormalinFixed ParaffinEmbedded FFPE tumor tissue and from peripheral blood PB by QIAamp DNA FFPE and AllPrep DNARNA kits respectively following the manufacturer™s instructions QIAGEN Milan Italy Hotspot TERTpmut were investigated by Sanger sequencing using ABI Genetic analyzer instrument Applied Biosystems Monza Italy Primers were reported in Table a0S1 Additional file a0 Table a0S1 and referred to GRCh37 genomic coordinate system NM_0000059 for regulatory core promoter a0 bp wwwncbinlmnihgovgene [] wwwensem blHomo_sapie ns [] Sequences™ alignments and their analyses were supported by Clustal wwwebiacukTools msaclust alo Ensembl Omega httpwwwensem blHomo_sapie ns and [] 0cPierini a0et a0al acta neuropathol commun Page of Table Epidemiological and a0clinical features of a0our cohort of a0patientsEpidemiologicalclinical dataTotal cohortGenderAge yearsDiagnosis WHO Common GliomasUncommon GliomasAnatomic locationMaleFemaleMFRangeMedian years‰¥ yearsDiffuse astrocytoma IDHwt grade IIDiffuse astrocytoma IDHmut grade IIAnaplastic astrocytoma IDHwt grade IIIAnaplastic astrocytoma IDHmut grade IIIGlioblastoma IDHwt grade IVGlioblastoma IDHmut grade IVOligodendroglioma IDHmut and 1p19qcodeleted grade IIAnaplastic oligodendroglioma IDHmut and 1p19qcodeleted grade IIIPilocytic astrocytoma grade IPleomorphic xanthoastrocytoma grade IIAnaplastic pleomorphic xanthoastrocytoma grade IIIFrontalFrontalparietalFrontaltemporalParietalParietaloccipitalTemporalTemporalparietalTemporaloccipitalOccipitalCerebellar hemisphereCorpus callosumThalamusPituitary glandInsularMulticentric pts pts pts pts pts patients wt wildtype mut mutantCOSMIC https cance rsange racukcosmi c websites []In silico TERTpmut functional analysis JASPAR toolThis bioinformatic tool estimates the binding affinity and the number of TFs binding sites for the input sequence provided in FASTA format A relative threshold score of and Δ relative score ‰¥ mutant™s relative score”wildtype™s relative score were chosen to define the statistically significant changes induced by TERTpmut as previously reported [] The JASPAR CORE predicted the effects of the four different TERTpmut that we detected in our patients ie the two new TERTpdup the TERTp and the TERTp146 on TFs binding capacity JASPAR CORE Collection httpjaspa rgener egnet 8th version [ ] JASPAR was also used to analyze two TERTpdup which have been previously reported in a case of MDS c1110_1101dup and in a case of thyroid cancer c1104_183dup [ ] According to JASPAR data we used the Venn diagram to plot TFs for which a significant enhanced probability of binding capacity or an 0cPierini a0et a0al acta neuropathol commun Page of Fig Histological and immunohistochemical analysis in patient UPN171 a HematoxylinEosin staining original magnification 200X enlarged neoplastic cells with multiple often bizarre hyperchromatic nuclei and high number of mitoses Vascular proliferation as seen in these œglomeruloids lower half of the image is a specific pattern of microvascular growth b HematoxylinEosin staining original magnification 400X multiple mitotic figures are evident in the middle field œGeographic pattern of necrosis detail in insert panel b c Positive GFAP staining highlights high neoplastic cells with astrocytic differentiation d Intense and diffuse nuclear TP53 stainingincrease of the number of binding sites was predicted httpbioin forma ticspsbugent bewebto olsVennIn vitro TERTpmut functional study luciferase assayTo study the effect of TERTpmut on the expression of TERT a luciferase assay was done for the TERTpdup detected in case UPN171 the TERTp146 UPN205 and the TERTp124 UPN216 The TERTdup of case UPN131 could not be studied due to lack of material A TERTp wildtype TERTpwt construct already available in the laboratory was also used as reference Additional file a0 Table a0S2 [] TERT core promoter a0bp was amplified with specific primers reported in Table a0 S3 Additional file a0 Table a0 S3 introducing cleavage sites for BglII forward and HindIII reverse restriction enzymes Then TERTpmut constructs were inserted in pGEMT easy plasmid Promega Madison WI USA and cloned in Electromax DH10BT1 cells Invitrogen Milan Italy to increase the amount of mutant DNA Finally the inserts were subcloned in pGL410[luc2] vectors Promega Madison WI USA upstream of LUC2 gene encoding for luciferase enzyme of Photinus Pyralis and resequenced An empty pGL410[luc2] vector was also used as negative control Luciferase assay was performed using the GBM U87MG cell line maintained in Dulbecco™s Modified Eagle Medium Thermo Fisher Scientific Monza Italy with fetal bovine serum and streptomycinpenicillin at a0°C5 CO2 U87MG cells were seeded in a 6multiwell plate — cellsml cotrasfected with a0 µg of modified pGL410[luc2] plasmids and with of pGL474[hRlucTK] a vector containing the luciferase gene of Renilla Reniformis by Viafect Transfection Reagent Promega Madison WI USA After 24h incubation cells were lysed and fluorescence emission was assessed using DualGlo Luciferase assay kit Promega following manufacturer™s instructions All experiments were performed in triplicate in three independent experiments 0cPierini a0et a0al acta neuropathol commun Page of ResultsNew somatic TERT promoter variantsTERTpmut were detected in cases including ODG DAAA and GBM Additional file a0 Table a0 S4 In GBM and DAAA TERTpmut were prevalent in IDHwt cases GBM IDHwt vs GBM IDHmut DAAA IDHwt vs DAAA IDHmut Chi square P Additional file a0 Table a0S5 Thus in agreement with the diagnostic criteria recommended by the cIMPACTNOW Update the DAAA IDHwt with TERTpmut were referred to as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV []In GBM TERTpmut there was a significant enrichment of cases harbouring EGFR amplification vs Chi square P andor monosomy 10PTEN deletions vs Chi square P Likewise EGFR amplification or gain of whole chromosome in combination with monosomy occurred in of TERTpmut DAAA IDHwtThe most common variant TERTp124 was detected in cases while the TERTp146 was found in cases TERTpmut were mutually exclusive heterozygous and equally distributed among the different histological subtypes Additional file a0 Table a0S5 Besides the TERTp124 and TERTp146 we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication occurring in a genomic region starting at and a0bp from the ATG starting site ie c1100_179dup TERTp10079 in case UPN131 and c1110_189dup TERTp11089 in case UPN171 Fig a02a b wwwncbinlmnihgovgene wwwensem blHomo_sapie ns cancersangeracukcosmic [ ] They shared a region of duplication of nucleotides from “ to “ nucleotides from the ATG start site The absence of TERTp10079 in the PB DNA demonstrated the somatic origin of this variant in case UPN131In silico analysis predicts TERTpmut effectsIn silico analysis predicted that both TERTpdup created new binding sites ie for TERTp10079 and for TERTp11089 which were respectively recognized by and TFs Instead TERTp124 and TERTp146 were predicted to increase the binding affinity for and sites and to enhance the probability of binding for and TFs respectively Additional file a0 Table a0S6 Although all TERTpmut affected the binding sites for diverse families of TFs the ETS group emerged as one of the most frequently involved in TERTp10079 for TERTp11089 in TERTp124 and in TERTp146 Fig a0 2c Additional file a0 Table a0 S7 Other recurrently involved TFs in TERTpdup variants were the Specificity ProteinKr¼ppelLike Factor SpKLF family ie in TERTp10079 and in TERTp11089 and the More than adjacent zinc finger factors family in TERTp10079 and TERTp11089 Additional file a0 Table a0S7The Venn diagram showed a close interrelationship between all TERTp mutations Namely all TERTp mutations shared an increase of the binding affinity or the number of binding motifs for common TFs Fig a03a including ETS members ETS1 ETS2 ERG ELK1 ETV6 FLI1 ELK4 SPIB ELF1 ELF3 ETV4 ETV1 FEV EHF ETV5 ELF5 SPI1 and GABPA and TEAD1 Fig a03a Additional file a0 Table a0S8 The Venn diagram also showed that the new TERTpdup were characterized by the exclusive involvement of common TFs Specifically there were SpKLF members ie KLF2 KLF3 KLF4 KLF5 KLF10 KLF11 KLF14 KLF15 KLF16 SP1 SP2 SP3 SP4 SP8 SP9 and EGR1 Fig a03a Additional file a0 Table a0 S8 and TFs that belong to different families Fig a03a Additional files and Tables S7 and S8 Matching our TERTpdup with the two cases of TERTpdup previously reported Additional files and Tables S9 and S10 [ ] JASPAR predicted that all variants determined an increase of binding sites for common TFs and confirmed that the SpKLF family was the most frequently involved Fig a0 3b Additional file a0 Table a0S11In vitro analysis confirms the a0increasing of a0TERT transcriptional activity induced by a0its promoter mutationsIn vitro luciferase assay was carried out to evaluate whether the new TERTp11089 variant induced an increase of TERT transcriptional activity enhancing its expression similarly to TERTp124 and TERTp146 [ ] In Table a0S12 Additional file a0 Table a0S12 we reported raw data referred to the fluorescence emission values expressed in Relative Luciferase Activity RLA of both Photinus Pyralis and Renilla Reniformis luciferase enzymes for all samples Our experiments demonstrated that all three variants caused a significant increase of TERT transcription by fold than wildtype TERTp11089 vs TERTpwt P TERTp124 vs TERTpwt P TERTp146 vs TERTpwt P Mann“Whitney U test Fig a0 On the other hand no differences on the levels of TERT expression were present between the diverse TERTp variants indicating they may all behave as gainoffunction mutations likely exerting the same consequences on TERT transcription 0cPierini a0et a0al acta neuropathol commun Page of Fig Schematic representation of TERTp mutations a TERT promoter electropherogram in case UPN131 The arrow indicates the start point of the c1100_179dup b TERT promoter electropherogram in case UPN171 The arrow indicates the start point of the c1110_189dup c Overview of all TERTp variants detected in our cases Upper arrow wildtype TERT core promoter with the normal location of ETS binding sites The vertical black lines indicate the genomic positions of TERTp variants Lower arrow positions and types of TERTp variants and their predicted effects on transcription factors binding sitesDiscussionAbnormal genomic events that alter telomere elongation are common in gliomas Particularly mutually exclusive mutations affect the TERT or the ATRX chromatin remodeler ATRX genes a critical regulator of telomere homeostasis by chromatin remodeling []Our studies on a cohort of patients confirmed previous data on the incidence and distribution of TERTpmut in diverse subtypes of CNS tumors As expected we found that TERTpmut were highly recurrent in ODG and GBM and less frequent in DAAA Additional file a0 Table a0 S4 TERTpmut were significantly enriched in GBM IDHwt cases Chi square P Additional file a0 Table a0S5 where they mainly occurred together with EGFR amplification Chi square P andor monosomy 10PTEN deletions Chi square P Similarly in DAAA TERTpmut were highly recurrent in IDHwt cases thus allowing the reclassification of of these subgroup of astrocytomas as œdiffuse astrocytic glioma 0cPierini a0et a0al acta neuropathol commun Page of ODG [] Afterwards TERTpdup were found in a case of myelodysplastic syndrome MDS c1110_1101dup and in a case of papillary thyroid carcinoma c1104_183dup [ ] Published TERTpdup as well as our cases are located in the same core promoter region that span a0 bp from the ATG start site Furthermore they are all located downstream TERTp124 and TERTp ie at “ nucleotides from TERTp124 and nucleotides from TERTp146 in a region that contains the binding sites for the TFs modulating TERT transcription Interestingly in silico analysis predicted these new TERTdup affect the transcriptional regulation of the gene through the creation of new binding sites for TFs that mainly belong to the ETS family Fig a02c Additional file a0 Table a0S7 Likewise an increased number of binding sites or an enhanced affinity for the ETS TFs has been previously reported in a thyroid cancer harbouring a TERTp c1104_183dup variant and in cases bearing TERTp124 or TERTp146 mutations [ ] Bioinformatic analyses were consistent with the luciferase data showing a significant increase of TERT expression in cells transfected with the new TERTp11089 variant as well as with the two recurrent TERTpmutThen we sought to assess the possible interrelationship between the four diverse TERTp mutations using the Venn diagram Fig a03a All four TERTp variants were predicted to share an increase binding capacity for ETS members Fig a03a Additional file a0 Table a0S8 which included GABPA a putative oncogene in GBM Namely in a0vitro studies on GBM cell lines have demonstrated that this transcription factor is needful in mediating the transcriptional reactivation of TERT dependent from TERTp or TERTp146 [ ] Besides ETS TFs all TERTp variants affected the binding capacity for TEAD1 a protein that belongs to TEF1related factors family and that has been demonstrated to act as a putative oncogene in GBM favoring cell infiltration in a0 vitroin vivo models []Although TERTp124 and TERTp146 and the new TERTp10079 and TERTp11089 variants shared the same effects on the binding capacity for ETS members the latters were characterized by the exclusive involvement of TFs mainly belonging to SpKLF family Fig a0 3a Additional files and Tables S7 and S8 SpKLF TFs are involved in a plethora of cellular processes ranging from proliferation and differentiation pluripotency and apoptosis in normal and tumoral tissues []Fig The Venn diagrams show all possible relations among a four TERTp variants reported in our cases refer to Additional file Table S8 and b TERTpdup described in this study c1100_179dup and c1110_189dup and those reported in literature c1104_183dup and c1110_1101dup refer to Additional file Table S11IDHwildtype with molecular features of glioblastoma WHO grade IV []Besides the two known TERTp124 and TERTp146 variants we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication sharing a duplicated region of nucleotides from “ to “ from the ATG start site Hitherto somatic TERTpdup has been reported in three human tumors The first one a duplication of nucleotides in the TERT core promoter was detected in a case of Altogether these data support the hypothesis that the recruitment of ETS family TFs plays a pivotal role in mediating the reactivation of TERT transcription in human tumors bearing different types of TERTpmut However they also indicate that slight differences mark TERTpdup variants whose activities appear to be 0cPierini a0et a0al acta neuropathol commun Page of Fig Luciferase assay The histogram reports the relative luciferase activities RLA of TERTp wildtype and for the variants c1110_189dup c1124 C T and c1146 C T p value refers to probability obtained using Mann“Whitney U testalso dependent from Kr¼ppelrelated factors Indeed among the TFs shared by all TERTpdup Fig a03b belonged to SpKLF family as reported in Tables S10 and S11 Additional files and Hence the precise definition of mutationspecific profiles would strengthen the definition of TERTdependent oncogenesis mechanismsOur study contributes to enrich the spectrum of recurrent somatic TERTpdup variants reporting for the first time two new gainoffunction mutations ie TERTp10079 and TERTp11089 in of GBM IDHwt cases These new mutations can be reliably detected by diagnostic assays used to investigate hotspot TERTp and TERTp146 Although the assessment of TERTp mutational status is not an essential diagnostic criterion it can be a relevant information to assist histological diagnosis [] As a matter of fact the status of TERTp together with IDH mutations and 1p19q codeletion classify gliomas in distinct subcategories ie triple negative triple positive cases with IDHTERT mutations and cases with a unique mutation either IDH or TERT that are typified by unique demographic clinical and biological characteristics [] Moreover TERTpmut has been proposed as one of the most relevant molecular marker to stratify DAAA IDHwt [] Thus we consider that molecular testing of TERTp mutations should be included in the clinical workup of GBM and DAAA in order to provide a precise diagnosis prospective multicentric studies on large cohort of patients will clarify the value of TERTp mutations as prognostic markerSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 Table a0S1 Primer set used for Sanger sequencing Additional file a0 Table a0S2Samples used for in vitro luciferase assay Additional file a0 Table a0S3 Primer set used to create constructs for luciferase assay Additional file a0 Table a0S4 Incidence and distribution of TERTp variants in the main glioma subgroups Additional file a0 Table a0S5 Incidence and distribution of TERTp variants in glioma subtypes according to WHO guidelines Additional file a0 Table a0S6 JASPAR analysis for the TERTp c1124 CT c1 CT and the new TERTpdupc1100_179dup c1110_189dup Additional file a0 Table a0S7 Transcription Factors predicted to be involved in TERTp variants Additional file a0 Table a0S8 Transcription Factors predicted to be involved in different TERTp variants Additional file a0 Table a0S9 JASPAR analysis for the two published TERTp duplications c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S10 Transcription factors predicted to be involved in the TERTpdup c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S11 Transcription factors predicted to be involved in all TERTp duplications Additional file a0 Table a0S12 Luciferase assay raw dataAbbreviationsTERT Telomerase Reverse Transcriptase TERTp TERT promoter TERTpmut TERT promoter mutation TERTpdup TERT promoter duplication TERTp124 c1124 TERT promoter mutation TERTp146 c1146 TERT promoter mutation bp base pair ETS Etwentysix transcription factor CNS central nervous system GBM glioblastoma ODG oligodendroglioma DA diffuse astrocytoma AA anaplastic astrocytoma GBM IDHwt glioblastoma IDHwildtype DA IDHwt diffuse 0cPierini a0et a0al acta neuropathol commun Page of astrocytoma IDHwildtype AA IDHwt anaplastic astrocytoma IDHwildtype TFs transcription factors DA IDHmut diffuse astrocytoma IDHmutant AA IDHmut anaplastic astrocytoma IDHmutant GBM IDHmut glioblastoma IDHmutant FFPE formalinfixed paraffinembedded PB peripheral blood MDS myelodysplastic syndrome TERTpwt TERTp wildtype TERTp10079 c1100_179dup TERTp11089 c1110_189dup SpKLF Specificity ProteinKr¼ppelLike Factor RLA relative luciferase activity ATRX ATRX chromatin remodelerAcknowledgementsNot applicable Authors™ contributionsTP RLS conceived the study planned the experiments and wrote the paper TP carried out and evaluated mutational analysis and in vitro functional studies CN made in silico analysis AGLF contributed in the analysis of in vitro luciferase assay MM and SA performed DNA extraction and FISH experiments FP VN and PG performed sequencing analysis PG SA and MEL provided the diagnosis and the tissue sections for molecularcytogenetic studies CC and RC ML GM and CM provided all clinical data VP GR and CM were involved in drafting the manuscript All the authors read and approved the final manuscript FundingThe project was supported by Comitato per la vita œDaniele Chianelli Perugia Italy Sergio Luciani Association Fabriano Italy and Fondazione Cassa di Risparmio Perugia Italy Grant numbers to RLS Availability of data and materialsAll data generated or analyzed during this study are included in this published [and in its supplementary information files]Ethics approval and consent to participateThis study was approved by the local ethic committee CEAS code number August 8th Consent for publicationAll participants signed an institutional informed consentCompeting interestsThe authors declare that they have no competing interestsAuthor details Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia Italy Hematology and Center of Bone Marrow Transplants Medicine and Surgery Department University and Hospital of Parma Via Gramsci Parma Italy Diagnostic Cytology and Histology Unit S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Radiotherapy S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Medical Oncology S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Neurosurgery S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Pathology Unit S Maria Hospital V Tristano di Joannuccio Terni Italy Received June Accepted August References Allory Y Beukers W Sagrera A Fl¡ndez M Marqu©s M M¡rquez M et al Telomerase Reverse Transcriptase promoter mutations in bladder cancer high frequency across stages detection in urine and lack of association with outcome Eur Urol “ Barthel FP Wei W Tang M MartinezLedesma E Hu X Amin SB et al Systematic analysis of telomere length and somatic alterations in cancer types Nat Genet “ Bell RJ Rube HT Kreig A Mancini A Fouse SD Nagarajan RP et al The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer Science “ Brat DJ Aldape K Colman H Holland EC Louis DN Jenkins RB et al cIMPACTNOW update recommended diagnostic criteria for œDiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV Acta Neuropathol “ COSMIC Catalogue of Somatic Mutations in Cancer Database Wellcome Sanger Institute Cambridge UK https cance rsange racukcosmi c Accessed May EckelPassow JE Lachance DH Molinaro AM Walsh KM Decker PA Sicotte H et al Glioma groups based on 1p19q IDH and TERT promoter mutations in tumors N Engl J Med “Ensembl DatabaseHomo Sapiens European Molecular Biology Laboratory™s European Bioinformatics Institute Cambridge UK httpwwwensem blHomo_sapie ns Accessed May Fornes O CastroMondragon JA Khan A van der Lee R Zhang X Richmond PA et al JASPAR update of the access database of transcription factor binding profiles Nucleic Acids Res 48D87“D92 https doi101093

Thyroid_Cancer

Relevance Function of LincROR inthe Pathogenesis of CancerWenjian Chen1  Junfa Yang23  Hui Fang4  Lei Li5 and Jun Sun1 Anhui Provincial Children™s Hospital Affiliated to Anhui Medical University Hefei China Key Laboratoryof Anti‚ammatory and Immune Medicine Ministry of Education Institute of Clinical Pharmacology Anhui MedicalUniversity Hefei China School of Pharmacy Anhui Medical University Hefei China Department of PharmacologyThe Affiliated Hospital of Hangzhou Normal University Hangzhou China The Affiliated Hospital of Anhui Medical UniversityHefei ChinaLong noncoding RNAs lncRNAs are the key components of noncoding RNAsncRNAs with a length of nucleotides They are transcribed from the socalledœdark matter of the genome Increasing evidence have shown that lncRNAs play animportant role in the pathophysiology of human diseases particularly in the developmentand progression of tumors LincROR as a new intergenic nonprotein coding RNAhas been considered to be a pivotal regulatory factor that affects the occurrence anddevelopment of human tumors including breast cancer BC colorectal cancer CRCpancreatic cancer PC hepatocellular carcinoma HCC and so on Dysregulation ofLincROR has been closely related to advanced clinicopathological factors predicting apoor prognosis Because lincROR can regulate cell proliferation apoptosis migrationand invasion it can thus be used as a potential biomarker for patients with tumorsand has potential clinical significance as a therapeutic target This reviewed therole of lincROR in the development of tumors its related molecular mechanisms andclinical valuesKeywords lncRNAs ncRNAs lincROR cancers biomarkerEdited bySridhar MuthusamiKarpagam Academy of HigherEducation IndiaReviewed byAnca Maria CimpeanVictor Babes University of Medicineand Pharmacy RomaniaOmar TorresQuesadaUniversity of Innsbruck AustriaCorrespondenceJun Sunsunjun500aliyuncomsunjun14190163com These authors have contributedequally to this workINTRODUCTIONSpecialty sectionThis was submitted toMolecular and Cellular Oncologya section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted July Published August CitationChen W Yang J Fang H Li L andSun J Relevance Functionof LincROR in the Pathogenesisof CancerFront Cell Dev Biol 103389fcell202000696Cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide According to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries Kumar and Sharawat Noorolyai Owing to a shortage in eï¬ective screening methods and lack of identificationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer Bray Koo Additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer Yan Therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer Currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy Nie Howeverin the past years the survival rate of patients with cancers remains dismal Nakashima Abbreviations BC breast cancer ceNAs competing endogenous RNA EMT epithelial“mesenchymal transition HCChepatocellular cancer iPSCs induced pluripotent stem cells lincROR Long intergenic nonprotein coding RNA regulatorof reprogramming lncRNAs Long noncoding RNAs PC pancreatic cancerFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORin the process of developing human antitumorThereforestrategiesto find new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantOver the past decades ncRNAs constitute more than of the RNAs made from the human genome but mostof the known noncoding RNAs ncRNAs havebeen discovered and remain largely unstudied Bhan Slack and Chinnaiyan Transfer RNA tRNA and ribosomal RNA rRNA constitute themajority of ncRNAsfollowed in abundance by messengerRNAs mRNAs Thus the remaining ncRNAs includingcircular RNA circRNA small nuclear RNA snRNA smallnucleolar RNA snoRNA microRNA miRNA and long nonfor ˆ¼ ofcoding RNA lncRNA together accounttotalncRNA Despite their low abundancethese ncRNAs havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modification andregulation of the cell cycle Huarte Kondo Peng 2017b Additionally because ncRNAs can be packagedinto extracellular vesicles EV including exosomes Meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of miRNAand lncRNA to recipient cells both locally and systemicallySun It is important to note that the expressionof ncRNAs their posttranscriptional modification particularlylncRNAs and their subcellular distribution have been shownto be important to when assigning their potential functionPalazzo and Lee Recently nextgeneration sequencingand bioinformatics technology have revealed that circRNAsplay crucial role in diagnosis and prognosis of various diseasesPamudurti Briefly circRNAs are singlestrandedtranscripts generated by backsplicing Jeck and Sharpless with covalently linked headtotail closed loop structures withneither 5cid48“3cid48 polarity nor a polyadenylated tail Memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear RNAs ChenJ and exhibiting a celltype or developmentalstagespecific expression pattern Barrett and Salzman Wang J J Many functions of circRNAs have alsobeen identified including their role as miRNA sponges bindingto RNAbinding proteins and protein decoys and functioningas regulators of transcription Hansen Du Yang Y Interestingly many circRNAs havebeen shown to be dysregulated in pathophysiological processesand circRNAs are known to regulate the expression of geneby acting as miRNA sponges in a mechanism that is termedas competitive endogenous RNA ceRNA mechanism Zheng Wang J J For example circMTO1have been demonstrated to harbor conventional miRNA bindingsites and has been identified as an inhibitor of miRNA9 inhepatocellular carcinoma HCC Han Additionallyour previous study has demonstrated that miRNA plays arole in limiting the development of liver fibrosis by markedlyblocking the activation and proliferation of hepatic stellatecells HSCs suggesting that miRNAs might be involved inthe development and progression of several forms of cancersYang J Yang Of notelncRNAswhich are mainly transcribed by RNA polymerase II are anew kind of ncRNA that are longer than nucleotidesMa Owing to the lack of reading frameslncRNAs have extremely limited or no protein coding capacityRuan Li J These new regulatorswere initially regarded as transcriptional noise with no specificbiologicalfunctions Kim and Sung Recently ourlaboratory found that epigenetic silencing of lncRNA ANRILpromoted the progression of liver fibrosis thereby indicating thatlncRNAs were associated with the progression of cancers Yang Interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncRNAsGuttman Additionally there has been new evidencesuggesting that lncRNAs may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modifications Davalos and Esteller Pang More importantlylncRNAs have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis Chen Q N Lu Subsequent studies have demonstrated thatlncRNAs may serve as ceRNAs for miRNAs and in chromatinremodeling during the development of cancers Huang Wang C J Figure illustrates the functionsof lncRNAs at the molecular level Regarding certain cancerassociated human lncRNAsit was demonstrated that lincROR was demonstrated to be predominantly upregulated intumors Peng 2017a The abnormal expression of lincROR in tumors has been suggested to be one of the mainleading factors driving the development The main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition EMT enhancement of the sensitivityto chemotherapy etc Chen 2016a Zhao Forexample the expression level of lincROR in HCC tissues wasinhibited compared with the adjacent tissues At the same timethe downregulation of lincROR was linked to the aggressiveprocess of the disease in patients with HCC Furthermore theability of migration and invasion of HCC cells may be delayedby the low expression level of lincROR In this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincROR in human tumors and discuss its prognostic andtherapeutic valuesOVERVIEW OF LincRORlincROR isAmong lncRNAsand importantcarcinogenic kb lncRNA located in chromosome which was initially identified as a highly expressed transcriptof pluripotent and embryonic stem cells Chen 2016bStudies found that the octamerbinding transcription factor a novelFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE Paradigms for the function of long ncRNAs Recent studies have identified a variety of regulatory paradigms for the mechanism by which long ncRNAsfunction many of which are highlighted here Transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of RNA polymerase II or inducing chromatin remodeling respectively An antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript Alternatively hybridization of the sense and antisense transcripts can allow Dicer to generate endogenous siRNAs By binding to specificprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerRNAprotein complex or alter where the protein localizes in the cell Long ncRNAs green can be processed to yield small RNAs such as miRNAspiRNAs and other less wellcharacterized classes of small transcriptsOCT4 SRYbox transcription factor SOX2 and Nanoghomeobox Nanog key pluripotency factors could regulatelincROR Wang HoweverlincROR was alsofound to be expressed in several ans including lungliver breast and colon Since its discovery research in thisfield has been extensively expanded during the past yearsrevealing the important role of lincROR in tumorigenesislincROR has been suggestedAdditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes Wang Li inMany reportsrecent years have revealed thatlincROR is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis TNM positivelymph node metastasis LNM and lower survival rate buthigher recurrence rateregarding lincROR and tumorigenesisthe upregulation ofCurrent evidence have strongly indicated thatlincRORmay exert an impact on a variety of cancers Pan Furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincROR via activation of theEMT in various cancers Hou Huang Zhan For example lincROR was demonstratedto be upregulated thereby promoting EMT in HCC Li J Besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was significantly aï¬ectedby lincROR Zhang Feng Moreimportantly the chemoresistance of pancreatic cancer PCand breast cancer BC Li as well as radioresistance of colorectal cancer CRC cells were observed to beelevated by lincROR Yang P Moreover lincROR has also been shown to exert a significantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofPC Recentlyit was also reported that lincROR could beused as a biomarker in the field of diagnosis and prognosisof BC and oral cancer Arunkumar Zhao Notably increasing studies showed that lincROR couldbe used as a ceRNA thus exerting its impact in the posttranscriptional network of tumor pathogenesis For examplein triplenegative BC lincROR has been shown to serve asa ceRNA therefore promoting the migration and invasion ofBC cells Signal Overall lincROR is a typicallncRNA that plays important regulatory roles in interactionwith miRNAs and maintenance of stem cell pluripotencytriggering the EMT as well Furthermore lincROR has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis Figure Therefore lincRORmay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer The transition of lincROR from basicresearch to clinical application requires further investigations asearly as possibleFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE LincROR is a typical lncRNA that plays important regulatory roles in interacting with miRNAs and maintaining stem cell pluripotency as well astriggering the EMT as well LincROR is also involved in various key roles under various stresses and in epigenetic regulationREGULATORY ROLE OF LincROR INVARIOUS TYPES OF CANCERIncreasing evidence has shown that the lincROR was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions Fu Spinelli Additionally studies found that the expression level of lincROR was substantially upregulated in samples of papillarythyroid carcinomas PTCs and PTCs cell lines as well as inmetastatic PTCs samples and PTCs cell lines Zhang Simultaneously cell migration and invasion could be regulatedby lincROR via aï¬ecting EMT Pastushenko and Blanpain More importantly studies demonstrated that lincRORwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors Hashemian Li 2020bcThis review summarizes the status of lincROR research invarious human cancers and discusses its mechanism and clinicalsignificance in the development and progression of tumor Theexpression pattern functional role and regulatory mechanism oflincROR are summarized in Table and depicted in Figure Breast CancerBC which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide Li Z In it was estimated that there would be million or so newly diagnosed cases of female BC Bray Hannafon The main risk factors forBC which is the difficult to change due to prolonged exposureto endogenous hormones is difficult to control Rudel Bray However comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with BC Rudel Goel Kumler Nevertheless it has been reported that aboutonethird of the patients with BC have the potential for cellmetastasis chemotherapy resistance and even recurrence Goel Kumler Hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of BCthe level ofthe level ofHou et althe expression ofinvestigated the expression level of lincRORin patients Hou Their results revealedlincROR was increased in BC tissuesthatMoreoverlincROR in theperipheral blood ofthe patients with BC was shown tobe closely related to TNM phase and LNM In additionthe woundhealing assay showed that overexpression of lincROR increased BC cells MCF10A mobility Transwell assayrevealed that lincROR overexpression remarkably increased themigration ability Hou More importantly theyfound that ectopic expression of lincROR induced an EMTprogram in MCF10A cells Fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype CD44highCD24low was elevated in MCF10A cellstransfected with lincROR plasmid Mechanistically the resultsof bioinformatic analysis and RNA immunoprecipitation analysisfrom Hou demonstrated thatlincROR functions asa ceRNA to regulate miR205 activity toward prevention ofthe degradation of transcripts of miR205 target genes suchas ZEB1 and ZEB2 from degradation Additionallyit wasshown that the expression levels of miR205 members weredecreased upon lincROR overexpression in MCF10A cellsHou More importantly Zhao recentlydemonstrated that CRISPRCas9generated BRCA1knockdownadiposederived stem cells stimulated a more aggressive behaviorFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORlateiLlateoaGlatenahZlatenehClateCiLlateihZlateiLnuSdnanaYlatenaPalategnahZlateuoHsecnerefeRBEZRmRORcniilMKPPBTPRmRORcniilgonaNRmRORcniilnirehdacEHZERORcnilSOSFDMNLegatsMNTTMEinosavniTMEinosavninoitargminoitarefiloPicnegocnOldetaugerpUamoncraciraulllecotapeHllseuceomyrotaugeRllnoitaerroclacniilCleorlanoitcnuFleoRinosserpxEsepytrecnaCsrecnacnamuhniRORcnLiELBATiRmRORcnilevrucCORMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnactsaerBTMEinosavnipBEZRORcnilliavvrusroopMNLegatsMNTnoitargminoitarefilorphtwicnegocnOldetaugerpUrecnaccitaercnaPMXOFpRmRORcniilNCSFRmRORcnipRmRORcniililiRmRORcnilecnatsserigurdTMEinosavniTMElecycllecinosavniliavvrusroopMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnacdoryhTiSOSFDMNLegatsMNTinoitargmecyclllecissotpopAicnegocnOldetaugerpUrecnacgnuLecnatsseriyparehtodariTMEinosavniSOSFDMNLegatsMNTnoitargmiissotpopanoitarefilorPicnegocnOldetaugerpUrecnaclatcerooClin BC cells than wildtype adiposederived stem cells Zhao Therefore we believe that CRISPRCas9 may beused to in the treatment of BC by inhibiting the expressionof lincROR during the progression of BC Conclusively thelincRORmiRNAs axis has been reported to closely aï¬ect theoccurrence and development of BCPancreatic CancerPC is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallySiegel Sabater The ˆ’year relativesurvival of patients with PC remained at approximately for“ Siegel Hence PC has been proposedto be one of the top two cancers in terms of fatalities in thenext decade Rahib Surgical resection remainsthe exclusive potential curative treatment Xiong However approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment Vincent Xiong A growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the efficient medical therapyof PC Vincent Boj Basuroy Thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in PCZhan have highlighted the oncogenic eï¬ectsof lincROR in the initiation and progression of PC Theirstudy demonstrated that the level of lincROR was significantlyelevated in PC tissues Zhan Moreoverthewoundhealing assay and Boyden™s chamber assay results showedthat lincROR silencing reduced the migratory capability andmetastasis of PC cells Zhan Another study byChen showed that the proliferation rates of shRORcellsin which the level of lincROR was suppressed were evidentlylower than those of shNCcells This result was confirmed bycolony formation assay suggesting that lincROR accelerated thegrowth of PC cells Chen 2016a Interestingly silencingof lincROR was shown to result in increased levels of theepithelial markers Ecadherin and αcatenin and decreased levelsof mesenchymal markers Ncadherin and vimentin indicatingthat lincROR plays an important role in the regulation ofEMT in PC cells Zhan Chen Moreimportantly microarray analysis identified ZEB1 as potentialtarget gene of lincROR Further the expression of lincRORand ZEB1 were observed to be negatively correlated with thatof p53 suggesting that lincROR might mediate migration andmetastasis in PC cells may partly via activation of ZEB1 throughthe inhibition of the expression of p53 Zhan Interestingly the fluorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincRORwas demonstrated to be negatively correlated to that of miR145MiR145 can induce posttranscriptional silencing of its targetedgenes by binding to the mRNA ™UTR or lincROR specificsites indicating that lincROR can act as a ceRNA to decreasemiR145 in PC cells thereby activating expression of Nanogthus leading to the proliferation of pancreatic cancer stem cellsPCSCs Gao Additionally Li furtherFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORFIGURE Underlying molecular mechanisms of lincROR in multiple cancers A LincROR binds to miR205 to upregulate ZEB2 while it also regulated theexpression of EMT markers B LincROR could interact with miR145 to inhibit FSCN1 and upregulated EMTassociated proteins while it decreases G0G1 arrestand facilitated drug resistance C LincROR facilitated EMT through upregulate EZHZ and regulated ZEB2 by competitively binding to miR145 and ZEB2overexpression leads to increased EMT In addition lincROR binds to miR8765p to upregulate FOXM1 D LincROR downregulated through EMT production andrepress the expression of miR145 E LincROR binds to miR68333p while also regulating the process of EMT F LincROR upregulated ZEB1 to attenuate theexpression of p53 while also decreasing the expression of miR145 to increase the level of Nanog and reduce that of miR124 to suppress PKM2 Detailedmechanisms of lincROR in other cancers are provided in the reviewproved that the impact of lincROR can be partly reversed byoverexpression of miR124 Consistently lincROR was observedto exhibited a negative correlation with the expression of miR Li Hence a lincRORmiR124PTBP1PKM2axis was identified in PC shedding new light on the lncRNAbased diagnosis and therapeutic approaches in PC Li 2020b Notably recent studies showed that PC cellderivedEVs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity Saari Additionally it was demonstrated that vesiclecontainingncRNAs could serve as EVassociated PC detection markersWorst Thus the presence of lincRORs in EVs frompatients with PC could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with PC this isworthy of further and wider research attentionHepatocellular CarcinomaAs the sixth most international commonly occurring cancer in HCC has become the fourth cause of cancerassociateddeaths worldwide It has been estimated that new casesand deaths will occur each year Bray BrieflyHCC has been reported to account for “ of all the livercancer cases half of which have been detected in China Omata Bray As such HCC poses a huge threatto the worldwide health especially that of the Chinese peopleOmata About of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor Vigano Thereforeon the basis of studying the pathogenesis of HCC it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of HCCLi C and Chen reported that theexpression level of lincROR was obviously elevated in HCCtissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincROR might be critical regulator in the progression ofHCC Furthermore biological function assay demonstrated thatlincROR could play promoting role in regulating migration andinvasion of HCC Chen Moreover downregulationof lincROR could result in a significant increase in G1G0phase and an obvious decrease in S phase Li C More importantly silencing of lincROR could lead to theincreased expression of Eˆ’cadherin and decreased expressionlevel of Nˆ’cadherin in HCC cell lines Li C furtherconfirmed that lincROR could bind to the zeste homolog EZH2 thereby aï¬ecting the expression of Ecadherin furtherindicating that lincROR could regulate the progression of EMTMoreover lincROR was further determined to be associatedwith DNA repair Currently mounting studies have identifiedreliable indicators of DNA damage such as phosphorylatedhistone H2AX γH2AX Chen uncover thatFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORlincROR could obviously decrease theoverexpression ofexpression level of γH2AX illuminating the suppressive eï¬ectsof the overexpression of lincROR on DNA repair in HCCFurther research demonstrated that lincROR could interactwith miRˆ’ and dramatically downregulate the expression ofmiRˆ’ in HCC cells Li C The abovementionedresults revealed that lincROR might play a promoting role inthe proliferation migration invasion and EMT of the HCC cellwhich was contrary to the ‚uence of miRˆ’ enrichmentLi C It suggested that overexpressed miRˆ’could eï¬ectively reverse the promotion of HCC tumorigenesisinduced by the overexpression of lincROR Li C Liand his colleagues proposed a mechanistic model that lincRORpromotes HCC tumorigenesis and autophagy partly throughnegatively regulating the expression of miRˆ’ Aside fromthat it has been reported that miR145 represses EMT tumormigration and invasion by directly targeting the ™UTRs ofZEB2 in the tumor The decrease in miRˆ’ and increase inZEB2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincROR knockdown Thereforeit was suggested that targeting the lincRORmiRˆ’145ZEB2axis might represent a novel therapeutic application in HCCLi C Similarly Zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincROR Moreover they further confirmedthat FOXM1mediated activation of lincROR contributes tothe poor sensitivity of HCC cells to sorafenib via partiallyregulating the miR8765pFOXM1 axis which forms a positivefeedback loop Further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of HCCZhi Consequently these studies may oï¬er newinsights regarding the pathology of HCC and provide potentialstrategies for lncRNAdirected treatment However both thein vivo ‚uence and other underlying mechanisms of lincRORstill remain to be determined and clarified in the future researchthe prognosisColorectal CancerThere are approximately million new CRC cases and CRCrelated deaths worldwide each year thus making CRC thethird most common cancer in the world Torre Although the treatment of CRC has significantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesBogousslavsky Torre Current studiesresults showed that approximately of cases with CRChave synchronous liver metastases during the time of diagnosisKawaguchi These patients have inherently lowsurvival rates of less than within years with an even worseprognosis Hu Kawaguchi Thus there isan urgent need to better understand the progression of CRC andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with CRCYang detected the expression of lincROR in CRCtissues compared to normal tissues by using qRTPCR Theyfound that the expression of lincROR was remarkably increasedin CRC tissues compared with normaltissues SimilarlylincROR was shown to be overexpressed in five CRC cell linesYan and Sun Li 2020a Then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincROR on proliferation viabilityapoptosis migration and invasion of CRC cells Knockdownof lincROR was shown to eï¬ectively inhibit the proliferationof CRC cells whereas its overexpression obviously increasedthe proliferative capacity of CRC cells Accordingly silencing oflincROR strongly inhibited the migratory and invasive abilitiesof CRC cells compared with that in the control cells Li et al2020a In contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincROR The MTS345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2Htetrazolium assay results showed thatthelincROR could enhance the viability ofoverexpression ofCRC cells Furthermore the flow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincRORoverexpression group was reduced by ± indicatingthat the overexpression of lincROR could inhibit apoptosisin the CRC cell lines Li 2020a More importantly arecent study revealed the role of lincROR in the EMT It wasrevealed that the upregulation of lincROR could increase theexpression of Ncadherin and Vimentin as well as decrease thelevel of Ecadherin leading to the promotion of the progressionof EMT Zhou Yan and Sun Meanwhilethe high expression of lincROR in CRC was also confirmedby Hu Mechanistically Li 2020a provedthat that lincROR could bind to miR68333p which wasdetermined to be significantly downregulated in CRC tissuesAdditionally a negative correlation was exhibited between theexpression of lincROR and miR68333p in BC tissues AntiAGO2 RNA immunoprecipitation assay further confirmed theseresults Li 2020a Besides rescue assays demonstratedthat downregulation of miR68333p could partly reversed theinhibition of tumorigenesis induced by lincROR knockdown inBC cells Li and his colleagues uncovered that lincROR exertedits oncogenic role through negatively regulating the expressionof miR68333p during the progression of CRC which mightgive new insights into molecular diagnosis and treatment Li 2020a In addition Li 2020a further uncoveredthat lincROR could mediate the expression level of SMC bysponging miR68333p in CRC cells thus promoting CRCprogression As for the eï¬ects of lincROR on radiotherapyresistance Yan and Sun showed that overexpression oflincROR increased the ability of CRC cells for radiotherapyresistance Collectively these findings indicated that lincRORmight be engaged in the metastatic process of CRC cells andcould promote the development of CRC through a variety ofmolecular mechanismsLung CancerLung cancer is the leading cause of cancerrelated deathsworldwide Bray Nonsmall celllung cancerNSCLC accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma Herbertz Bray Brainard and Farver Although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival OS rate for patients with advanced lung cancer is lesslarge cellFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChen et alRelevance Function of LincRORthan Zhou Therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and efficient tumor markers is very urgentIn recent yearslincROR has appeared as an importantregulator oflung cancer Research from Qu demonstrated that the expression of lincROR was increasedin NSCLC tissues compared to that in the normal tissuesThe overexpression of lincROR was shown to be closely relatedto the poor prognosis of LNM histological grade and stageof TNM Pan Qu Another s

Thyroid_Cancer

predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

Thyroid_Cancer

"Severe COVID19 has a high mortality rate Comprehensive pathological descriptions of COVID19 are scarce and limited in scope We aimed to describe the histopathological findings and viral tropism in patients who died of severe COVID19Methods In this case series patients were considered eligible if they were older than years with premortem diagnosis of severe acute respiratory syndrome coronavirus infection and COVID19 listed clinically as the direct cause of death Between March and April full postmortem examinations were done on nine patients with confirmed COVID19 including sampling of all major ans A limited autopsy was done on one additional patient Histochemical and immunohistochemical analyses were done and histopathological findings were reported by subspecialist pathologists Viral quantitative RTPCR analysis was done on tissue samples from a subset of patientsFindings The median age at death of our cohort of ten patients was years IQR “ Thrombotic features were observed in at least one major an in all full autopsies predominantly in the lung eight [] of nine patients heart five [] and kidney four [] Diffuse alveolar damage was the most consistent lung finding all ten patients however anisation was noted in patients with a longer clinical course We documented lymphocyte depletion particularly CD8positive T cells in haematological ans and haemophagocytosis Evidence of acute tubular injury was noted in all nine patients examined Major unexpected findings were acute pancreatitis two [] of nine patients adrenal microinfarction three [] pericarditis two [] disseminated mucormycosis one [] of ten patients aortic dissection one [] of nine patients and marantic endocarditis one [] Viral genomes were detected outside of the respiratory tract in four of five patients The presence of subgenomic viral RNA transcripts provided evidence of active viral replication outside the respiratory tract in three of five patientsInterpretation Our series supports clinical data showing that the four dominant interrelated pathological processes in severe COVID19 are diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Additionally we report here several novel autopsy findings including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activation which require additional investigation to understand their role in COVID19Funding Imperial Biomedical Research Centre Wellcome Trust Biotechnology and Biological Sciences Research CouncilCopyright The Authors Published by Elsevier Ltd This is an Access under the CC BY licenseIntroductionIn the UK the death toll from severe COVID19 is among the highest worldwide1 Severe COVID19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 Pathological correlates are required to understand the pathophysiology of COVID19 Autopsybased histopathological analysis is crucial in this respect In anticipation of the COVID19 pandemic our group produced national guidelines for autopsy performance in suspected COVID19 cases3COVID19 is caused by infection with severe acute respiratory syndrome coronavirus SARSCoV245 Although SARSCoV2 and its predecessor SARSCoV causing severe acute respiratory syndrome [SARS] are toll similar on a molecular and clinical level COVID19 has a lower death rate for COVID19 vs for SARS and a substantially higher death deaths worldwide from COVID19 as of Aug vs from SARS than SARS due to a higher basic reproduction number1 The postmortem findings in patients with SARSCoV infection included diffuse alveolar damage DAD splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular SARSCoV detected in the lungs kidney brain and haematological ans6 Various autopsy series on COVID19 have begun to emerge in the literature7“ Here we document the major pathological Lancet Microbe Published Online August 101016 S2666524720301154Department of Cellular Pathology Northwest London Pathology B Hanley MBBCh Prof K N Naresh MD C Roufosse PhD J Weir FRCPath Prof R Goldin MD P Viola MD M Osborn FRCPath and Department of Hepatology P Manousou PhD Imperial College London NHS Trust London UK Centre for Haematology B Hanley Prof K N Naresh and Centre for Inflammatory Diseases C Roufosse Department of Immunology and Inflammation Department of Infectious Disease Prof G S Cooke FRCP A Abdolrasouli PhD O C Swann MRes L Baillon BSc R Penn MSc Prof W S Barclay PhD and Department of Metabolism Prof M Thursz MD Faculty of Medicine Imperial College London London UK Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute Imperial College London London UK Prof A G Nicholson DM Renal and Transplant Centre Hammersmith Hospital Imperial College Healthcare NHS London UK R Corbett PhD Department of Neuropathology Kings College Hospital London UK Prof S AlSarraj FRCPath Death Investigation Committee Royal College of Pathologists London UK M Osborn and Nightingale NHS Hospital London UK M Osborn Correspondence to Dr Brian Hanley Department of Cellular Pathology Northwest London Pathology Charing Cross Hospital campus London W6 8NA UK bhanleyimperialacukwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cResearch in contextEvidence before this studyCOVID19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce We reviewed the literature available on COVID19 autopsy findings up to and including May For this we searched PubMed and Google Scholar databases with no language restrictions using the search terms œCOVID19 œSARSCoV2 œhistology œautopsy and œpostmortemAdded value of this studyOur series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal COVID19 and correlating these findings with data on viral tropism The most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion Several novel autopsy findings in patients with COVID19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationImplications of all the available evidenceOur study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in COVID19 Other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation The presence of ongoing viral replication in late stage COVID19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantSee Online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed COVID19MethodsPatient selectionFor this study eligible patients were older than years with premortem SARSCoV2 infection and COVID19 listed clinically as the direct cause of death under part on the Medical Certificate of Cause of Death [MCCD] Consent was obtained for all included patients according to the Human Tissue Authority codes of practice by a member of the Trust Core PostMortem Consent Team Consent rate was · ten of patients Exclusion criteria included extended postmortem interval before autopsy days and patients with COVID19 contributing but not directly leading to death under part of the MCCD Patients were from Imperial College National Health Service NHS Trust nine patients London UK and Royal Brompton Harefield Foundation NHS Trust one patient London UK Premortem SARSCoV2 infection was identified using the Coronavirus Typing multiplextandem PCR HighPlex System Aus Diagnostics Chesham UK Ethical approval for this project was provided by the Imperial College Healthcare Tissue Bank R20012Autopsy proceduresFull autopsies were done on nine patients PM1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance PM10 Full postmortem examinations included standard sampling and were done according to Royal College of Pathologists guidelines3 Eight different regions of the brain were sampled for each full neuropathological examination All tissue samples were fixed in formalin for a minimum of h before embedding Histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung AGN and PV haem ato pathology and immune pathology KNN liver RG gastrointestinal MO neuropathology SAS and renal pathology CR Integrated interpretation was done by a subspecialty autopsy pathologist BH and MO All cases were reviewed independently by at least two pathologistsPCR proceduresFresh tissue for quantitative RTPCR qRTPCR analysis was processed within the biosafety level facilities at St Mary™s Hospital London UK approved by the UK Health and Safety Executive and in accordance with local rules at Imperial College London Total RNA was obtained from fresh tissue samples by use of TRIzolchloroform extraction followed by precipitation and purification using the RNeasy kit Qiagen Hilden Germany qRTPCR against E gene RdRp and subgenomic RNA was done as described elsewhere1617 In patient PM5 total fungal genomic DNA was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block Purified DNA was amplified with PCR for panfungal and Mucoralesspecific targetsStatistical analysisAll data was analysed using SPSS software version and expressed using median IQR and percentageRole of the funding sourceThe funder of the study had no role in study design data collection data analysis data interpretation or writing of the The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationResultsBetween March and April ten patients were included in the study The median age at death was years wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cIQR “ Seven of ten patients were men three were women and most patients were White or Asian nine [] Hypertension four [] patients and chronic obstructive pulmonary disease three [] were the most common contributing factors to death according to MCCD All ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation Of eight patients assessed for inflammatory markers all had elevated inflammatory markers These features were either apparent upon presentation to hospital eight [] of ten patients or developed in an inpatient two [] patients PM8 and PM9 Most patients died within weeks of symptom onset seven [] patients and were not intubated or ventilated six [] patients Four patients were intubated during their presentation PM2 for days PM5 for days PM6 for less than day and PM7 for days The median bodymass index BMI was in the obese range · IQR ·“· and more patients were obese according to BMI at post mortem five [] of nine than indicated on the MCCD one [] of ten The median interval between death and postmortem examination was days IQR ·“· although the limited post mortem had a shorter interval less than h after death Detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p All patients had DAD six showed purely exudative phase DAD and four showed a mixture of exudative and anising DAD appendix p figure Three of four patients with anisingphase DAD had spent a substantial period on a ventilator days days and days Florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three [] of ten patients and patchy acute bronchopneumonia three [] patients were observed Interstitial macrophages were prominent Macrophages were accompanied by scattered plasma cells Mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients We noted that lymphocytes in the lung were predominantly CD4positive T cells CD56positive natural killer cells were rarely found Occasionally a patient had small aggregates of small B cells Chronic bronchiolitis was seen in most patients nine [] of ten No granulomas or viral inclusion were seen Invasive mucormycosis was noted in one patient PM5 figure and confirmed with Mucoralesspecific PCR The mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientMacroscopic two [] of nine patients and microscopic eight [] of nine pulmonary thromboemboli were frequent observations appendix p figure Both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure External examination findings of deep venous thrombosis were not noted Very focal lymphocytic vasculitis was identified in one patientThrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an One of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis Coronary artery disease was negligible or mild in most patients seven [] of nine Acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2A PM1 A mottled myocardium and subendocardial contraction band necrosis was noted in a ACEBDF 03m µm µmFigure Pulmonary pathological findings in patients with COVID19A Macroscopic subpleural petechial haemorrhage in a 24yearold man PM6 B Hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman PM9 at 20x magnification C CD61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries PM6 D Squamous metaplasia in a 61yearold man PM1 with exudative phase diffuse alveolar damage at × magnification E Interstitial multinucleated giant cells in a 79yearold man PM7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive CD68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins F Periodic acid Schiff staining indicating wide irregular aseptate and ribbonlike hyphae with angle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man PM5 the insert is a Grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0csecond patient PM2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p PM1 and PM2 were the two patients with the highest active viral load detected in the heart A single patient had a right atrial thrombus Pericarditis was ACEGBDFH µm µm 03m µmFigure Thrombotic features identified at autopsy in patients with COVID19A Macroscopic right coronary artery thrombosis arrow in a 61yearold man PM1 with exudative phase diffuse alveolar damage B Macroscopic pulmonary thromboembolism arrow in a 97yearold man PM8 C Thrombus in the lung of a 79yearold woman PM9 on haematoxylin and eosin staining at 20X magnification the insert shows CD61 immunohistochemistry indicating moderate staining for platelets D Plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man PM1 the insert shows strong CD61 staining for platelets Periodic acid Schiff staining showing a glomerular microaneurysm arrow E and microthrombi within glomerular capillary loops arrow F at 40X magnification indicative of thrombotic microangiopathy in a 97yearold man PM8 Macroscopic splenic G and hepatic H infarction in a 22year old man PM5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae PM5 while the other showed only microscopic acute pericarditis appendix p figure The median heart weight was high g and four of nine patients had left ventricular hypertrophy Nonbacterial thrombotic marantic endocarditis was noted in one patient PM5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure PM5 had disseminated mucormycosis and numerous other thrombotic features appendix p Cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients PM8 appendix p Lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure Increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four [] of seven patients sinus histiocytes of hilar lymph nodes in three [] of six and bone marrow four [] of eight Phagocytosis was identified in at least one of these ans in six of nine patients Bone marrow haemophagocytosis was prominent in two patients PM4 and PM8 and focal in two patients PM7 and PM9 Depletion of periarteriolar Tcell sheaths within the white pulp was observed figure Red pulp was generally congested showing reduced numbers of CD8positive T cells Plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents Both IgMpositive and IgGpositive plasma cells were identified and they were polytypic for lightchain expression figure Lymph nodes showed preservation of follicles and relative depletion of paracortical areas Medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses Bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding A necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on Ziehl Neelson staining appendix p All spleen and lymphoid material examined with immuno histochemistry were negative for EpsteinBarr virus and cytomegalovirusPancreatitis was noted in two of eight patients PM5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure No fungal hyphae were noted in the pancreas PM8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure A third of patients three [] of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure No wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cadrenalitis was noted Two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainMedian combined kidney weight was within normal range at g IQR “ Salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients PM6 24yearold man of arterial intimal thickening than expected for that age appendix p We observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher Large droplet fatty change was seen in most patients seven [] of eight Cirrhosis or bridging hepatic fibrosis were noted in three patients No liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure The median liver weight was g IQR “ and three of nine patients showed hepatomegaly liver weighing g Two patients PM4 and PM7 showed marked autolysis and were not included in analysisModerate to intense microglial activation was the most prominent pathological feature in the CNS five [] of five patients Mild Tcell infiltration was noted around blood vessels and capillaries in all five patients but B cells were absent We found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by BAPP β amyloid precursor protein stain However no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arteryTissues from five patients were analysed for presence of viral genomes against E gene and indications of viral replication against subgenomic RNA transcripts by qRTPCR Viral RNA was present in respiratory tract samples including lung of all five cases analysed In addition two of three patients had detectable viral RNA in the nasal epithelium and four of five patients in the trachea Evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5A Viral genomes were also detected using a different qRTPCR targeted at RdRp gene and patterns were consistent between the two sets of primers data not shown A third primer set that detected subgenomic RNA indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5BACE µmBDF µm µmFigure Other notable autopsy findings in patients with COVID19A Contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man PM5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10X magnification B Adrenocortical microinfarcts in a 79yearold woman PM9 with reendothelialising thrombus in small adrenal vessels highlighted by CD34 insert bottom left and haematoxylin and eosin insert top right Marantic endocarditis C highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis D in a 22yearold man PM5 with COVID19 and a secondary fungal lung infection E Periodic acid Schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man PM6 20X magnification F Microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man PM8 20X magnificationDiscussionIn this series we have described the major pathological findings identified at autopsy in ten patients who died of severe COVID19 The most consistent findings were DAD thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to SARSCoV2 infection were also identifiedDAD was the most consistent and prominent feature in our series and others78 The specific phase of DAD probably represents the degree and chronicity of the offending insult SARSCoV2 infection in relation to the time of death This is similar to previous coronavirus epidemics6 The conclusion by Copin and colleagues7 that COVID19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe Published online August 101016S2666524720301154 s 0cADBC µm µm µm µm µmEF µm µm µmFigure Pathological findings in haematological ans in patients with COVID19Tcell depletion in the spleen of a 79yearold woman PM9 with COVID19 haematoxylin and eosin staining of the spleen at 10X magnification A CD20 staining of spleen indicating presence of B cells B 10X magnification with the insert showing the same region at higher power 20X magnification and CD3 staining of spleen indicating depletion of T cells C 10X magnification with the insert showing the same region at higher power 20X magnification Bone marrow phagocytosis in a 97yearold man PM8 with COVID19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis D 40X magnification and CD68PGM1 staining of bone marrow indicating presence of phagocytosis 20X [E] and 40x [F] magnificationchronicity five patients had spent approximately weeks on a ventilator Barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy Reports of lung histology in early COVID19 also suggest a degree of lymphocytic pneumonia although DAD is probably superimposed on this over time in the majority of fatal cases7 Pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ Definite evidence of in COVID19 will require quantitative analysis comparing tissues from COVID19 patients with DAD associated with other conditions and unaffected tissues Several cases of invasive pulmonary aspergillosis have been reported in patients with severe COVID19 pneumonia18 To our knowledge this is the first description of histologically proven mucormycosis in patients with COVID19 and suggests that other human fungal pathogens including members of Mucoromycotina can complicate COVID19associated infectionslymphocyte depletion tissuerelated Numerous clinical features including raised serum Ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with COVID192 Thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other COVID19 autopsy series15 In a retrospective study of autopsies in patients with acute respiratory distress syndrome and DAD of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 Another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 Whether thrombosis in COVID19 is more common than in other causes of DAD remains uncertain however our data support thrombosis as being a striking feature in these patients A study suggested endotheliitis as a prominent feature in patients with severe COVID1910 but this was not a prominent feature in our patients Importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death The extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the MCCDA raised cytokine profile has been documented in a subset of patients with severe COVID192 Consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 Of the four patients with bone marrow haemophagocytosis one patient PM7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°C however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe Published online August 101016S2666524720301154s 0cA substantial feature in COVID19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings When compared with those with mild disease patients with severe COVID19 tend to have a higher neutrophil to lymphocyte ratio and higher CD4positive to CD8positive Tcell ratio22 Additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 We have corroborated this evidence by documenting a low number of T cells especially CD8positive T cells and FOXP3positive regulatory T cells in the spleen and lymph nodes in severe fatal COVID19 Notably normal plasma cell both IgM and IgG positive response was present in haematological ans in most patientsThe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance We report here evidence of viral genomic RNA outside the respiratory tract This finding is in agreement with several previous studies that have identified viral genomes by qRTPCR in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 We also report detection of subgenomic RNA a product that is only produced in actively infected cells A report identified low viral load in the brain of three of patients with COVID19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 Although we cannot exclude that the RNAs detected in our series were similarly carried to the site of sampling in blood the distribution of RNA in different tissues varied widely between postmortem casesPM3 and PM4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas PM3 and PM4 died after long stays in intensive care units and had either lower overall viral RNA PM5 or higher viral RNA outside the respiratory tract PM2 PM1 and PM2

Thyroid_Cancer

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

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

Thyroid_Cancer

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

Oral squamous cell carcinoma OSCC is a common kind of squamous cell carcinoma of the head and neck which is a threat to public health Long noncoding RNAs lncRNAs are associated with the development of various diseases including cancers LncRNA titin antisense RNA TTN‘AS1 is known as a crucial regulatory factor in several cancers Nevertheless the specific functions of TTN‘AS1 in OSCC remains obscureMethods The expression of TTN‘AS1 in OSCC samples or cells was analyzed through qRT‘PCR Colony formation assay EdU assay flow cytometry assay TUNEL assay and wound healing assay were conducted to estimate the func‘tions of TTN‘AS1 in OSCC cells RIP and luciferase reporter assays were utilized to detect the interaction between TTN‘AS1 and miR‘‘3p as well as between miR‘‘3p and NFAT5Results TTN‘AS1 expression was stronger in OSCC cells Knockdown of TTN‘AS1 effectively restrained cell prolifera‘tion and migration but had inductive role in apoptosis Moreover TTN‘AS1 could function as the miR‘‘3p sponge in OSCC and miR‘‘3p exerted the inhibitory functions on OSCC cell growth In addition NFAT5 was proven as the target of miR‘‘3p Rescue assay indicated that overexpressing NFAT5 could reverse the inhibitory function of TTN‘AS1 depletion on cell growthConclusion lncRNA TTN‘AS1 contributed to the progression of OSCC via miR‘‘3pNFAT5 axisKeywords TTN‘AS1 miR‘‘3p NFAT5 Oral squamous cell carcinomaBackgroundOral squamous cell carcinoma OSCC is one of the commonest squamous cell carcinomas occurs in the head and neck It ranks sixth in occurrence and had a high mortality rate [ ] According to many years of investigation and research the pathogenesis of OSCC is related to the internal factors such as drinking and smoking but its specific pathogenesis is still unclear [ ] Although the surgery for OSCC is effective the situation for the overall survival of OSCC patients is still unfavorable [ ] Thus Correspondence fusuwei2009163comDepartment of Stomatology Henan Provincial People™s Hospital People™s Hospital of Zhengzhou University No7 Weiwu Road Zhengzhou Henan Chinaindepth study of the potential molecular mechanisms of OSCC is of great significance for developing new therapeutic strategiesLong noncoding RNAs lncRNAs are classified as the subgroup member of noncoding RNAs family with over nucleotides in length which are not able to encode proteins [ ] Recently lncRNAs are confirmed to involve in different cell progression such as cell proliferation and cell apoptosis Moreover the crucial functions of lncRNAs in the occurrence and development of assorted cancers have also been reported through a flow of researches [ ] Different kind of lncRNAs exerted different functions in cancers For example PVT1 accelerated esophageal carcinoma cell migration and invasion via sponging miR145 and regulating FSCN1 [] 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 0cFu a0et a0al Cancer Cell Int Page of SARCC alters he androgen receptormiRNA1433p signals thereby suppresses the progression of renal cell carcinoma [] And GAPLINC facilitated gastric cancer cell growth through serving as a sponge of miR378 to regulate MAPK1 [] Titin antisense RNA TTNAS1 is a novel lncRNA that takes part in the regulation of cancer development in accordance with existing researches For illustration TTNAS1 with high expression in lung adenocarcinoma cells can expedite cellular functions of lung adenocarcinoma through serving as a sponge of miR1425p to regulate CDK5 [] Nevertheless its specific function of TTNAS1 in OSCC remains unclearHere we selected TTNAS1 as the object of our research and investigated the regulatory mechanisms and functions in OSCCMethodsTissues samplesPaired tissues adjacent normal and tumor were collected from patients with OSCC who were diagnosed at Henan Provincial People™s Hospital Patients participated in this study didn™t receive any kind of therapy before surgery All patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial People™s Hospital Samples were stored at ˆ’ a0 °C until useCell linesHuman normal squamous epithelial cell line NOK obtained from Shanghai Honsun Biological Technology CoLtd Shanghai China human tongue squamous carcinoma cell lines including SCC4 SCC9 CAL27 procured from ATCC Manassas VA USA and BICR cell obtained from European Collection of Authenticated Cell Cultures ECACC UK were used in current study NOK cell was cultured in DMEM Gibco Rockville MD USA with antibiotics and FBS Gibco CAL27 cell was cultured in DMEM containing FBS SCC4 cell was cultured in DMEM F12 Medium containing a0ngml hydrocortisone and FBS SCC9 cell was cultured in DMEM F12 Medium containing a0mM a0lglutamine a0gL sodium bicarbonate a0mM HEPES a0 mM sodium pyruvate supplemented with a0 ngml hydrocortisone and FBS BICR16 cell was cultured in DMEM with 500ugml G418 and FBS Cell culture was conducted under a condition with CO2 and a0°CTotal RNA extraction and a0qRT‘PCRTRIzol Reagent Invitrogen Carlsbad CA was responsible for total RNA extraction from samples or cells Afterwards RNA samples were converted into cDNA Japan PowerUp„¢ SYBR® Green Master Mix Life Techby employing Reverse Transcriptase Kit Takara Shiga nologies Grand Island NY USA was utilized for PCR analysis [] After amplification ˆ’ΔΔCt method was applied to quantify PCR products U6 snRNA or GAPDH was used as the internal control for lncRNA mRNA or miRNA All primers used in this experiments were provided in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateTransfectionsThe shRNAs designed for TTNAS1 or NFAT5 and nonspecific shRNAs as well as pcDNA31NFAT5 and empty vector theses transfection plasmids were procured from GenePharma Shanghai China In addition the miR4113p mimicsinhibitor and NC mimicsinhibitor were procured from Genechem Shanghai China SCC4 and SCC9 cells were collected for a0h of plasmid transfections by use of Lipofectamine Invitrogen Sequence for all plasmids used in current study were listed in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateCCK‘ assayAs previously described [] CCK8 Kit Beyotime Shanghai China was applied to detect cell viability under manufacturer™s protocols Cells cellswell were planted in 96well plates After and a0h the CCK8 reagents were added into each well Cell viability was detected using a microplate reader to measure the absorbance at the wave length of a0nm Each samples were assayed for more than triplicateColony formation assayAfter indicated transfections SCC4 and SCC9 cells were planted into 6well plates with cells in each well Following 14day of cell culture the resulting colonies were fixed using PFA for a0min stained using crystal violet solution for a0min and finally counted manually [] Each samples were assayed for more than triplicateEdU assayfor cell proliferation detection by use of BeyoClick„¢ EdU assay was undertaken in cells of SCC4 and SCC9 EdU Cell Proliferation Kit Beyotime Shanghai China with Alexa Fluor [] The DAPI staining solution was acquired from Beyotime for detecting cell nucleus After washing in PBS cells were studied using inverted microscope Olympus Tokyo Japan Each samples were assayed for more than triplicate 0cFu a0et a0al Cancer Cell Int Page of Flow cytometryCell apoptosis of transfected SCC4 and SCC9 cells was assayed employing the flow cytometer BD Biosciences Franklin Lakes NJ in the presence of Annexin VPI double staining kit Invitrogen Cell samples were collected from 6well plates via centrifugation then stained in Binding Buffer and assayed with flow cytometry [] Each samples were assayed for more than triplicateTUNEL assayThe transfected cell samples of SCC4 and SCC9 were washed employing PBS and fixed using PFS for TUNEL assay [] in the presence of TUNEL assay reagent Merck KGaA Darmstadt Germany Following addition of DAPI staining solution cell samples were analyzed using optical microscopy Olympus Each samples were assayed for more than triplicateWound healingThe transfected cell samples of SCC4 and SCC9 were seeded in 6well plates and cultivated until confluence [] Then the artificial wounds were created with a0μL of pipette tip At and a0h after incubation in serumfree medium the distance of wound healing were imaged under microscope Olympus Each samples were assayed for more than triplicateSubcellular fractionationThe TTNAS1 content in cytoplasmic and nuclear fracPARIS„¢ Kit Invitrogen as requested by provider Cell tions of SCC4 and SCC9 cells was studied by use of samples were lysed with cell fractionation buffer and cell disruption buffer then centrifuged for separating cell cytoplasm and cell nucleus [] For quantification GAPDH and U6 served as the cytoplasmic indicator and nuclear indicator respectively Each samples were assayed for more than triplicateFISHThe subcellular location of TTNAS1 in SCC4 and SCC9 cells was also studied with FISH assay using the deigned specifically TTNAS1probe Ribobio Guangzhou China After fixation the digested cells were airdried and cultured with probes in the hybridization buffer then treated in DAPI staining buffer [] Olympus fluorescence microscope was used for imaging Each samples were assayed for more than triplicateApplying the Magna RIP„¢ RNABinding Protein Immunoprecipitation Kit [] RIP assay was conducted RNA immunoprecipitation RIPfor RNA interaction in SCC4 and SCC9 cells as guided by provider Millipore Bedford MA RIP lysis buffer Thermo Fisher Scientific Waltham MA USA was applied to obtain the lysates Lysis was incubated with the magnetic beads Invitrogen Carlsbad CA USA conjugated with antiAgo2 antibody or antiIgG antibody at a0 °C overnight Complex was washed and purified according to the protocol of RIP kit used in this experiment The enrichment of RNAs were examined via RTqPCR Each samples were assayed for more than triplicateLuciferase reporter assayTTNAS1 fragment covering wildtype or mutant miR4113p binding sites were employed to construct TTNAS1WT or TTNAS1Mut vectors by use of the pmirGLO dualluciferase vectors Promega Madison WI SCC4 and SCC9 cells were cotransfected with miR4113p mimics or NC mimics and TTNAS1WT or TTNAS1Mut vectors for a0h followed by analysis of dualluciferase reporter assay system Promega [] Renilla luciferase activity was used as the internal control Each samples were assayed for more than triplicateWestern blotCells were lysed via RIPA buffer BCA Protein Assay kit Pierce Biotechnology Rockford IL was used to assess the concentration of protein Separation of equal amount of proteins was conducted via SDSPAGE BioRad Laboratories Hercules CA followed by the transformation to PVDF membranes Millipore Bedford MA The membranes were blocked with skim milk and incubated with primary and secondary antibodies All antibodies were obtained from Abcam Cambridge MA USA Protein bands were detected using a ECL detection kit Pierce Biotechnology Rockford IL Each samples were assayed for more than triplicateAnimal studySix 4weekold BALBc nude mice Shanghai Laboratory Animal Center was subjected to animal study in line with the ethical standards and guidelines of Henan Provincial People™s Hospital SCC6 cells × stably transfected with shNC or shTTNAS11 were injected into the right dorsal flanks of six mice Tumor sizes and volume were monitored by a caliper every a0days Four weeks later the mice were killed followed with the resection of tumors for measuring tumor weightStatistical analysesData of three or more independent assays were exhibited as the mean ± SD In addition Student™s ttest or onewaytwoway ANOVA followed by Tukey post hoc test 0cFu a0et a0al Cancer Cell Int Page of use of GraphPad Prism ® GraphPad Software Inc La was employed for comparing the group difference by Jolla CA USA Experimental data were collected when p ResultsKnockdown of a0TTN‘AS1 restrains the a0proliferation and a0migration of a0OSCC cellsAt first the relative higher level of TTNAS1 was observed in OSCC samples rather than adjacent normal ones Additional file a0 Fig S1A Next we detected the expression of TTNAS1 in OSCC cells through qRTPCR analysis We discovered that TTNAS1 expression was extremely high in OSCC cells in comparison of normal human squamous epithelial cell NOK cell Fig a01a At the same time we also found that TTNAS1 expression in SCC4 and SCC9 cells was highest Thus we knocked down TTNAS1 expression in SCC4 and SCC9 cells and identified that the TTNAS1 expression was exactly declined Fig a0 1b Following functional experiments were implemented to test the influence of inhibiting TTNAS1 on cells proliferation apoptosis and migration CCK8 assay unveiled that TTNAS1 depletion had significantly suppressive effect on cell viability Additional file a0 Fig S1B The number of colonies and EdU positive cells were reduced after silencing TTNAS1 indicating that cell proliferation could be restrained by TTNAS1 depletion Fig a01c d Then it was found by flow cytometry and TUNEL experiments that apoptosis was accelerated when decreased the level of TTNAS1 Fig a01e f Finally wound healing assay revealed that the migrated capability of SCC4 and SCC9 cells was hampered by silencing TTNAS1 Fig a0 1g In a word knockdown of TTNAS1 restrained cell proliferation and migration of OSCCTTN‘AS1 acts as a0miR‘‘3p sponge in a0OSCCThen we tested the distribution of TTNAS1 in SCC4 and SCC9 cells The results indicated that TTNAS1 tended to be located in the cytoplasm of SCC4 and SCC9 cells Fig a0 2a b indicating the potential posttranscriptional regulatory role of TTNAS1 in OSCC A flow of evidence suggested that lncRNA could serve as a ceRNA to regulate mRNAs through sponging miRNAs at posttranscriptional level [ ] Then we utilized starBase website to predict the possible miRNA which could have the binding site of TTNAS1 and one potential miRNA miR4113p was found out Fig a02c Then qRTPCR analysis was implemented to test the expression of miR4113p in OSCC samples and cells And the results indicated that miR4113p expression was lower in OSCC tissues and cells Additional file a0 Fig S1C and Fig a0 2d The lowest level of miR4113p was detected in SCC4 and SCC9 cells After that we discovered the binding site of miR4113p and TTNAS1 from starBase website Fig a02e and conducted Ago2RIP assay to evaluate the binding possibility of them We discovered that miR4113p and TTNAS1 were markedly enriched in antiAgo2 group Fig a02f and Additional file a0 Fig S1D which indicated that they were coexisted in RISC Following we overexpressed miR4113p and conducted the luciferase reporter assay We discovered that miR4113p overexpression caused a notable reduction on the luciferase activity of TTNAS1WT while the luciferase activity of TTNAS1Mut displayed no visible change Fig a02g h indicating that TTNAS1 could bind to miR4113p Overall TTNAS1 sponges miR4113p in OSCCUpregulation of a0miR‘‘3p represses OSCC cell growth and a0migrationIn order to search the role of miR4113p in OSCC functional experiments were implemented Firstly colony formation and EdU assays indicated that overexpressing miR4113p suppressed the proliferation of SCC4 and SCC9 cells Fig a0 3a b Moreover apoptosis of SCC4 and SCC9 cells was accelerated by miR4113p mimics through flow cytometry analysis and TUNEL assays Fig a03c d As illustrated in Fig a03e overexpression of miR4113p visibly reduced cell migration Taken together overexpression of miR4113p suppressed growth and migration in OSCCNFAT5 is a0the a0downstream target of a0miR‘‘3p in a0OSCCFor the sake of further verifying ceRNA hypothesis we searched the targets of miR4113p Combining the searching results from miRmap microT and PicTar databases candidate target genes were found under the condition Program number programs Fig a0 4a Then qRTPCR assay was applied to detect the influence of miR4113p overexpression and TTNAS1 inhibition on the levels of these mRNAs The results displayed a significant downregulation of mRNAs TLL2 MGAT4A RAB21 and NFAT5 when miR4113p was overexpressed and TTNAS1 was knocked down while other mRNAs were almost unchanged Fig a0 4b Then we tested the expressions of TLL2 MGAT4A RAB21 and NFAT5 in OSCC cells through qRTPCR for further detection We discovered that only NFAT5 displayed a high expression in OSCC cells Fig a04c High level of NFAT5 was further determined in OSCC tissues compared to adjacent normal ones Additional file a0 Fig S2A Thus we selected NFAT5 to conduct the further experiments Following we discovered the binding site of NFAT5 and miR4113p from starBase Fig a04d And RIP assays were implemented to evaluate the relationship of TTNAS1 NFAT5 and miR4113p The results 0cFu a0et a0al Cancer Cell Int Page of Fig Knockdown of TTN‘AS1 restrains the proliferation and migration of OSCC cells a The expression of TTN‘AS1 was tested through qRT‘PCR in OSCC cells b The interference efficiency of TTN‘AS1 was detected by qRT‘PCR in SCC‘ and SCC‘ cells c d Cell proliferation ability was measured by colony formation and EdU experiments when TTN‘AS1 was inhibited e f Cell apoptosis was evaluated through flow cytometry and TUNEL experiments after silencing TTN‘AS1 g Wound healing assays were utilized to estimate cell migration when TTN‘AS1 was subjected to knockdown P P 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 acts as the miR‘‘3p sponge in OSCC a b The cellular location of TTN‘AS1 was identified in SCC‘ and SCC‘ through Subcellular fractionation and FISH c StarBsae website was utilized to predict the possible miRNAs that could bind with TTN‘AS1 d MiR‘‘3p expression was detected through qRT‘PCR in OSCC cells e The binding site of TTN‘AS1 in miR‘‘3p f RIP assay was utilized to evaluate the relationship between miR‘‘3p and TTN‘AS1 g The efficiency of miR‘‘3p overexpression was tested through qRT‘PCR h Luciferase reporter assays were conducted to verify the correlation of miR‘‘3p and TTN‘AS1 P P showed that TTNAS1 NFAT5 and miR4113p were enriched in Ago2 indicating that TTNAS1miR4113pNFAT5 axis combined with RISC Fig a04e and Additional file a0 Fig S2B Then miR4113p was silenced and the interference efficiency was detected We could observe that miR4113p expression exactly declined after inhibition Fig a04f Following we detected the expression of NFAT5 when TTNAS1 and miR4113p were inhibited through qRTPCR Results indicated that NFAT5 expression could be hampered by TTNAS1 depletion but then recovered by miR4113p inhibition Fig a0 4g and Additional file a0 Fig S2C It demonstrated that NFAT5 and TTNAS1 were positively associated while NFAT5 and miR4113p were negatively correlated Then we investigated the function of NFAT5 in OSCC cells Firstly we knocked down the expression of NFAT5 in SCC4 and SCC9 cells and tested the knockdown efficiency Fig a04h and Additional file a0 Fig S2D NFAT5 expression could be hampered effectively after knockdown Then colony formation and EdU assays were carried out and the 0cFu a0et a0al Cancer Cell Int Page of Fig Upregulation of miR‘‘3p represses cell proliferation and migration in OSCC a b Cell proliferation was estimated through colony formation and EdU experiments when miR‘‘3p was overexpressed c d Flow cytometry and TUNEL experiments were implemented to measure cell apoptosis after overexpressing miR‘‘3p e Wound healing assays were adopted to test cell migration ability when miR‘‘3p was subjected to upregulation P See figure on next pageFig NFAT5 is a target gene of miR‘‘3p in OSCC a mRNAs which had the binding site with miR‘‘3p were predicted by starBase b The qRT‘PCR analysis was utilized to screen out the mRNAs which could be inhibited by NFAT5 depletion and miR‘‘3p overexpression c The expressions of TLL2 MGAT4A RAB21 and NFAT5 in SCC‘ and SCC‘ cells through qRT‘PCR d The binding site of NFAT5 and miR‘‘3p e RIP assay was adopted to test the relationship between TTN‘AS1 miR‘‘3p and NFAT5 f The interference efficiency of miR‘‘3p was tested by qRT‘PCR analysis g The expression of NFAT5 was detected when NFAT5 and miR‘‘3p was silenced h The interference efficiency of NFAT5 was tested by qRT‘PCR analysis i j Cell proliferation was evaluated through colony formation and EdU experiments when NFAT5 was knocked down k l Cell apoptosis was measured through flow cytometry and TUNEL experiments after inhibiting NFAT5 m Wound healing assays were carried out for estimating cell migration after NFAT5 was subjected to inhibition P 0cFu a0et a0al Cancer Cell Int Page of result indicated that silencing NFAT5 repressed the proliferation of SCC4 and SCC9 cells Fig a0 4i j Moreover cell apoptosis capability was expedited by NFAT5 depletion in flow cytometry and TUNEL assays Fig a04k l Finally wound healing assays indicated that silencing NFAT5 could hamper cell migration capability Fig a04m 0cFu a0et a0al Cancer Cell Int Page of Collectively NFAT5 was a target gene of miR4113p in OSCC and it accelerated the progression of OSCCTTN‘AS1 promotes OSCC progression via a0miR‘‘3pNFAT5 axisFor the sake of proving whether TTNAS1 could accelerate OSCC progression via miR4113pNFAT5 axis rescue assays were implemented Ahead of rescue assays qRTPCR was adopted to test the overexpression efficiency of NFAT5 in SCC4 and SCC9 cells The results displayed that NFAT5 expression was visibly increased after transfecting with pcDNA31NFAT5 Fig a05a Next we detected the mRNA and protein levels of NFAT5 in SCC4 and SCC9 cells after transfection It was uncovered that NFAT5 levels decreased by TTNAS1 depletion were rescued by the inhibition of miR4113p or the upregulation of NFAT5 Additional file a0 Fig S2E Then colony formation and EdU rescue assays were conducted we discovered that cell proliferation was hampered by TTNAS1 depletion but then it was recovered by NFAT5 overexpression or miR4113p inhibition Fig a0 5b c Through flow cytometry and TUNEL assays we found that knockdown of miR4113p or upregulation NFAT5 could reverse the cell apoptosis ability which was accelerated by TTNAS1 depletion Fig a05d e In the end it was indicated through wound healing assay that the inhibited cell migration caused by knockdown of TTNAS1 was restored by NFAT5 overexpression or miR4113p inhibition Fig a05f Thus we confirmed that TTNAS1 promoted OSCC cell growth and migration by miR4113pNFAT5 axisTTN‘AS1 promoted OSCC cell growth in a0vivoIn vivo study was conducted to support above in a0 vitro findings We observed that tumor size volume and weight in shNC group were all smaller than those in shTTNAS11 group Fig a06a“c Importantly IHC staining indicated that silencing of TTNAS1 caused a reduction in the positivity of Ki67 and PCNA Fig a06d All these experiments unveiled that TTNAS1 promotes OSCC progression via miR4113pNFAT5 axisDiscussionOral squamous cell carcinoma OSCC is a common squamous cell carcinoma of the head and neck It has a relatively high incidence worldwide As the regulatory functions of lncRNA in assorted cancers are constantly being explored lots of lncRNAs have also been confirmed to play a crucial role in promoting the development of OSCC For example PLAC2 could promote cell growth through activating wntβcatenin pathway in OSCC [] CEBPAAS1 was considered to correlate with the bad prognosis and it also could facilitate tumorigenesis through CEBPABcl2 in OSCC [] Moreover P4713 was reported to contribute to the malignant phenotypes of OSCC through activating the JAKSTAT3 pathway [] In our research we investigated the functions of TTNAS1 in OSCC TTNAS1 was a novel lncRNA and it served as the oncogene in lung adenocarcinoma [] In this study TTNAS1 was discovered to be highly expressed in OSCC cells And TTNAS1 depletion impaired cell proliferation and migration but it accelerated cell apoptosis in OSCC Overall TTNAS1 exerted the carcinogenic effect in OSCCMiRNAs are small RNAs with “ nucleotides in length without ability of coding protein [] In recent years an increasing number of evidences discovered that lncRNA could function as a crucial element of competing endogenous RNA ceRNA network by sponging miRNA to regulate mRNA so as to take part in the regulation of cancer progression [ ] For example lncRNA ATB functioned as a ceRNA to expedite YAP1 through sponging miR5905p in malignant melanoma [] PAGBC acted as a sponge of miR133b and miR and accelerated gallbladder tumorigenesis [] AFAP1AS1 could act as a ceRNA of miR4235p to expedite nasopharyngeal carcinoma progression [] In our research we utilized bioinformatics tools to find the possible miRNA which could bind to TTNAS1 After screening miR4113p was selected With the conduction of RIP and luciferase experiments we proved that TTNAS1 could act as ceRNA to sponge miR4113p in OSCC MiR4113p was verified as the tumor suppressor gene in ovarian cancer and it could restrain cell proliferation migration and invasion of ovarian cancer [] Thus we investigated the functions of miR4113p in OSCC As we expected miR4113p could repress cell proliferation and migration but accelerate cell apoptosis in OSCC In short our research confirmed that TTNAS1 sponged miR4113p and overexpressing miR4113p could repress the progression of OSCCNFAT5 is a mRNA and it has been reported to be associated with several cancers For example NFAT5 was proved to conduce to the glycolytic phenotype rewiring and pancreatic cancer progression through transcription of PGK1 [] Moreover NFAT5 cpuld also promote glioblastoma celldriven angiogenesis through EGFL7 which was mediated via SBF2AS1 and miR3383p [] In our research we discovered that NFAT5 was highly expressed in OSCC cells And based on the mechanism experiments we also proved that NFAT5 was the target of miR4113p and overexpressing it could accelerate the progression of OSCC Rescue experiment indicated that upregulation of NFAT5 could offset TTNAS1 knockdownmediated functions on the progression of OSCC 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 promotes OSCC progression via miR‘‘3pNFAT5 axis a The qRT‘PCR analysis was utilized to examine the overexpression efficiency of NFAT5 in SCC‘ and SCC‘ cells b c Cell proliferation capability in SCC‘ and SCC‘ cells was measured by colony formation and EdU assay in different groups d e Cell apoptosis was tested through flow cytometry and TUNEL assays in different groups f Wound healing assays were implemented to detect the cell migration ability in different groups P 0cFu a0et a0al Cancer Cell Int Page of Fig TTN‘AS1 promoted OSCC cell growth in vivo a Tumors removed from the mice injected with sh‘NC‘transfected cells or sh‘TTN‘AS11‘transfected cells b c Volume and weight in different groups were measured d IHC staining of tumor tissues collected from different groups with anti‘Ki‘ and anti‘PCNA P proving the functions of TTNAS1miR4113pNFAT5 axis in OSCCtransfected with sh‘TTN‘AS11 was examined by qRT‘PCR and western blot analyses after co‘transfection with miR‘‘3p inhibitor or pcDNA31NFAT5 P ConclusionTaken together TTNAS1 could contribute to the progression of OSCC via miR4113pNFAT5 axis which may provide the new idea for the exploration of OSCC treatmentsSupplementary informationSupplementary information accompanies this paper at https doi101186s1293 ‘‘ ‘Additional file a0 Sequence for all plasmids used in current studyAdditional file a0 Figure S1 A TTN‘AS1 expression in adjacent normal and tumor tissues was examined by qRT‘PCR analysis B CCK‘ assay was applied to analyze the viability of SCC‘ and SCC‘ cells transfected with sh‘NC sh‘TTN‘AS11 or sh‘TTN‘AS12 C The level of miR‘‘3p was assessed in pairs of OSCC tissues and adjacent normal tissues D Agarose gel electrophoresis for the Ago2‘RIP assay in Fig 2F P Additional file a0 Figure S2 A NFAT5 expression in paired tissues obtained from OSCC patients B Agarose gel electrophoresis for the Ago2‘RIP assay in Fig 4E C Protein level of NFAT5 in cells transfected with sh‘NC sh‘TTN‘AS11 or co‘transfected with sh‘TTN‘AS11 and miR‘‘3p inhibitor D Protein level of NFAT5 in cells transfected with sh‘NC sh‘NFAT51 and sh‘NFAT52 E mRNA and protein level of NFAT5 in cells AbbreviationsOSCC Oral squamous cell carcinoma TTN‘AS1 Titin antisense RNA lncRNAs Long non‘coding RNAs ceRNAs Competing endogenous RNAs miRNAs microRNAs mRNA Messenger RNA ATCC American type culture collection DMEM Dulbecco™s modified Eagle™s medium FBS Fetal bovine serum RIPA Radioimmunoprecipitation assay SDS‘PAGE Sulphate‘polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride RT‘qPCR RNA extraction and quantitative real‘time polymerase chain reaction HRP Horseradish peroxidase FISH Fluorescence in situ hybridization WT Wild‘type Mut Mutant SD Stand‘ard deviation ANOVA Analysis of varianceAcknowledgementsWe appreciate all the people involved in this studyAuthors™ contributionSF project administration study design and review experiments YZ SL and ZS methods investigation data JZ and QH preparation draft manuscript All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsNot applicable 0cFu a0et a0al Cancer Cell Int Page of Ethics approval and consent to participateAll patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial People™s HospitalConsent for publicationAuthors confirmed that this work can be published The content of this manu‘script is original and it has not yet been accepted or published elsewhereCompeting interestsNo competing interest existReceived February Accepted June References Krishna Rao SV Mejia G Roberts‘Thomson K Logan R Epidemiology of oral cancer in Asia in the past decade“an update ‘ Asian Pac J Cancer Prev APJCP “Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol ““Sacco AG Cohen EE Current treatment options for recurrent or metastatic head and neck squa

Thyroid_Cancer

suffer from a high false positive rate because predicted expression of different genes may behighly correlated due to linkage disequilibrium between eQTL We propose a novel statistical method Gene Score Regression GSR to detect causal gene sets for complex traitswhile accounting for genetogene correlations We consider noncausal genes that arehighly correlated with the causal genes will also exhibit a high marginal association with thecomplex trait Consequently by regressing on the marginal associations of complex traitswith the sum of the genetogene correlations in each gene set we can assess the amountof variance of the complex traits explained by the predicted expression of the genes in eachgene set and identify plausible causal gene sets GSR can operate either on GWAS summary statistics or observed gene expression Therefore it may be widely applied to annotateGWAS results and identify the underlying biological pathways We demonstrate the highaccuracy and computational efficiency of GSR compared to stateoftheart methodsthrough simulations and real data applications GSR is ly available at githubcomlilabmcgillGSRIntroductionGenomewide association studies GWAS have been broadly successful in associating geneticvariants with complex traits and estimating trait heritabilities in large populations [“] Overthe past decade GWAS have quantified the effects of individual genetic variants on hundredsof polygenic phenotypes [ ] GWAS summary statistics have enabled various downstreamanalyses including partitioning heritability [] inferring causal single nucleotide polymorphisms SNPs using epigenomic annotations [] and gene sets enrichment analysis fora1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Zhang W Li SY Liu T Li Y Partitioning genebased variance of complex traitsby gene score regression e0237657 101371journalpone0237657Editor F Alex Feltus Clemson University UNITEDSTATESReceived March Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237657Copyright Zhang 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 of the data used inthis paper are described under subsection Realdata application in the manuscript and pastedbelow as reference We applied our approach toinvestigate pathway enrichment for complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressiontraits Fig 2b using publicly available summarystatistics and genotypeexpression weights basedon GTEx whole blood samples wwwgtexportalhomedatasets2 The GWASsummary statistics were downloaded from publicdatabase databroadinstitutealkesgroupsumstats_formatted We downloadedexpression weights and reference LD structureestimated in Genomes using Europeanindividuals from the TWASFUSION websitehttpgusevlabprojectsfusion Franke labcelltypespecific gene expression dataset wereobtained from databroadinstitutempgdepictdepict_downloadtissue_expression Inaddition we applied GSR to test for gene setenrichment in three wellpowered types of cancerbreast invasive carcinoma BRCA cases and controls thyroid carcinoma THCA casesand controls and prostate adenocarcinomaPRAD cases and controls using geneexpression datasets from The Cancer GenomeAtlas TCGA Uniformly processed normalizedand batcheffect corrected gene expressiondatasets from TCGA and GTEx were obtained fromfigsharecomsData_record_3 Gene expression and phenotype werestandardized before supplying to the GSR softwareStandard GSEA was also performed forcomparison Gene sets were downloaded from theMSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combinedBIOCARTA KEGG and REACTOME to create a totalof gene sets We also downloaded the GO biological process terms as additional genesets as well as the gene sets pertaining tooncogenic signatures for the TCGA data analysisFunding The research is supported by CanadaFirst Research Excellence Fund CFREF HealthyBrains Healthy Life HBHL New Investigator fund at McGill University and Mon trealNeurologic Institute MNI and NSERC DiscoveryGrant RGPIN20190621 The funders had nocomplex traits [] However it remains challenging to link these genetic associations withknown biological mechanisms One main reason is that the majority of the GWAS loci are notlocated in known genic regions of the human genomeTranscriptomewide association studies TWAS [“] offer a systematic way to integrateGWAS and the reference genotypegene expression datasets such as the GenotypeTissueExpression project GTEx [] via expression quantitative loci eQTL In TWAS we couldfirst quantify the impact of each genetic variant on expression variability in a population andobtain predicted gene expression levels based on new genotypes Then we could correlate thepredicted gene expression with the phenotype of interest in order to identify pivotal genes[] Moreover when individuallevel genotypes and gene expression levels are not availablewe could still quantify genetophenotype association ie TWAS statistics using only themarginal effect sizes of SNPs on the phenotype and on gene expression respectively []These concepts and implementations have largely facilitated explanation of genetic associationfindings at the gene or the pathway levelHowever as depicted in Fig TWAS are often confounded by the genetogene correlationof the genetically predicted gene expression due to the SNPtoSNP correlation ie linkage disequilibrium LD [] Consequently relying on the TWAS statistics may lead to false positivediscoveries of causal genes and pathways One approach to address this problem is to finemapcausal genes by inferring the posterior probabilities of configurations of each gene being causalin a defined GWAS loci and then test gene set enrichment using the credible gene sets of prioritized genes [] However this approach is computationally expensive restricted to GWASloci and sensitive to the arbitrary thresholds used for determining the credible gene set andthe maximum number of causal genes per locusAnother method called PASCAL [] projects SNP signals onto genes while correcting forLD and then performs pathway enrichments as the aggregated transformed gene scoreswhich asymptotically follows a chisquare distribution However PASCAL does not leverage the eQTL information for each SNP thereby assuming that a priori all SNPs have thesame effect on the gene Stratified LD score regression LDSC offers a principle way to partition the SNP heritability into functional categories defined based on tissue or celltypespecific epigenomic regions [] or eQTL regions of the genes exhibiting a strong tissue specificity [] Although LDSC is able to obtain biologically meaningful tissuespecific enrichments it operates at the SNP level rendering it difficult to assess enrichment of gene setsMoreover neither PASCAL nor LDSC is able to integrate the observed gene expression datameasured in a disease cohort rather than the reference cohort that are broadly availableacross diverse studies of diseases including cancers such as The Cancer Genome AtlasTCGA []role in study design data collection and analysisAlthough expressionbased methods such as gene set enrichment analysis GSEA aredecision to publish or preparation of themanuscript No author received a salary from anyof the fundersCompeting interests The authors have declaredthat no competing interests existoften adopted in combination with the observed gene expression and phenotypes [] theygenerally do not account for the genetogene correlation While this type of correlation is usually caused by shared transcriptional regulatory mechanisms across genes GSEA still likelyproduces false positives in identifying causal pathwaysIn this study we present a novel and powerful genebased heritability partitioning methodthat jointly accounts for genetogene correlation and integrates information captured at eitherthe SNPtophenotype or the SNPtogene level We utilize this method to identify plausiblecausal gene sets or pathways for complex traits We showcase its high accuracy and computational efficiency in various simulated and real scenariosPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Overview of confounding effects on pathway analysis a A hypothetical example illustrates the confounding issue when using the geneticallypredicted transcriptome to assess the pathway enrichments for a target phenotype The causal gene set includes a causal gene which is linked to noncausal gene via their respective causal SNPs and which are in strong linkage disequilibrium b A GWAS locus SNP associations with the targetphenotype are summarized The causal SNP for the causal gene is in red The SNPs that drive noncausal genes are in blue The rest of the SNPs are ingreen SNPs exhibit correlated signals due to the linkage disequilibrium LD as displayed by the upper triangle of the SNPSNP Pearson correlationmatrix c A TWAS locus The genetogene correlation is partly induced by the SNPtoSNP correlation and partly due to intrinsic coregulatoryexpression program d Pathway associations based on averaged gene associations101371journalpone0237657g001MethodsPartitioning genebased variance of complex traitsWe assume gene expression has linearly additive effects on a continuous polygenic trait yyi ¼XAijaj þ �ijð1Þwhere Aij denotes the expression of the jth gene in the ith individual for i N individuals and j G genes αj denotes the true effect size of the jth gene on the trait andj Þ �i denotes the residual for the ith individual in this linear model andaj � Nð0 s2�i � Nð0 s2� ÞPiyi ¼ N yy ¼ Here we further assume that both y and A are standardized such that NPiAij ¼ and j Aj ¼ for j GN ANPLOS ONE 101371journalpone0237657 August PLOS ONE 0cWe define the estimated marginal effect size of the jth gene on the trait as ajGene score regressionaj ¼NAj y¼NAj ðXAkak þ �ÞkX¼kNAj Akak þNAj �X¼krjkak þ �ð2Þð3Þð4Þð5Þwhere � ¼ N Aj � withVarð�0Þ ¼N Aj Varð�ÞAj ¼N s2�and rjk ¼ and the kth geneN Aj Ak is the estimated Pearson correlation in gene expression between the jth geneWe define w2j ¼ Na2j Then if we further assume α r and � are independent we haveE½w2j � ¼ E½Na2j �X¼ NE½Ã°rjkak þ �0Þ2�k¼ NE½r jk�E½a2k� þ s2�Xkð6Þð7Þð8ÞNow consider C gene sets Cc where c C and denote the proportion of total trait Here Cc denotes the numVarðajÞvariance explained by the cth gene set as τc with tc ¼ber of genes in the cth gene setPjCcjj2CcConsequentlyE½a2k� ¼ VarðakÞ ¼Xck2CctcBy approximating E½r N we have thatXjk� with r E½w2jk þ j � ¼ NE½r jk�E½a2k� þ s2�k¼ NXtcXr jk þXtc þ s2�ck2Ccc¼ NXtclðj cÞ þ cwhere we define gene score as lðj cÞ ¼tinuous trait is normalizedPr jk and VarðyÞ ¼k2Ccð9Þð10Þð11ÞPctc þ s2� ¼ since the conPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionTherefore if we are able to obtain estimates for w2j and C gene score lj c for j Gand c C we will be able to perform linear regression of the estimated w2j on lj cand derive regression coefficient that is an estimate for each τc c C respectivelyThese are available from GWAS summary statistics of SNPtotrait effect sizes eQTL summary statistics of SNPtogene expression effect sizes and a reference LD panel Specifically Suppose we have estimated effect sizes βp× of p SNPs based on a GWAS including Ngwassamples ieb ¼NgwasXywhere XNgwas×p is the standardized genotype Meanwhile we have the eQTL summary statistics W estimated usingAeQTL ¼ XeQTLWTherefore the predicted gene expression in GWAS is given bySinceA ¼ XWj ¼ Na2w2j¼ NðNAj yÞ2¼ NðNWj XyÞ2¼ NðW j bÞ2the required w2j can be estimated without accessing any individuallevel data Furthermore a reference LD panel Sp×p summarizing SNPtoSNP correlation in thematched population with the GWAS study can provide estimates for rjk asR ¼ ½rjk�¼NAA¼NW XXW¼NW SWPLOS ONE 101371journalpone0237657 August ð12Þð13Þð14Þð15Þð16Þð17Þð18Þð19Þ PLOS ONE 0cGene score regressionIt is noteworthy that with individuallevel gene expression data we can also easily obtainthe required w2j and R [rjk] by definitionIn practice many gene sets are not disjoint and share common genes with each otherTherefore we regress one gene set at a time along with a œdummy gene set that include theunion of all of the other genes The dummy gene set is used to account for unbalanced genesets and to stabilize estimates of τc We also include an intercept in the regression model toalleviate nongeneset biases for example positive correlation between gene scores and truegene effect sizes that could lead to intercept greater than and negative correlation betweengene scores and true gene effect sizes could lead to intercept smaller than Simulation designTo assess the accuracy of our GSR approach we simulated causal SNPs for gene expression aswell as causal gene sets for a continuous trait based on real genotypes and known gene setsfrom existing databases Our simulation included two stages At stage we first simulatedgene expression based on reference genotype panel We then estimated SNPgene effects W gfor each gene g based on the simulated gene expression and genotype which were then used topredict gene expression At stage separately we simulated the a continuous trait using simulated gene expression based on genotype and estimated the marginal SNPphenotype effectsSimulation step simulating gene expression To simulate individual genotype we first partitioned genotype data for individuals ofEuropean ancestry obtained from the Genomes Project [] into independent LD blocks as defined by LDetect [] We then randomly sampled LD blocks and used only those LD blocks for the subsequent simulation We used LD blocks as opposed to whole genome to reduce computational burden required for multiple simulation runs For LD block j j of an individual i i we randomly sampledfrom the available samples for block j and concatenated these sampled LD blocks for this individual We repeated this procedure to simulate genotype Xref forNref individuals as a reference population We standardized the simulated genotype Xref We randomly sampled k incis causal SNPs per gene within ± kb around the genewhere k default We also experimented different number of causal SNPs k allin cis SNPs We sampled SNPgene weights Wg � N ð0 h2g kÞ where gene expression heritability h2 default which is the variance of gene expression explained by genotype We alsoexperimented different gene heritability h2g ¼ f02 05gg ¼ We then simulated gene expression Agref Xref Wg � where � � N ð0 s2� Þ and� ¼ s2Nrefk XrefWgk2ð h2g 00 1ÞINrefto match the desired heritability 00 h2gh2g¼s2�kXref Wg k2Nref Finally we applied LASSO regression Agref � �XWg to get Wg for each geneSimulation step simulating phenotypePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regression We simulated another Ngwas GWAS individuals by the predefined LD blocksamong the Europeans in Genome data following the same procedures as decribedabove We then standardized the simulated genotype Xgwas We then sampled a causal pathway Cc from MSigDB such that all of the Gc � jCcj genes inCc were causal genes for the phenotype For each noncausal pathway we removed genes that were also present in the causal pathway We removed noncausal pathways containing fewer than five genes afterwardsdefault Alternatively in more realistic scenarios we allowed for sharing genes with causalpathways by noncausal pathways We sampled genephenotype effect a � N ð0 s2aGcIGcÞ where the phenotypic varianceexplained by gene expression s2s2a f01 05ga ¼ default We also experimented different We simulated gene expression Ac as in step for the Ngwas individuals and standardized itto obtain �Ac We simulated a continuous trait using causal gene expression y ¼ �Acα þ �y where�y � N ð0 s2Þ Here s2�yof variance explained 00 s2s2a�ya¼s2�yk �Acαk2Ngwas¼ Ngwask �Acαk2ð s2a 00 1ÞINgwasto match the predefined proportion Lastly we computed GWAS summary SNPtotrait effect size b ¼ N XgwasyWe repeated these simulation procedures times Unless otherwise stated while we wereexperimenting various settings we kept the other settings at their default values k causalSNP per gene gene expression variance explained per causal SNP h2ance explained per gene s2a ¼ one causal pathway Using these obtained summary statistics we were able to perform GSR PASCAL LDSC and FOCUS in each simulated scenarioApplying existing methods PASCAL PASCAL was downloaded from www2g ¼ 01k phenotypic variunilchcbgindexphptitlePascal [] We executed the software using default settings LDSCStratified LD score regression software was downloaded from githubcombulikldsc[] Because LDSC operates on SNP level we considered SNPs located within ± kbaround genes in each pathway to be involved in the corresponding pathway Then for eachpathway we computed the LD scores over all chromosomes We experimented the options ofrunning LDSC with and without the baseline annotations using our simulated data Wefound that LDSC running without the baseline worked better in our case One possible reason is that the baseline annotations cover genomewide SNPs whereas there are much fewerSNPs in the simulated pathways FOCUS We downloaded FOCUS [] from githubcombogdanlabfocus We used FOCUS to infer the posterior probability of each gene beingcausal for the phenotype across all of the LD blocks We then took the credible gene set asfollows We first summed all of the posteriors over all of the genes We then sorted the genesby the decreasing order of their FOCUSposteriors We kept adding the top ranked the geneinto the credible gene until the sum of their posteriors was greater than or equal to the of the total sum of posteriors We used the credible gene set for hypergeometric testfor each pathway to compute the pvalues We also tried other thresholds for credible setsranging from including the fewest genes to including the most genes GSEAGSEA software was obtained from oftwarebroadinstitutegsea [] We used thePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Gene scores correlated with marginal TWAS summary statistics a Gene scores were correlated with marginal TWAS chisquare statistics ofSchizophrenia We grouped genes into bins by their gene scores to reduce noise For each bin we calculated the average gene scores and chisquarestatistics χ2 An unbinned version is supplied in S1 Fig in S1 File b Summary of Pearson correlation between marginal TWAS summary statisticsand gene scores for traits The negative correlation for T2D indicates that this trait possibly due to complicated genetic architecture andconfounding genetoenvironment interaction and drug effects is not suitable for using our approach101371journalpone0237657g002commandline version of GSEA to test for gene set enrichments using the observed geneexpression and phenotype dataReal data application We applied our approach to investigate pathway enrichment for complex traits Fig 2b using publicly available summary statistics and genotypeexpressionweights based on GTEx whole blood samples The GWAS summary statistics were downloaded from public database databroadinstitutealkesgroupsumstats_formatted[] We downloaded expression weights and reference LD structure estimated in Genomesusing European individuals from the TWASFUSION website httpgusevlabprojectsfusion [ ] Franke lab celltypespecific gene expression dataset were obtainedfrom databroadinstitutempgdepictdepict_downloadtissue_expressionIn addition we applied GSR to test for gene set enrichment in three wellpowered types ofcancer breast invasive carcinoma BRCA cases and controls thyroid carcinomaTHCA cases and controls and prostate adenocarcinoma PRAD cases and controls using gene expression datasets from The Cancer Genome Atlas TCGA Uniformlyprocessed normalized and batcheffect corrected gene expression datasets from TCGA andGTEx were obtained from figsharecomsData_record_35330593 [] Geneexpression and phenotype were standardized before supplying to the GSR software StandardGSEA was also performed for comparisonGene sets were downloaded from the MSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combined BIOCARTA KEGG and REACTOME to create atotal of gene sets We also downloaded the GO biological process terms as additional gene sets as well as the gene sets pertaining to oncogenic signatures for the TCGAdata analysisResultsGene scores were correlated with TWAS statistics in polygenic complex traitsOur method GSR is built on the hypothesis that the marginal gene effect sizes on the phenotype should be positively correlated with the sum of correlation with other genes whichPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressioninclude causal genes To validate this hypothesis we defined gene score for each gene as thesum of its squared Pearson correlation with all of the other genes derived from gene expression levels We calculated TWAS marginal statistics as the product of GWAS summary statistics β and eQTL weights W derived from the GTEx whole blood samples Eq To assessthe impact of genetogene correlation on TWAS statistic we correlated the gene scores withthe TWAS marginal statistics for complex traits Overall most traits had Pearson correlation between the gene score and the marginal TWAS statistic above For instance thecorrelation in schizophrenia was InterQuartile Range ” based on permutations Fig This implies a pervasive confounding impact on the downstream analysisincluding gene set or pathway enrichment analysis causal gene identification etc using theTWAS summary statistics while assuming independence of genes Fig GSR improved pathway enrichment powerIn simulated scenarios with default settings Methods compared to PASCAL and LDSC GSRdemonstrated hugely improved computational efficiency Table superior sensitivity indetecting causal pathways with an improved statistical power as well as competitive specificityin controlling for false positives Fig Specifically in simulations GSR achieved an overall area under the precisionrecall curve AUPRC of and identified the true causal pathway as the most significant one times compared to times by PASCAL which onlyachieved an overall AUPRC of Notably the FOCUSpredicted crediblegene sets were also significantly enriched for causal pathways Fig We then varied four different settings a the number of causal SNP per gene SNPgene heritabilities genephenotype variance explained overlapping causal pathway Wefocused our comparison with PASCAL because it directly tested for pathway enrichment andhas been demonstrated to outperform other relevant enrichment methods [] In all simulation settings GSR demonstrated an improved power in detecting the causal pathways S2 Figin S1 File as it was able to detect causal pathways when multiple SNPs influenced geneexpression when the proportion of variance explained by the gene expression was low orwhen the causal and noncausal pathways were allowed to overlap In contrast a lot of causalpathways were not deemed significant by PASCAL based on a pvalue threshold of which was equivalent to a Bonferronicorrected pvalue threshold of after correcting formultiple testing on approximately pathways tested per simulationImproved power in pathway enrichment leveraging observed geneexpressionOne unique feature of GSR is the ability to run not on only the summary statistics but also onobserved gene expression where the genegene expression correlation is directly estimatedTable Comparison of existing methods with GSRMethodPASCAL []LDSC []FOCUS []GSEA []GSRGWASTWASMeasured expressionRunning timesum stat �sum statsum statsum statsum statindividual expressionindividual expression m h   h m min� Summary statistics  For custom gene sets the main computation time for LDSC is calculating the LD score for all of the Genome SNPs101371journalpone0237657t001PLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Evaluation of power and robustness of GSR in detecting causal pathways a Precisionrecall curves for GSR and PASCAL summarizingresults from simulations b Summary of pvalues obtained by running GSR along with PASCAL LDSC and FOCUS times For each methodthe enrichment significance for causal pathways and noncausal pathways are displayed We experimented FOCUS with and crediblesets for the pathway enrichments For the ease of comparison we plotted the yaxis on a squareroot negative logarithmic scale Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g003from the insample gene expression To evaluate the accuracy of this application we simulatedgene expression and phenotype for individuals which were provided as input to GSR forpathway enrichment analysis As a comparison we applied GSR to the summary statistics generated from the same datasetAs in the simulation above the SNPexpression weights were estimated from a separate setof reference individuals whereas the SNPphenotype associations were estimated fromonly individuals Notably the sample size for the GWAS cohort is much smaller than theprevious application to mimic the real data where usually fewer than individuals haveboth the RNAseq and phenotype available eg TCGA Additionally we applied standardGSEA [] to the same dataset with the observed gene expression We observed an improvedpower of GSR when using the observed gene expression over GSR using the summary statisticsFig whereas GSEA had a comparable performance as the latter Specifically all causal pathways in the simulated replicates had a pvalue below with the largest pvalue being × ˆ’ as determined by GSR using observed gene expression while no causal pathwayreached this level of significance with the smallest pvalue being × ˆ’ determined byGSEA We also compared the performances of GSR using observed gene expression to GSEAin various simulation settings and obtained consistent conclusions S3 Fig in S1 FileGene set enrichments in complex traitsApplying GSR to complex traits we revealed various pathways where the enriched gene setswere biologically meaningful For example the enriched gene sets for high density lipoproteinHDL predominantly involve lipid metabolism In contrast for Lupus gene sets wereenriched in interferon signalling pathways a known immunological hallmark We listed thetop enrichments over gene sets from MSigDB and Gene Ontology terms for HDL and theautoimmune trait Lupus in S1 Table in S1 FileAdditionally we applied GSR to test celltypespecific enrichments using cell types of which were derived from GTEx and cell types were derived from Franke lab datasets[] We observed biologically meaningful cell typespecific enrichment for the complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Comparison of pathway enrichment determined by GSR using or not using observed gene expression information and by GSEA NominalNOM pvalues yielded by GSEA were summarized Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g004traits Fig In particular central neural system cellspecific gene sets were enriched forschizophrenia immune cellspecific gene sets for lupus immune cellspecific and digestivetract cellspecific gene sets for Crohn™s disease and cardiac cellspecific gene sets for coronaryartery disease Lastly we correlated traits based on their gene set enrichments and observedmeaningful phenotypic clusters suggesting shared biological mechanisms by the related phenotypes S4 Fig in S1 File For example Crohn™s disease and ulcerative colitis two subtypes ofinflammatory bowel disease formed a cluster Neurological diseases schizophrenia and bipolardisorder formed a cluster Moreover lipid traits including LDL HDL and Triglyceridesformed their own clusterApplication on observed gene expressionLastly using expression profiles of BRCA THCA and PRAD from TCGA and GTEx [] wetested the enrichments of oncogenic gene sets as well as gene sets from BIOCARTAKEGG and REACTOME in each type of tumor Overall we observed a significantly strongerenrichments for the oncogenic signatures with higher p values compared to the more generalgene sets across all three tumour types ttest pvalue × ˆ’ × ˆ’ and × ˆ’for BRCA PRAD and THCA respectively S5 Fig in S1 File As a comparison we also ranstandard GSEA and observed qualitatively similar enrichments S5 Fig in S1 FilePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Celltypespecific enrichment of gene sets for representative complex traits GSR was applied to each complex trait in order toidentify significantly enriched gene sets among predefined celltypespecific gene sets represented by nine different colors Gene setswere indicated by dots and were aligned in the same order on the xaxis Red lines indecate Bonferronicorrected pvalue threshold 101371journalpone0237657g005DiscussionIn this work we describe GSR an efficient method to test for gene set or pathway enrichmentsusing either GWAS summary statistics or observed gene expression and phenotype information We demonstrate robust and powerful detection of causal pathways in extensive simulation using our proposed method compared to several stateoftheart methods Whenapplying to the real data we also obtained biologically meaningful enrichments of relevantgene sets and pathways These features warrant GSR a widely applicable method in variousstudy settings with an aim to interpret association test results and capture the underlying biological mechanismsOur approach has superior computational efficiency In particular GSR took only minutes running on the full summary statistics and less than minutes on the full gene expressiondata with one million SNPs and genes to test for enrichments of over gene sets Inour simulations it is not surprising that FOCUS can accurately finemap causal genes as thesimulation designs followed similar assumptions adopted by FOCUS [] However FOCUSis at least times slower than GSR For the simulated data FOCUS took minutes to finemap all of the genes in GWAS loci whereas GSR took under three minutes to test for pathwayenrichments on the same machine Additionally the computational cost of FOCUS is exponential to the number of causal genes considered within each locus whereas GSR is not affectedPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionby the number of causal genes Also because GSR operates at genomewide level no thresholdis needed to decide which genes to be included whereas FOCUS needs userdefined thresholdfor constructing the credible gene set for the subsequent hypergeometric enrichment testGiven these advantages we envision that GSR will be a valuable tool for the bioinformaticcommunity and statistical genetic community as a fast way to investigate the functional implications of complex polygenic traitsIn different simulation settings GSR exhibits improved pathway enrichment power overPASCAL and LDSC two popular methods for partitioning heritability and identifying causalgene sets Since GSR leverages SNPtogene association summaried by eQTL weights whileeither PASCAL or LDSC operate

Thyroid_Cancer

"Lactation has a negative effect on female sexual function Hormonal changes during lactation causechanges which might lead to dyspareunia lack of libido and anasmia There are various pharmacological andnonpharmacological approaches to treat sexual dysfunction While pharmacological treatment has multipleunwanted side effects nonpharmacological therapies such as complementary medicine are a potential saferalternative The aim of this study is to evaluate the effect of ear acupressure on sexual function of lactating womenMethodsdesign This is a randomized clinical trial with a parallel sham control group In this study lactatingwomen between months and year after childbirth were referred to health care centers in Qazvin City andwould be invited to participate Participants will be divided into intervention n and control n groupsusing simple block randomization Both intervention and sham control groups will be visited over sessionswithin a 4day interval At each visit the adhesives containing Vaccaria seed will be adhered for the interventiongroup while nonlatexbased adhesives with no Vaccaria seeds will be placed on the same ear acupoints for thesham control group Selected ear acupoints include genitalia two ear points pelvic point master shoulder andposterior pituitary gland The women will be asked to hold the seeds on their ears for days and press each earpoint three times a day for s After days they will be asked to remove the seeds from their ears and rest for day Sexual function as primary outcome in both groups will be assessed using the Female Sexual Function Indexbefore and immediately after and months after the intervention Also Sexual Quality of Life as secondaryoutcome will be assessed using Sexual Quality of LifeFemale SQOLF before and months after intervention Datawill be analyzed using repeated measure ANOVA at the significant level of Discussion This study is expected to support the impact of ear channel ear acupressure on sexual function inlactating womenTrial registration Iranian Clinical Trial Registration Center IRCT20190626044028N1 Registered on August Keywords Ear acupressure Sexual function Lactation Correspondence nbahramiqumsacir2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin IranFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBarghamadi Trials Page of BackgroundThe postchildbearing period is a crucial stage in thewoman™s life that involves physical hormonal mentalsocial and cultural changes [] Fatigue insomnia somestressors such as child care and changes in the bodyimage along with hormonal changes can reduce interestin sexual life and decrease the number of sexual intercourses during this period [] Endocrine function of lactation has a negative effect on sexual function becauseprolactin decreases sexual hormones such as androgenand estrogen This mechanism reduces sexual functionbecause of vaginal dryness vaginal epithelium atrophyand dyspareunia [] About two thirds of women duringlactating period experience at least one sexual problemsuch as decreased libido lack of sexual pleasure dyspareunia and vaginal dryness which usually are resolvedwithin year after childbirth [ ] The prevalence ofsexual dysfunction increases from to in the prepartum period [] to to in the postpartum period[] The results of various Iranian studies confirmed thehigh prevalence to of sexual dysfunctionduring lactation [ ] Therefore sexual dysfunction is acommon problem during lactation that needs moreattention from health care providersIn the postchildbearing period most women try to return to their sexual life within to weeks after childbirth Howeverit may take up to year to resumesexual relationships with the same quality of beforepregnancy [] Psychotherapy and pharmacotherapy aretwo main methods for treating female sexual dysfunctionFSD [] However the US Food and Drug Administration FDA does not recommend any foods or medicines for the treatment of FSD [] Therefore nonpharmacological therapies such as complementary medicine are a potentially safer alternative to pharmacologicaltherapy Acupressure is one of the noninvasive complementary methods oftraditional Chinese medicineTCM based on the principles of acupuncture []Acupuncture stimulates medical points and meridiansthat are distributed throughout the body to regulatephysiologic reactions [] Acupressurelike acupuncture modulates a person™s vital energy by stimulatingacupoints and meridians that are distributed throughoutthe body []The external ear is one of the several somatotopicmicrosystems that can be used in acupressure Ear acupressure also known as auriculotherapy or auricularacupressure [ ] was first presented in ancient Chinese medicine to years BC but Dr Paul Nogierwas the first Western physician who put forward auriculotherapy in a scientific way [ ] Accordingto this theory each part of the body is associated with aspecific part of the ear that reflects the physiological orpathological state of the body [] In this method theouter surface of the ear auricle can be stimulated to reduce pathological conditions in other parts of the body[] Ear stimulation can be performed in various wayssuch as manual finger pressure electrical stimulation lasers differenttypes of needles magnetic seeds andseeds to strengthen neural connections [] The WorldHealth anization WHO has recognized ear acupuncture as a promising therapeutic approach becauseof its efficacy in managing health disorders [] Ear acupuncture has been recognized as a form of acupuncturethat can affect all body ans [] Ear acupressure isan easy noninvasive and safe therapy [ ] It is easyto use for mothers who need to take care of their childin the postchildbearing period []There are several studies supporting the effects of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] However the effect of ear acupressure on sexualfunction has not been studiedResearch aimThis study is designed to investigate the effect of earacupressure on the sexual function of lactating womenMethodsDesign and settingThis is a randomized clinical trial with a parallel shamcontrolled group The study is designed in accordancewith the CONSORT standards Lactating women referred to health care centers in Qazvin City Iran will beinvited to participate Figure shows the CONSORTflow diagram The current protocol is anized basedon the SPIRITTCM extension []ParticipantsIn this study breastfeeding women between monthsand year after childbirth will be referred to health carecenters in Qazvin City Iran and they will be invited toparticipate in the study Inclusion criteria will be willingness to participate in the study being literate beingprimiparous breastfeeding fullterm singleton deliverylesionfree auricle no ulcer and pain in the ear and ability to present for all intervention sessions Exclusion criteria will be being away from their spouse for more than month having complications during pregnancy orhaving postpartum depression in the postchildbearingperiod These criteria were diagnosed using the Edinburgh Postpartum Depression Scale substance abuse inthe individual or the spouse and history of illnessesaffecting sexual function in a woman or her spouse egpremature ejaculation cardiovascular mental health disorders thyroid diseases any form of cancer or injuries 0cBarghamadi Trials Page of Fig The CONSORT flow diagramof the genital area The use of drugs affecting sexualfunction such as psychotropic cardiovascular neurological and hormonal drugs will also be excluded Afterthe initial screening the Demographic and ObstetricQuestionnaire and Female Sexual Function Index FSFIwill be completed by the participants before anyinterventionRole and qualification of practitionersThe research team consists of one specialist in auriculotherapy TO two specialists in reproductive health ZAand NB and one midwifery postgraduate student Theprotocol of intervention was designed based on the literature review and expert opinion of TO who is an expert inauriculotherapy and is a journal editor for several international journals Other team members have been qualified in this technique before designing present researchParticipant screening and care providing will be done bySB and ZA and NB will monitor her during the first sessions to ensure the fidelity of providing interventionSample size calculationSample size is estimated according to the study ofBokaie with the mean and standard deviation of 0cBarghamadi Trials Page of total sexual function score has been reported as ± and ± in the intervention telephonecounseling group and control groups respectively []type error α Therefore considering the first confidence the second type error β power and error d and the possibility of loss tofollowup individuals for each group are requiredSo the total sample size will be peopleInterventionEar acupressure groupThe researcher will clean the auricle using alcoholand then place Vaccaria seeds using nonlatexbased adhesives on ear acupoints for genitalia pelvis shoulderand the posterior pituitary gland Figure illustrates theintended ear acupoints Intervention is designed for sessions within a 4day interval During these sessionsthe seeds are placed on the designated ear acupressurepoints The women will be instructed to keep the seedson their ears for days during which each point shouldbe compressed three times a day for s The compression should be performed with moderate stimulationthrough pressing steadily and slightly tighter until feelinga slight tingling and discomfort After days they willbe asked to remove the seeds from their ears and restfor a day [] The women will be reminded daily bysending the text and will be reminded by phone for thenext interventional session This procedure will be performed for ten sessions for each participant It should benoted that if a participant has problems during the useof the method for any reason displacement of seedsdiscomfort etc she can remove the previous seed andask the researcher to reattach new seedsSham control groupThe control group will have sessions that will be thesame as the intervention group except that no Vaccariaseeds will be placed on the ear acupointsMeasuresIn this studycollectionfour measures will be used for dataDemographic and Obstetric QuestionnaireThe demographic section includes questions such asage education level occupation place of residence economic status age at marriage and length of marriageThe obstetric section includes questions regarding thenumber of pregnancies type of contraceptive methoddelivery date type of delivery perinealinstrumental delivery history of painful intercourse in prenatalperiod infant gender birth weight first lactation number of intercourse per month before pregnancy onset offirst intercourse after delivery and average number ofintercourses per week during lactation Validity of thisquestionnaire will be confirmed by some of the facultymembers ofthe midwifery department of QazvinUniversity of Medical Sciences IraninjuryFig The selected ear acupoints 0cBarghamadi Trials Page of Female Sexual Function Index FSFIIt has been designed by Rosen to evaluate sexualfunction in women over the past weeks [] It consistsof items subdivided into six subcategories of sex desire items arousal items lubrication items asm items satisfaction items and pain itemsThese subcategories have response ranges from or to with higher scores indicating better sexual performance [] The questionnaire has been used in manystudies abroad and has shown to have a high degree ofinternal consistency reliability and validity [] Thefindings of Fakhri and Mohammadi™s study showed thatthe Farsi version of FSFI FSFIIV is a reliable and validinstrument with appropriate psychometric properties toevaluate women™s sexual function [ ] Reliability ofthe scale has been calculated through stability analysisor calculation of the internal consistency coefficient TheCronbach™s alpha coefficient was and was higher forall domains and for the whole scale [] In addition reliability was confirmed by the testretest method and thecorrelation coefficient was reported high indicating thatFSFIIV is reusable within a 4week interval [ “]The maximum score for each domain is and for thewhole scale is Also any score less than will beused to indicate sexual dysfunction []The Edinburgh Postpartum Depression Rating ScaleThis 10item scale has been designed to detect depression from weeks after delivery The Edinburgh Scalescores vary between and with cutoff point andabove to detect postpartum depression [] Psychometric evaluation of the Farsi version of this scale is carriedout in [] The Cronbach™s alpha for the Edinburgh Scale has been reported as Validity of the Edinburgh Beck Scale has been reported as Thefindings of the study confirmed the high validity of theFarsi version of the Edinburgh Scale for the diagnosis ofpostpartum depression []The Sexual Quality of LifeFemale SQOLFIt is a short tool that specifically assesses the relationshipbetween sexual function and women™s quality of life Ithas been developed by Symonds [] This 18itemquestionnaire focuses on sexual selfesteem emotionalaffairs and relationships Each item is responded with aLikert scale of strongly agree score to strongly disagree score Higher scores indicate better quality ofsexual life for women [] Validity and reliability of theFarsi version ofthis scale have been confirmed byMaasoumi []Primary outcomePrimary outcome of this study is to investigate changesof sexual function using the FSFISecondary outcomeThe secondary endpoint is investigating the quality ofwomen™s sexuallife which will be assessed using theSexual Quality of Life Questionnaire Any harm or sideeffects will be also reportedStudy procedureAfter obtaining the necessary permissions from the Ethics Committee of Qazvin University of Medical Sciencesregistration at the Iranian Clinical Trial RegistrationCenter IRCT and obtaining the permission from Qazvin Nursing and Midwifery School the researcher willattend for recruitment in Qazvin Health Centers Thelactating women of each center will be invited for participation The process and purpose of the research willbe explained to those who meet the inclusion criteriaAlso they will be ensured that their information will bekept confidential and that the questionnaires will be anonymous and coded in accordance with the researchethics They will enter into the study after signing thewritten consent form The researcher will ensure thatthey can leave the study at any time After enrolling eligible individuals a simple block randomization will beused Participants will be randomly assigned to the intervention and control groupsThe questionnaires will be filled out by the participants before the intervention Then the interventionacupressure will be performed as mentioned aboveEvaluation of sexual function in both the interventionand control groups will be performed using the FSFIquestionnaire immediately and months after theintervention Figure provides the timeline of recruitment allocation and assessmentStatistical analysisData will be analyzed using the chisquare test Fisherexact test and independent t test via the SPSS v24 software The KolmogorovSmirnov and Shapiro tests willalso be used to check for the normal distribution of sexual function scores Descriptive statistics number meanand standard deviation will be used to describe thedemographic characteristics of the participants The repeated measure ANOVA will be used to compare themean sexual function of women before and after theintervention The significance level of all tests is set asp Methods to protect against biasRandomization and allocationSampling will be made using a twostep samplingmethod In the first step Qazvin City will be consideredas five geographical districts Next two health centerswill be randomly selected in each of these five districtsof Qazvin City Secondly women referred to selected 0cBarghamadi Trials Page of STUDY PERIODEnrolment AllocationPostallocationCloseoutTIMEPOINT1t1t2t3t4t5t6t7t8t9t10t11t12t13tXENROLLMENTEligibility screenInformed consentRandomized by independent personAllocationINTERVENTIONSGroup A Auricular acupressureGroup B ControlASSESSMENTSFSFISQOLXXXXXXX X XXFig Schedule of enrolment interventions and assessmentshealth centers will be assessed for eligibility and in caseof willingness to participate in the study will be recruited They will be randomly assigned to two groupsas follows One letter is assigned to each group A intervention group B control group and all possible conditions for the 4block will be written and numbered asfollows 1AABB 2ABAB 3BBAA 4BABA 5ABBA6BAAB In a simple block randomization method using thetable of random numbers numbers of blocks will be selected until the specified sample size is achieved Therandom allocation sequence is specified For example ifthe numbers given are and respectively theallocation sequence will be as follows AABB ABABABAB BBAA As a result each participant will have aunique code n Allocation concealmentFor this purpose after preparing the allocation sequencethe sequence is annotated on paper and placed inopaque sealed envelopes which will be numbered consecutively The questionnaires will be coded in the samemanner A questionnaire with the same code will becompleted by the person who receives code intervention The assignment sequence and its concealment willbe performed by someone outside the research teamBlindingParticipants outcome assessor and statistical analyzerwill be blind to which group a participant was placedThe individual entering the data from the participantquestionnaires on demographic information postpartumdepression female sexual functioning and female qualityof life will be blind to which participant was assigned tothe auricular acupressure seed group or the sham control groupTreatment fidelityOne of the researchers SB is responsible to performintervention She has participated specific course on auriculotherapy and is supervised by acupuncturist MHAabout how to perform the intervention Approximately of intervention sessions will be run under supervision of the acupuncturist to ensure that all acupoints arechosen correctlyData managementOne of the researchers SB will be responsible for collecting data at each study stage and will assign anotherindividual for the task of data entry into the SPSS software This process will be performed under supervisionof NB and ZA 0cBarghamadi Trials Page of Ethical and safety issuesThe research protocol will be reviewed and approved bythe Ethics and Human Research Committee of QazvinUniversity of Medical Sciences decree code IRQUMSREC1398056 It is registered on the Iranian ClinicalTrial Registration Center underofIRCT20190626044028N1 In addition the following ethical considerations will be considered throughout this research obtaining written informed consent the volunteernature of participation in the study ability to withdrawfrom the study at any time and confidentiality of information even during the publication of findings All ethical issues related to clinical trials will be considered Informedconsent during recruitment phase will be obtained by SBdecreecodeDiscussionTo the best of our knowledge this study is the first randomized clinical trial investigating the effect of ear acupressure on the sexual function of lactating women Thestrength of this study is the randomized design with parallel treatment procedures and a large sample size butdue to the importance of the treatment practitionerknowing whether they are placing an adhesive patchwith a Vaccaria seed or without one a true double blindstudy is not possible Acupressure has no known side effects is noninvasive and is easy to use [ ] Therefore the therapist and clients can develop a sense ofclosenesstrust and confidence [] Several studieshave shown promising results about the effect of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] In the study of Oakley acupuncture wasaffective in premenopausal women with sexual function[] In line with previous literature this study wouldprovide insights about the impact of ear acupressure onsexual function in lactating womenAbbreviationsCONSORT Consolidated Standards of Reporting Trials SQOLF Sexual Qualityof LifeFemaleAcknowledgementsNot any in this stageTrial statusThe recruitment has not yet begun and necessary permissions are acquiredthe estimated date of recruitment is November The expected timefor completing the recruitment is March Protocol version registered on August Authors™ contributionsAll authors SB ZA NB and TO contributed to the design of the study SBNB and ZA drafted the preliminary manuscript TO revised the manuscriptand prepared the final version of the manuscript All authors revised themanuscript agreed to be fully accountable for ensuring the integrity andaccuracy of the study and read and approved the final version of themanuscript to be published All the authors met the criteria for authorshipand listed as coauthors on the title pageFundingThe research deputy of Qazvin University of Medical Sciences will providefinancial support Study funders have no role in the study design thecollection management analysis and interpretation of data the writing ofthe report and the decision to submit the report for publicationAvailability of data and materialsAnalyzed data and materials will be deidentified and publishedEthics approval and consent to participateResearch protocol is approved by the Ethics and Human ResearchCommittee of Qazvin University of Medical Sciences decree codeIRQUMSREC1398056 Permissions from responsible authority will beobtained before recruitment Informed consent will be acquired beforeparticipationConsent for publicationNot applicableCompeting interestsNone to declareAuthor details1Student Research Committee Qazvin University of Medical Sciences QazvinIran 2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin Iran 3Emperor™s College of TraditionalOriental Medicine Santa Monica CA USAReceived November Accepted August ReferencesAbdelhakm EM Said AR Elsayed DMS Effect of PLISSIT model sexualcounseling program on sexual quality of life for postpartum women Am JNurs Sci Avery MD Duckett L Frantzich CR The experience of sexuality duringbreastfeeding among primiparous women J Midwifery Womens Health“Acele EÖ Karaçam Z Sexual problems in women during the firstpostpartum year and related conditions J Clin Nurs ““ Mohammed YF Hassan HM AlDinary AM Rashed NS Sexual function afterchild birth according to the mode of delivery AAMJ “Si H Kumamoto Y Sato Y Masumori N Horita H Kato R Prevalenceof female sexual dysfunction symptoms and its relationship to quality of lifea Japanese female cohort study Urology “ Williams A HerronMarx S Carolyn H The prevalence of enduring postnatalperineal morbidity and its relationship to perineal trauma Midwifery “Ahmad Shirvani M Bagheri NM Sexual dysfunction and related factorsamong breast feeding women Iran J Obstet Gynecol Infertility “Tork Zahrani S Banaei M Ozgoli G Azad M Investigation of the postpartumfemale sexual dysfunction in breastfeeding women referring to healthcarecenters of Bandar Abbas Iran J Obstet Gynecol Infertility “Yörük F Karaçam Z The effectiveness of the PLISSIT model in solvingpostpartum sexual problems experienced by women Athens J Health “ Davis SR Van Der Mooren M van Lunsen RH Lopes P Ribot J Rees M et alEfficacy and safety of a testosterone patch for the treatment of hypoactivesexual desire disorder in surgically menopausal women a randomizedplacebocontrolled trial Menopause “ Belkin ZR Krapf JM Goldstein AT Drugs in early clinical development forthe treatment of female sexual dysfunction Expert Opin Investig Drugs“ Ozgoli G Mobarakabadi SS Heshmat R Majd HA Sheikhan Z Effect of LI4and BL32 acupressure on labor pain and delivery outcome in the first stage 0cBarghamadi Trials Page of of labor in primiparous women a randomized controlled trial ComplementTher Med “ Oleson T Auriculotherapy manual Chinese and Western systems of ear Mohammadi KH Heydari M Faghihzadeh S The Female Sexual FunctionIndex FSFI validation of the Iranian version Health Monit J Iran Inst HealthSci Res Burri A Cherkas L Spector T Replication of psychometric properties of theFSFI and validation of a modified version FSFILL assessing lifelong sexualfunction in an unselected sample of females J Sex Med “Sidi H Abdullah N Puteh SEW Midin M The female sexual function indexFSFI validation of the Malay version J Sex Med “Sun X Li C Jin L Fan Y Wang D Development and validation of Chineseversion of female sexual function index in a Chinese population”a pilotstudy J Sex Med “Ter Kuile MM Brauer M Laan E The female sexual function index FSFI andthe female sexual distress scale FSDS psychometric properties within aDutch population J Sex Marital Ther “ Cox JL Holden JM Sagovsky R Detection of postnatal depressiondevelopment of the 10item Edinburgh Postnatal Depression Scale Br JPsychiatry “ Ahmadi kani Golzar A GoliZadeh Z Validation of Edinburgh PostpartumDepression Scale EPDS for screening postpartum depression in Iran J NursEduc “Symonds T Boolell M Quirk F Development of a questionnaire on sexualquality of life in women J Sex Marital Ther “ Maasoumi R Lamyian M Montazeri A Azin SA AguilarVafaie ME HajizadehE The sexual quality of lifefemale SQOLF questionnaire translation andpsychometric properties of the Iranian version Reprod Health Oakley SH WaltherLiu J Crisp C Pauls R Acupuncture in premenopausalwomen with hypoactive sexual desire disorder a prospective cohort pilotstudy Sexual medicine 201643e176“e81Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacupuncture Yeh ML Chang YC Huang YY Lee TY A randomized controlled trial ofauricular acupressure in heart rate variability and quality of life forhypertension Complement Ther Med “Tan JY Molassiotis A Wang T Suen LK Current evidence on auriculartherapy for chemotherapyinduced nausea and vomiting in cancer patientsa systematic review of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chien LC Chiang YC Lin SW Huang CK Ren D Reduction innausea and vomiting in children undergoing cancer chemotherapy byeither appropriate or sham auricular acupuncture points with standard careJ Altern Complement Med “ Abbate S Chinese auricular acupuncture Florida Routledge Yeh TL Chen HH Pai TP Liu SJ Wu SL Sun FJ The effect ofauricular acupoint stimulation in overweight and obese adults a systematicreview and metaanalysis of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chiang YC Hoffman SL Liang Z Klem ML Tam WW Efficacyof auricular therapy for pain management a systematic review and metaanalysis Evid Based Complement Alternat Med Kim JY Ryu HS Nam SH Park KS Effects of auricular acupressure therapy onnocturia and insomnia in the elderly Korean J Rehabil Nurs “ Wang YJ Hsu CC Yeh ML Lin JG Auricular acupressure to improvemenstrual pain and menstrual distress and heart rate variability for primarydysmenorrhea in youth with stress Evid Based Complement Alternat MedShergis JL Ni X Jackson ML Zhang AL Guo X Li Y A systematicreview of acupuncture for sleep quality in people with insomniaComplement Ther Med “ Dantas KKdL Auriculoterapia chinesa com o uso de sementes de colza nadismenorreia primaria relato de caso Universidade Federal do Rio Grandedo Norte Portman DJ Bachmann GA Simon JA Group OS Ospemifene a novelselective estrogen receptor modulator for treating dyspareunia associatedwith postmenopausal vulvar and vaginal atrophy Menopause “ Oleson T Flocco W Randomized controlled study of premenstrualsymptoms treated with ear hand and foot reflexology Obstet Gynecol“Kwon SJ Park JS Effects of auricular acupressure on chemotherapyinducednausea vomiting and serum serotonin level Korean J Adult Nurs “ Di YM May BH Zhang AL Zhou IW Worsnop C Xue CC A metaanalysis ofearacupuncture earacupressure and auriculotherapy for cigarette smokingcessation Drug Alcohol Depend “ Huang ETY Di PhD YM Acupuncture therapies for chronic obstructivepulmonary disease a systematic review of randomized controlled trialsAltern Ther Health Med Dai L Cheng CW Tian R Zhong LL Li YP Lyu AP Standard ProtocolItems for Clinical Trials with Traditional Chinese Medicine recommendations explanation and elaboration SPIRITTCM extension Chin J Integr Med “ Bokaie M Hajimaghsoudi S Dehghani A Hosseini F The effect of sexualhealth counselling on the sexual function and satisfaction of breastfeedingwomen in the form of group consultation and telephone consultancyJ Adv Pharm Educ Res 20199S2191“ Rosen CB Heiman J Leiblum S Meston C Shabsigh R Ferguson DD'Agostino R The Female Sexual Function Index FSFI a multidimensionalselfreport instrument for the assessment of female sexual function J SexMarital Ther “Fakhri A Pakpour AH Burri A Morshedi H Zeidi IM The Female SexualFunction Index translation and validation of an Iranian version J Sex Med“ Wiegel M Meston C Rosen R The female sexual function index FSFI crossvalidation and development of clinical cutoff scores J Sex Marital Ther“ 0c"

Thyroid_Cancer

Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c'

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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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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

Hepatocellular carcinoma HCC is a high mortality disease the fifth most general cancer worldwide and the second leading to cancer‘related deaths with more than new patients diagnosed each year First the high expression of centromere M CENPM in mammary gland tissue of b‘catenin transformed mice was identifiedMaterials and methods In our study we evaluated the expression of CENPM in hepatocellular carcinoma based on data obtained from an online database Multivariate analysis showed that the expression of CENPM and M classifica‘tion was an independent prognostic factor for patients with hepatocellular carcinomaResults Survival analysis showed that patients with high CENPM had a worse prognosis than patients with low CENPM P A multivariate Cox regression hazard model showed that B cells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P Using the network the most frequently changed neighbor genes of CENPM were shown and the most common change was RAD21 Conclusion Our study found that the expression of CENPM was significantly increased in patients with hepatocellu‘lar carcinoma and it was related to a variety of clinical characteristics its correlation with the level of immune infiltra‘tion and poor prognosis so CENPM can be used as a useful prognosis for patients™ markers and HCCKeywords Hepatocellular carcinoma Centromere protein M Data mining PrognosisBackgroundHepatocellular carcinoma HCC a high mortality disease which is the fifth most general cancer in the world and the second most common lead to cancerrelated deaths with over new patients diagnosed each year [ ] Viral hepatitis and nonalcoholic steatohepatitis are the most common causes of cirrhosis and approximately of cases develop to HCC [] Due to the recurrence of HCC the prognosis of HCC remains discouraging and the 5year overall survival rate which Correspondence wawang123soutlookcomDepartment of Infectious Diseases Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Chinais only to [] Despite the rapid development of advanced medical technology there are still no useful curable strategies for HCC patients [] Byeno et a0al [] reported that based on longterm survival data serum OPN and DKK1 levels in patients with liver cancer can be deemed as novel biomarkers that show prognostic useful for liver cancer Other serum markers such as alphafetoprotein AFP and alkaline phosphatase ALP or AKP are proverbially used in clinical but they lack sufficient sensitivity and specificity [] Therefore finding useful biomarkers is indispensable for diagnosis and treatment for HCC patientsPosttranscriptional modifications are essential for tumorigenesis and development Centromere protein M CENPM otherwise called PANE1 CENPM and 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 0cWu a0and Yang Cancer Cell Int Page of C22orf18 which encodes a kinetic protein binds to spindle microtubules to regulate chromosomal separation during cell division [] Expression of the PANE1 gene was found preferentially in immune cells involving tumor tissues and tumor derived cell lines and leukemias and lymphomas [] Brickner et a0 al [] found highly expressed in B lineage chronic lymphocytic leukemia BCLL cells and resting CD19 B cells may be a potential therapeutic target for BCLL Bierie et a0al [] also demonstrated that human CENPM transcript cRNA was detected only in a0vivo or in a0vitro in activated B cells and T cells These studies suggested CENPM may play critical role in tumor immune response and may be deemed to therapeutic target for immunotherapy However the role of CENPM in HCC prognostic remains unclear In our study we evaluated the expression of CENPM in HCC based on data from an online database to further understand the biological pathway of CENPM related to the pathogenesis of HCC In addition we also analyzed the connection between CENPM expression and clinical features as well as the correlation of its expression with immune infiltration level in HCC comes an online tumor infiltrating immune cells analysis toolMaterials and a0methodsData collectionInformation on RNAsequencing data tissues workflow type HTSeqCounts and comparative clinical data patients data format BCR XML were identified and got from the level standardized FPKM of the TCGAHCC cohort Use boxplots to imagine expression differences for discrete variables [] The clinical factors included gender stage age grade Tphase Mphase Nphase survival status and number of days of survival Data analysis were checked by R version and R Bioconductor software packagesGSEA enrichmentGene Set Enrichment Analysis GSEA created a list of all gene permutations related to CENPM expression The samples were then divided into a high CENPM group and a low CENPM group as training sets to distinguish potential functions and use GSEA to clarify significant survival differences Genome replacement is performed multiple times with each exam The degree of expression of CENPM was used as a phenotypic marker Normalized enrichment scores NES and nominal Pvalues have been used to classify the pathways of enrichment in each phenotypeImmune infiltrates analysisTIMER [] is a comprehensive database for the systematic study of immune infiltration in various malignant tumor types The abundance of immune infiltrates CD8 T cells B cells CD4 T cells macrophages neutrophils and dendritic cells was evaluated by our statistical methods and has been estimated using pathology Methods evaluated it The network also enables users to explore the clinical relevance of one or more tumor immune subpopulations and has the flexibility to correct multiple covariates in a multivariate Cox proportional hazard model Meanwhile we contrast the differential level of CENPM between tumors and normal on all TCGA tumorsUALCAN and a0c‘BioPortal analysisUALCAN [] is a userfriendly intelligent network asset for analyzing discovering cancer data and indepth analysis of TCGA gene expression information One of the highlights of the portal is that it allows users to found between biomarkers or computer approval of potential genes of interest and to evaluate genes in different clinical subgroups such as gender age race tumor grade etc expression cBioPortal [] is an online free asset that can visualize analyze and download largescale cancer transcription datasets The portal included cancer studies The tab biological interaction network of CENPM and its coexpressed genes was got and neighboring genes with altered frequencies were containedTargetScan analysisTargetScan [] is a web for predicting potential biological targets of miRNAs TargetScanHuman deems that the match to human ²UTR and its orthologs is estimate by a UCSC genomewide adjustment As an alternative they are ranked according to their predicted conservative positioning possibilities FunRich [] is a tool designed to process varieties of geneprotein datasets in spite of the anism and used for functional enrichment analysis We used Funrich tools for miRNA enrichment analysis including analysis of biological pathways biological processes BP cellular components CC and molecular functions MFStatistical analysisScatter plots and paired plots visualize the differences between normal and tumor samples Use delete ways to handle disappeared data and if any individual value is disappeared the data will exclude the full sample The relationship between clinical factors and CENPM was used by logistic regression Wilcoxon rank sum test and Kruskal test Multivariate Cox analysis was used to assess the effect of CENPM expression on survival and other clinical factors such as age gender stage distant metastasis Benjamini“Hochberg™s means of converting P values to FDR 0cWu a0and Yang Cancer Cell Int Page of ResultsPatients™ characteristicsThe TCGA database contains patients The clinical and pathological properties of these samples are shown in Table a0 The middle age at diagnosis in TCGA was a0 years old range “ a0 years and median finally contact for subjects was a0 months range “ a0months Meanwhile followup for subjects conformed alive and death patients Our study cohort included female and Table TCGA hepatic carcinoma patient characteristicsClinical characteristicsAge at diagnosis yearFutime monthGender Female MaleStage I II III IV NAGrade G1 G2 G3 G4 NAT‘classification T1 T2 T3 T4 TX NAM‘classification M0 M1 MXN‘classification N0 N1 NX NAStatus Alive DeathData express as mean min“maxTotal “ “ male patients Stage I was located in patients stage II in stage III in and stage IV in Tumor stage was found T1 in patients T2 in T3 in and T4 in Node stage contained N0 in and N1 in of cases had distant metastases All the subjects were adenomas or adenocarcinomasCENPM expression and a0clinical factorsScatter plot showing difference in CENPM expression among normal and tumor samples P we then use paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P Fig a01a b The outcomes suggested that the expression of CENPM was significant difference The expression of CENPM correlated significantly with the patient grade P clinical stage P and Tclassification P Fig a01d“f Univariate analysis utilizing logistic regression uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors Table a0 CENPM expression in HCC as appreciably connected with grade OR CI “ G1 vs G3 stage OR CI “ I vs III and Tclassification OR CI “ T1 vs T3 indicated that patients with low CENPM expression are inclined to advance to a further advanced stage than those with high CENPM expressionSurvival and a0multivariate analysisSurvival analysis found that HCC with CENPMhigh had a worse outcome than that with CENPMlow P Fig a0 1c The univariate analysis suggested that CENPM linked essentially to stage HR CI “ P and Tclassification HR CI “ P Table a0 Multivariate analysis showed that the expression of CENPM HR P and M classification HR P were independent prognostic factors for patients with HCC Table a0GSEA analysisTo identify useful pathways that may be differentially initiated in liver cancer we performed a GSEA analysis between low and high CENPM expression datasets We chose the most abundant signaling pathway depending on the standardized enrichment score NES Table a0 The results showed that CENPM high expression differentially enriched cell cycle DNA replication RNA degradation certain cancers phagocytosis P53 signaling pathway and purine metabolism Fig a0 0cWu a0and Yang Cancer Cell Int Page of Fig CENPM expression and the association among clinicopathologic factors a The scatter plot showed the difference of CENPM expression between normal and tumor samples P b paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P c Survival analysis P d Grade e Stage f T‘stageTable CENPM expression associated with a0pathological characteristics logistic regressionclinical Clinical characteristicsTotal NAge vs ‰¤ Gender female vs maleGrade G1 vs G3Stage I vs IIIT‘stage T1 vs T3Odds ratio in a0CENPM expression “ “ “ “ “P‘valueCategorical dependent variable greater or less than the median expression levelImmune infiltrates related to a0CENPM in a0HCCThe correlation between CENPM liver cancer in expression and the abundance of immune infiltrates was statistically significant P Fig a0 3a A multivariate Cox proportional hazard model showed that Bcells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P indicating that these immune cells significantly affect the prognosis it is worth further research and exploration Table a0 At the same time the expression of CENPM was also statistically significant P Finally we compared CENPM expression between various tumors and normal tissues The results showed that CENPM was overexpressed in various cancers P Fig a03bAssociations survival Table a and a0 clinicopathologic characteristics in a0 TCGA patients using Cox regression b Multivariate survival model after a0variable selectionwith a0overall Clinicopathologic variableHR CIP‘valuea Age continuous Gender female vs male Stage IIIIIIIV Grade G1G2G3G4 T‘classification T1T2T3T4 Distant metastasis M0M1MX Lymph nodes N0N1NX CENPM expression high vs lowb Distant metastasis M0M1MX CENPM expression high vs low “ “ “ “ “ “ “ “ “ “UALCAN and a0c‘BioPortal analysis in a0HCCIn the age subgroup normal age “ a0years normal age “ a0years normal age “ a0years and normal age “ a0 years among patients with liver cancer CENPM has substantially higher transcription levels than healthy individuals Analysis in the weight subgroup gender subgroup ethnicity subgroup tumor grade subgroups analysis also showed significantly higher CENPM in HCC patients Fig a0 In order to determine the 0cWu a0and Yang Cancer Cell Int Page of Table Gene sets enriched in a0phenotype highGene set nameKEGG_CELL_CYCLEKEGG_DNA_REPLICATIONKEGG_RNA_DEGRADATIONKEGG_BLADDER_CANCERKEGG_NON_SMALL_CELL_LUNG_CANCERKEGG_THYROID_CANCERKEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSISKEGG_P53_SIGNALING_PATHWAY KEGG_PURINE_METABOLISMSizeNESNOM P‘valFDR q‘valNES normalized enrichment score NOM nominal FDR false discovery rate Gene sets with NOM Pval and FDR qval are considered as significantbiological interaction network of CENPM in liver cancer we used the network in the Network tab in cBioPortal showing the most frequently changed neighbor genes in CENPM and the most common change was RAD21 Fig a0 and Table a0miRNAs related to a0CENPMAccording to the online database the top of the miRNA families are hsamiR13075p hsamiR449b3p and hsamiR67785p related to the gene CENPM The conserved sites of the miRNA family that are widely conserved in vertebrates Fig a06a Using the Funrich database to explore the function of the identified miRNAs BP are significantly enriched in the regulation of nucleobases signal transduction cell communication transport regulation of gene expression and anogenesis CC are mainly concentrated in the nucleus cytoplasm Golgi apparatus endosome actin cytoskeleton and early endosome The MF are mainly transcription factor activity transcription regulation activity protein serine GTPase activity and ubiquitinspecific protease activity rich biological pathways in the ErbB receptor signaling network TRAIL signaling pathway Glypican pathway and syndecan1 mediated signaling events and signal transduction events mediated by hepatocyte growth factor receptor cMet Fig a06b“eDiscussionIn this work we performed a detailed assessment of CENPM expression in hepatocellular carcinoma based on the TCGA database and explored its relationship with clinicopathological features survival function immune infiltration and expression differences Understanding whether higher expression biomarkers in tumors are directly related to hepatocellular carcinoma can help us understand the mechanism of the observed clinical survival patterns In our findings the significant expression of CENPM suggests that CENPM may play an important CENPM is an role in regulating cancer progression This should draw attention to current views on the improvement of liver cancer and may reveal potential biomarkers or indicators to determine prognosisindispensable centromere protein involved in centromere assembly which regulates mitochondrial protein assembly and chromosome segregation [] Huang et a0 al [] cloned and identified the cDNA sequence of porcine PANE1 and found that porcine PANE1 gene was expressed differently in seven different tissues with the highest expression in lymph nodes and the lowest expression in kidney Until now the expression of CENPM and its potential prognostic effect on hepatocellular carcinoma has not yet been investigated our outcomes showed that the expression of CENPM in hepatocellular carcinoma was related to advanced clinical pathologic factors grade clinical stage Tclassification survival time and poor prognosis Univariate analysis uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors and Mclassification may play an indispensable role in the inclined to advance to a further advanced stage The univariate and multivariate analysis also suggested CENPM still remained freely connected with OS and recommended that CENPM may act as a potential prognostic biomarker of prognosis and therapeutic target in hepatocellular carcinoma but more researches needed to conduct for further study In addition we further analyzed various clinicopathological features of HCC samples using the UALCAN database and all of them showed high transcription of CENPMTo identify differential signaling pathways in liver cancer GSEA analysis results show that cell cycle DNA replication RNA degradation some cancers phagocytosis P53 signaling pathway and purine metabolism are differentially enriched in CENPM high expression phenotype CENPM may influence cell cycle DNA replication RNA degradation then controls the begins and development 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment plots from gene set enrichment analysis GSEAof cancer cells Kim et a0al [] was identified CENPM as a key gene that mediates the anticancer effect of garlic and cisplatin on bladder cancer and showed that patients with low CENPM expressed better progressionfree survival than patients without high expression Studies also found the CENPM genes encode a human minor histocompatibility antigen expressed by tumor cells [ ] Yu et a0al [] found CENPM could as AFPrelated diagnostic biomarkers in HCC and validate the results using quantitative realtime PCR Our study for the first time investigated the CENPM mRNA expression and its prognostic significance in hepatocellular carcinoma Chen et a0al [] demonstrated that LHX6 can inhibit the proliferation invasion and migration P53 signaling pathways during hepatocarcinogenesis Qin et a0 al [] found that P53stabilizing and activating RNA can strengthen the interaction between hnRNP K and P53 which ultimately leads to the accumulation and transactivation of P53 So CENPM may play a role via P53 signaling pathway and more researches needed to conduct in the future 0cWu a0and Yang Cancer Cell Int Page of Fig Immune infiltrates correlation with CENPM in HCC a Correlation between CENPM in HCC expression and abundance of immune infiltrates P b CENPM expression between various tumor and normal tissueTable Multivariate survival model analysis based on a0TIMER online toolClinicopathologic variableCoefHR CIP‘value SigAgeGender MaleRace BlackRace WhiteStage IIStage IIIStage IVPurityB cellsCD cellCD4 T cellsMacrophages “ˆ’ “ “ˆ’ “ “ “ “ “ˆ’ “ˆ’ “ˆ’ “ “NeutrphilsDendriticCENPMPvalue significant codes ‰¤ ‰¤ ‰¤ ‰¤ · ˆ’ “ “ “·Previous studies demonstrated that human CENPM transcript cRNA was only detected in activated B and Tcells either in a0vivo or in a0vitro These studies suggested CENPM may play important role in tumor immune response so we used an online tool to analysis immune infiltrates correlation with CENPM in HCC Multivariable Cox proportional hazard model showed that B cells CD8 T cells macrophages and dendritic cells of immune infiltrates statistically significant P in HCC indicating that these immune cells significantly affecting the prognosis A latest study showed CD8 CD68 and FoxP3 immune cells were associated with HCC particularly in the invasive margin [] Macrophages not only promote the proliferation colony formation and migration of HCC cells but also maintain tumor growth and metastasis by secreting hepatocyte growth factor HGF [] Pang et a0 al [] proposed that fusion of dendritic cells DC with tumor cells can effectively activate antitumor immunity in the body and affect tumor progression [] These studies indicate that CENPM may play an important role in tumor immune response and can be a good therapeutic target for immunotherapy 0cWu a0and Yang Cancer Cell Int Page of Fig Boxplot showing relative expression of CENPM in subgroups of patients with HCC UALCANTo determine the biological interaction network of CENPM in liver cancer we applied the most frequently changed neighbor genes of CENPM on the Network tab in cBioPortal and the most frequent change was RAD21 RAD21 is a nuclear phosphoprotein which becomes hyperphosphorylated in cell cycle M phase One study found that depletion of RAD21 resulted in reduced levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters resulting in enhanced selfrenewal of hematopoietic stem and progenitor cells HSPC [] Recent studies have shown that removing RAD21 in a background lacking Pds5 can rescue the phenotype observed only in the absence of Pds5 [] Our study may provide information on adhesion kinetics in replication fork studies in patients with liver cancer Our study also used the Targetscan online tool to distinguish CENPMrelated miRNAs To check the function of the identified miRNAs bioenrichment was performed through the Funrich database It is rich in ErbB receptor signaling network TRAIL signaling pathway Glypican pathway syndecan1 mediated signaling events and biological pathways of hepatocyte growth factor receptor cMet signaling events Studies have reported that selective cMet inhibitors have antitumor activity in HCC and have acceptable safety and tolerability in Child“Pugh A liver function patients [] A recent study found that abnormal HGFcMet upregulation and activation are often observed in bladder cancer [] Studies have also found that metastasis associated with colon cancer MACC1 regulates PDL1 expression and tumor immunity in gastric cancer GC cells through the cMetAKTmTOR pathway [] We hypothesized that CENPM may regulate the expression of cMet leading to the occurrence of HCC and more related research Fig The network for CENPM and the most frequently altered neighbor genesTable The type and a0frequency of a0CENPM neighbor gene alterations in a0HCC cBioPortalGene symbolRAD21RPS27AHCTF1NUF2PMF1Amplification Homozygous deletionMutation Total alteration 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment analysis of the miRNA altered in the CENPM in HCC Funrich and Targetscan a Conserved sites for miRNA families broadly conserved among vertebrates b Cellular components c KEGG pathway analysis d Biological processes e Molecular functionsis needed To date this study demonstrates for the first time the important role of CENPM in the prognosis of hepatocellular carcinoma However future clinical trials are needed to validate these results and promote the use of CENPM in the prognostic evaluation of hepatocellular carcinomaConclusionsOur study found that the expression of CENPM was significantly increased in patients with hepatocellular carcinoma and was related to a variety of clinical features its correlation with the level of immune infiltration and poor prognosis so CENPM may become a useful biomarker for the prognosis of patients with liver cancerKEGG Kyoto encyclopedia of genes and genomes BP Biological processes CC Cellular components MF Molecular functions OS Over survivalAcknowledgementsNot applicableAuthors™ contributionsWZH designed and analyzed the research study WZH wrote and revised the manuscript YDL and WZH collected the data and all authors contributed to final manuscript All authors read and approved the final manuscriptFundingThis work is not supported by grantsAvailability of data and materialsRNA‘seq data and corresponding clinical data were acquired from the data portal for TCGA https porta lgdccance rgovEthics approval and consent to participateNot applicableAbbreviationsHCC Hepatocellular carcinoma CENPM Centromere protein M GSEA Gene set enrichment analysis TCGA Cancer genome atlas GO Gene ontology Consent for publicationNot applicable 0cWu a0and Yang Cancer Cell Int Page of Competing interestsThe authors declare that they have no competing interestsReceived January Accepted August References Torre LA Bray F Siegel RL Ferlay J Lortet‘Tieulent J Jemal A Global cancer statistics CA Cancer J Clin “Tang Y Wang H Ma L et al Diffusion‘weighted imaging of hepatocellular carcinomas a retrospective analysis of correlation between appar‘ent diffusion coefficients and histological grade Abdominal Radiol “ Coskun M Hepatocellular carcinoma in the cirrhotic liver evaluation using computed tomography and magnetic resonance imaging Exp Clin Transplant 201715Suppl Lang H Sotiropoulos GC Brokalaki EI et al Survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers J Am Coll Surg “Jiao Y Fu Z Li Y Meng L Liu Y High EIF2B5 mRNA expression and its prognostic significance in liver cancer a study based on the TCGA and GEO database Cancer Manag Res “ Byeon H Lee SD Hong EK et al Long‘term prognostic impact of osteo‘ pontin and Dickkopf‘related protein in patients with hepatocellular carcinoma after hepatectomy Pathol Res Pract “Shen Y Bu L Li R et al Screening effective differential expression genes for hepatic carcinoma with metastasis in the peripheral blood mononu‘clear cells by RNA‘seq Oncotarget “ Renou JP Bierie B Miyoshi K Cui Y Djiane J Reichenstein M Shani M Hennighausen L Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated beta‘catenin Onco‘gene “ Bierie B Edwin M Melenhorst J et al The proliferation associated nuclear element PANE1 is conserved between mammals and fish and preferentially expressed in activated lymphoid cells Gene Expr Patterns “ Brickner AG The PANE1 gene encodes a novel human minor histocom‘patibility antigen that is selectively expressed in B‘lymphoid cells and B‘CLL Blood “ Kruppa J Jung K Automated multigroup outlier identification in molecu‘lar highthroughput data using bagplots and gemplots BMC Bioinf Li T Fan J Wang B et al TIMER a web server for comprehensive analysis of tumor‘infiltrating immune cells Cancer Res 20177721e108“e110110 https doi1011580008‘5472CAN‘‘ Chandrashekar DS Bashel B Balasubramanya SAH Creighton CJ Rodri‘guez IP Chakravarthi BVSK Varambally S UALCAN a portal for facilitat‘ing tumor subgroup gene expression and survival analyses Neoplasia “ https doi101016jneo201705002 Gao et al Sci Signal Cerami et al Cancer Discov when publishing results based on cBioPortal https doi1011582159‘ Agarwal V Bell GW Nam J Bartel DP Predicting effective microRNA target sites in mammalian mRNAs eLife 20154e05005 https doi107554eLife Pathan M Keerthikumar S Ang CS Gangoda L Quek CY Williamson NA Mouradov D Sieber OM Simpson RJ Salim A Bacic A FunRich an open access standalone functional enrichment and interaction network analysis tool Proteomics “ https doi101002pmic20140 Foltz DR Jansen LE Black BE Bailey AO Yates JR III Cleveland DW The human CENP‘A centromeric nucleosome‘associated complex Nat Cell Biol “ Huang H Deng H Yang Y et al Molecular characterization and associa‘tion analysis of porcine PANE1 gene Mol Biol Rep “ Kim WT Seo SP Byun YJ et al The anticancer effects of garlic extracts on bladder cancer compared to cisplatin a common mechanism of action via centromere protein M Am J Chin Med “ Yu Z Wang R Chen F et al Five novel oncogenic signatures could be uti‘lized as AFP‘related diagnostic biomarkers for hepatocellular carcinoma based on next‘generation sequencing Dig Dis Sci “ Chen HQ Zhao J Li Y et al Epigenetic inactivation of LHX6 mediated microcystin‘LR induced hepatocarcinogenesis via the Wntβ‘catenin and P53 signaling pathways Environ Pollut 2019252Pt A216“ Qin G Tu X Li H et al lncRNA PSTAR promotes p53 signaling by inhibit‘ing hnRNP K deSUMOylation and suppresses hepatocellular carcinoma Hepatology https doi101002hep30793 Ihling C Naughton B Zhang Y et al Observational study of PD‘L1 TGF‘β and immune cell infiltrates in hepatocellular carcinoma Front Med Laus‘anne Dong N Shi X Wang S et al M2 macrophages mediate sorafenib resistance by secreting HGF in a feed‘forward manner in hepatocellular carcinoma Br J Cancer “ Pang YB He J Cui BY et al a potential antitumor effect of dendritic cells fused with cancer stem cells in hepatocellular carcinoma Stem Cells Int Janco JMT Lamichhane P Karyampudi L Knutson KL Tumor‘infiltrating dendritic cells in cancer pathogenesis J Immunol “ Fisher JB Peterson J Reimer M et al The cohesin subunit Rad21 is a negative regulator of hematopoietic self‘renewal through epigenetic repression of HoxA7 and HoxA9 Leukemia Carvajal‘Maldonado D Byrum AK Jackson J et al Perturbing cohesin dynamics drives MRE11 nuclease‘dependent replication fork slowing Nucleic Acids Res “ Bouattour M Raymond E Qin S et al Recent developments of c‘met as a therapeutic target in hepatocellular carcinoma Hepatology “ Sim WJ Iyengar PV Lama D et al c‘Met activation leads to the establish‘ment of a TGFβ‘receptor regulatory network in bladder cancer progres‘sion Nat Commun Tong G Cheng B Li J et al MACC1 regulates PDL1 expression and tumor immunity through the c‘MetAKTmTOR pathway in gastric cancer cells Cancer Med “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

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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

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

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi

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

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

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

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

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

Thyroid_Cancer

99mtc‘labeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodes”the first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1“ It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6“ Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15“ This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator œ99mTcGTTOM produced by Tomsk Polytechnic University TPU”Tomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agent”tin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2ˆ™2H2O “ a0mgml in different ratios and then adding a a0ml of eluate of 99mTc “ a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of “ a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl2ˆ™2H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer œSympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc ˆ’ A1Avc C100 A1 ˆ’ A2A1 C50 A2 ˆ’ A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThin‘layer chromatography TLC procedure To determine radiochemical purity of 99mTc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia — a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl2ˆ™2H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTc“nanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTc“nanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing “ a0g Injection of RP in a dose of “ a0MBq was performed between the first and second fingers of the rat™s hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œEuropean Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package œStatistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agent”tin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of “ a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is “ Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Al2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Colloid yield “ a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n DTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n Yield of colloid “ a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTc“nanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is “ Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A œ99mTcAl2O3 B œ99mTcFeC C œ99mTcDTPAmodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of “ a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP œ99mTcDTPAmod and RP œ99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol “ Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 “ Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer “ Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c“ Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med “ Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med “ Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging “ Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol “ Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med “ Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun “ RuizDom­nguez J M IbarzServio L Garc­ade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol “ Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys “ O™Connor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras “ Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med “ Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol “ Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem “ Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett “ Lukasz K Thin Layer Chromatography in Drug Analysis “ CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med “ Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J “ AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures „– Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c 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 license and indicate if changes 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 If material 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 obtain 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Thyroid_Cancer

"Postoperative pain in ambulatory surgery is a multifactorial issue affecting patient satisfaction time ofdischarge and rehospitalization This study evaluated the efficacy and safety of nalbuphine for the treatment ofpostoperative pain after ambulatory surgery relative to tramadolMethods This multicenter randomized double blind and controlled study was conducted at centers Inaccordance with the inclusion criteria ambulatory surgery patients were recruited These patients hadmoderate to severe pain after ambulatory surgery with a visual analogue scale VAS score cm They wererandomly divided into an experimental n or control n group and treated for analgesia with mgkg of nalbuphine or mgkg of tramadol respectively VAS scores adverse events and vital signs of the patientswere recorded before administration baseline T1 and min T2 h T3 h T4 and h T5 after administrationof analgesia A decrease in pain intensity of more than compared with the baseline was used as an indicatorof analgesic efficacy The experimental and control groups were compared with regard to this indicator of efficacyat each timepointResults The VAS scores of the experimental and control groups were statistically comparable at timepoints T1T4At T5 the VAS scores of the experimental group were significantly lower than that of the control The pain intensitywas significantly higher in the experimental group compared with the control at T2 and T3 Adverse events andvital signs were similar for the two groups at each timepointConclusions Nalbuphine can provide effective and safe pain relief in patients after ambulatory surgeryTrial registration The registration number is ChiCTRIOR16010032 the date of registration was Keywords Nalbuphine Tramadol Ambulatory surgery Postoperative analgesia Anesthesia Pain Correspondence qulianguohotmailcom chengzg2004hotmailcom1Department of Anesthesiology Xiangya Hospital of Central South UniversityNo Xiangya Road Changsha Hunan 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 0cGuan BMC Anesthesiology Page of BackgroundPostoperative pain is a multifactorial issue that may result in patient dissatisfaction delayed discharge and unanticipated hospital admission after ambulatory surgery[] Both delayed discharge and unanticipated hospitaladmission have the undesirable effect ofincreasinghealthcare costs [] In the postoperative period moderate to severe pain are frequently observed during thefirst to h after ambulatory surgery []hassurgerysignificantlyPatient recovery after ambulatory surgery has improvedsince the introduction of the concept of enhanced recovery after surgery a multimodal perioperative care pathwaydesigned to achieve early recovery after surgery []Ambulatoryshortenedhospitalization accelerated turnover and reduced hospitalcosts and rates of nosocomial infections [] However theshortened hospitalization and increased mobility of surgical patients have necessitated the need to improve theefficacy of anesthesia and perioperative managementTherefore postoperative pain and the complicationsarising from its treatment are important considerationsfor patients undergoing ambulatory surgeryVarious drugs have been used to prolong postoperativeanalgesia such as tramadol [] ketorolac [] dexmedetomidine [] ketamine [] and nalbuphine [] Nalbuphine a synthetic opioid agonistantagonist analgesic isprimarily a kappa κ agonist and a partial mu μ antagonist It has a better safety profile with fewer side effectscompared with other opioids because of its agonist andantagonist activities [] Nalbuphine [] exerts its analgesic and hypnotic effects through its κ opioid receptorwhich may reduce μ opioid receptorrelated adverseevents Numerous studies [ ] have reported its advantages in pain managementThere have been few studies in China of nalbuphinefor the treatment of postoperative pain after ambulatorysurgery The present study evaluated the analgesic efficacy and safety of intravenous nalbuphine hydrochloriderelative to tramadol for the treatment of postoperativepain aftera noninferiority control trialambulatoryincludingsurgeryMethodsParticipantsThis study was approved by the Ethics Committee ofXiangya Hospital of Central South University IRB Written informed consent was obtainedfrom all subjects participating in the trial The trial wasregistered prior to patient enrollment at chictrcnChiCTRIOR16010032 Principalinvestigator QulianGuo Date of registration A multicenter prospectiverandomized parallelcontrolled doubleblinded study for pain managementafter ambulatory surgery in adult patients was undertakenin hospitals Patients were screened at each center Thestudy was reported in accordance with the guidelines ofthe Consolidatedof Reporting TrialsCONSORTStandardsThe patient inclusion criteria were as follows to years old ASA American Society of AnesthesiologistsIII with postoperative pain after surgeries of the breastexcept radical surgery for mastocarcinoma or thyroidor hysteroscopy or laparoscopic cholecystectomy operative time h visual analog scale VAS score cm before the surgery and VAS score cm after recoveryfrom anesthesia body mass index BMI “ kgm2and signed informed consentPatients were excluded from this study if they wereallergic to the medication or any of the excipients inthe product Patients with current or histories of anyof the following were also excluded opioid allergyacute or chronic alcoholism or drug addiction neurological disease opioid used within the last monthsparalytic ileus increased intracranial pressure or headinjury chronic opioid use taking opioids for morethan months hypotension hypothyroidism asthmato be avoided during seizure hypertrophy oftheprostate epilepsy coronary heart disease bronchialasthma respiratory insufficiency or respiratory failurePatients taking or who had taken monoamine oxidaseinhibitor or antidepressants within the past dayswere excluded Patients with abnormal preoperativeliver and kidney function were also excluded definedas abnormal alanine aminotransferase ALT asparticaminotransferase AST blood urea nitrogen BUNor creatinine Cr ALT and AST times the normallimit and BUN and Cr higher than the normallimit coronary heart disease bronchial asthma respiratory insufficiency or respiratory failure or poorlycontrolled or difficult hypertension The latter wasdefined as systolic blood pressure SBP ‰¥ mmHgor diastolic pressure DBP ‰¥ mmHg In additionpatients with any ofthe following were excludedpregnancy abnormal coagulation function participation in another medication trial within the previous days unable to express their intention correctlypoor compliance unable to complete the study program or anyone the researchers considered inappropriate to participateTrial designPatients were randomly assigned to receive either the experimental group E or control group C treatment inthe postoperative period Group E was treated with nalbuphine hydrochloride Yichang HumanwellPharmaceutical diluted with saline to mgL Group Cwas administered tramadol hydrochloride diluted withsaline to mgL 0cGuan BMC Anesthesiology Page of The study medication was selected and prepared according to a random number list nalbuphine hydrochloride or tramadol hydrochloride The study wasblinded by excluding the researcher who prepared thepostoperative medications from participating in test observations and followups The researchers involved inobservation and evaluation of the experiment and patients and doctors were blinded throughout the studybeforeinductionofdexamethasoneInterventionsAll patients were administered intravenously with mgofgeneralanesthesia and mg of ondansetron at the time of surgery completion to prevent postoperative nausea andvomiting The bispectralindex BIS value was maintained between and during the operationAnesthesia induction was performed using sufentanil μgkg and propofol “ mgkg with cisatracurium “ mgkg given when necessary Anesthesiawas maintained by simultaneous infusion of propofol“ μgkgmin An additionaland remifentanil mgkg of cisatracurium was added intraoperativelywhen required Intraoperative fluid infusion and otheranesthetic management were performed routinelyAfter the surgery patients who were fully awake andfeeling pain for the first time were assessed for painwhile at rest using the VAS If the VAS score was cm the patients were included in the study and the painscore was used as the baseline T1 The test medicationsnalbuphine hydrochloride or tramadol hydrochloridewere administered at mLkg The VAS at rest wasused to evaluate the efficacy of the medications and wasrecorded before administration T1 and after administration at min T2 h T3 h T4 and h T5The following vital signs were recorded at each timepoint SBP DBP mean arterial pressure MAP heartrate and respiratory rate Adverse events and any medications used were also recordedWithin h after administration of the medications ifthe VAS score was cm it was deemed that the analgesic effect was invalid and the patient was discontinuedfrom the trial One hundred milligrams of flurbiprofenaxetil was infused intravenously as a rescue analgesiaand the name and dose were recorded The use of otheranalgesics aside from those involved in the study suchas opioidstranquilizers anesthetics and antiemeticswere prohibited during the study period If other analgesics were required to control the pain the patient wasdiscontinuedOutcomesPrimary outcomeThe pain intensity was measured using the VAS A decrease in VAS score of more than compared with thebaseline was used as an indicator of analgesic efficacy []The VAS score was also compared between groups E andC at all timepoints to determine any differences in the efficacy and duration of the analgesic effectsSecondary outcomeThe vital signs SBP DBP respiratory rate and heartrate were measured and used as safety indicators Thevital signs were also compared between groups E and Cand within each group at each timepoint Any differences observed could be used as a secondary indicatorto determine analgesic efficacyAdverse eventsAdverse events such as medication extravasation dizziness nausea vomiting and hidrosis were recorded during the study The rates of adverse events was comparedbetween groups E and C to determine the effects of thetreatmentsSample sizeSample size was calculated by VAS at rest at each timepoint Based on a previous report [] a single intravenous injection of tramadol was administered to patientswith postoperative pain after day surgery and the VASscore was cm at min after administration Assuming that the analgesic effect of nalbuphine was betterthan tramadol with α and β the VAS scoredifference between the two groups μA ˆ’ μB would be and the standard deviation σ The samplesizen was calculated using the formula [] 14 12n ¼ α zþ z1 ˆ’ ⠈’ α2 13 15Þð μˆ’ μBAEach group required subjects and with consideration of the estimated dropout rate patients wereincluded in each group Therefore patients were recruited in this study with patients in each centerStatistical methodsDescriptive statistics were used to describe all demographic data The ttest was applied to analyze thechanges in VAS scores between the two treatmentgroups at each timepoint and at different timepointsrelative to the baseline The Wilcoxon test was used toanalyze the pain classification of patients at each observation timepoint The pain intensity between the twogroups was compared using the chisquared χ2 testP was considered statistically significant The incidence of adverse events changes in blood pressure respiratory rate and heart rate relative to the baseline at 0cGuan BMC Anesthesiology Page of each timepoint and differences between the groupswere analyzed using the ttestResultsParticipantsThe study population comprised randomly codedpatients recruited from centers Fig However patients were excluded as they did not meet theeligibility criteria of a VAS score cm Thusthetrial consisted of patients in group E and in group CBaseline dataThe differences in age and gender between groups Eand C were not statistically significant Table Theresults of the preoperative test physical examinationand medical histories of the two groups were relatively similar with no statistical difference Therewere no statistically significant differences in the typesof surgery between the two groups Table Therewere also no differences in the use of opioids including sufentanil and remifentanil between the twogroups during surgeryDuring the observation period and patients in groups E and C respectively weretreated with rescue analgesic medication consistingof mg of flurbiprofen axetil There was no statistically significant difference between the two groupsTable Patient demographics of groups E and CaSubjects nGender n MaleFemaleAge yBMI kgm2Respiration rpmHeart rate bpmHeart rhythm n NormalAbnormalSBP mmHgDBP mmHgGroup E Group C ± ± ± ± ± ± ± ± ± ± ± ± P ± MAP mmHgaGroup E was treated with nalbuphine hydrochloride diluted with saline to mgL Group C was administered tramadol hydrochloride diluted with salineto mgL ± with regard to the percentage using rescue analgesicmedication χ2 P There was no significant deviation from the regimen for all concomitantnostatistically significant difference between the twogroupscombination medicationsandandFig CONSORT flow diagram of progress through the phases of a randomized doubleblinded parallel controlled trial of the groups 0cGuan BMC Anesthesiology Page of Table Types of surgery n aGroup ETotal nBreast Thyroid Hysteroscopy LC Others Group CaReported as n unless indicated otherwise Group E was treated with nalbuphine hydrochloride diluted with saline to mgL Group C was administeredtramadol hydrochloride diluted with saline to mgL Other surgeries included lumbar disc exploration laparoscopic gastric perforation repair endoscopic sinussurgery surgical removal of internal fixation of fractured bonesLC laparoscopic cholecystectomy OutcomesPrimary outcomeA pairwise comparison of the VASs determined at rest atdifferent timepoints between groups E and C revealed nodifference between the VAS scores at T1 T2 T3 or T4 respectively However at T5 the VAS at rest of group E wassignificantly lower than that of group C Fig A decreasein pain intensity of more than compared with the baseline T1 was used as an indicator of analgesic efficacyTable The analgesic efficacy experienced by group E atT2 and T3 was significantly higher than that of group CAdverse eventsAdverse events occurred in subjects in group Eand subjects in group C with no serious adverse events or deaths occurring in either group Thenumber of adverse events was higher in group E compared with group C but the difference was not statistically significant Table Secondary outcomeThe vital signs SBP DBP respiratory rate and heart rateof groups E and C at all timepoints were statisticallyFig The VAS at rest in the experimental Group E and controlgroup Group C Time points T1 before administration T2 afteradministration at min T3 after administration at h T4 afteradministration at h T5 after administration at h aAfteradministration the VAS of Group E was lower than that of Group Cfrom T2T5 bthere was a statistically significant difference in VASbetween the groups at T5 Data are expressed asmean ± standard deviationsimilar Table For both groups the mean SBP DBPand heart rate at each of the timepoints T2 T3 T4 and T5were significantly lower than at T1 However the bloodpressures at T2 to T5 were comparable to that at admission T0 and there was no significant difference in respiratory ratesDiscussionIn this prospective multicenter study patientswere randomized to receive either nalbuphine groupE or tramadol group C to treat pain after ambulatory surgery Group E experienced significantly longerduration of analgesia compared with group C Ateach timepoint the vital signs SBP DBP respiratoryrate and heart rate of the groups were statisticallycomparable However within each group there weresignificant differences in SBP DBP and heart rate atT2 T3 T4 and T5 relative to T1 Overall the analgesic effect of nalbuphine was comparable to that oftramadol with nalbuphine having a longer durationof analgesiaIn China the number of day surgeries is increasingdue to improvements in surgery and anesthesia withshorter recovery time and patients discharged within h after surgery Therefore there is a higher demand forTable Pain reduction when compared to baseline T1 n adT2eT3fT4gT5EffectiveGroup Eb Noneffective Group Cc χ2PEffective Noneffective Effective Noneffective Effective Noneffective aEffective pain reduction is defined as a decrease in pain intensity compared with the baseline T1 Noneffective is defined as a decrease in painintensity compared with the baseline T1bGroup E n was treated with nalbuphine hydrochloride diluted withsaline to mgLcGroup C n was administered tramadol hydrochloride diluted withsaline to mgLdT2 after administration at mineT3 after administration at hfT4 after administration at hgT5 after administration at h 0cGuan BMC Anesthesiology Page of Table Patients experiencing adverse events nTotal subjects experiencing adverse eventsVasculitis medication extravasationDizziness nausea vomitingHidrosisGroup EGroup Canesthesia and a need to improve the quality of analgesics While achieving rapid recovery patients also needto avoid complications related to surgery and anesthesiasuch as pain nausea and vomiting Numerous studies[ ] have shown that after day surgery nearly ofpatients experience pain Postoperative pain not only affects patients™ rehabilitation and prolongs hospitalizationit can also result in progression from acute to chronicTable Vital signs at each timepoint aT0T1T2T3T4T5Vital signsSBPDBPGroup Eb ± ± Respiratory rate ± Heart rate ± Group Cc ± ± ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± SBPDBP ± ± ± ± Respiratory rate ± ± Heart rate ± ± PaT0 at admission T1 before administration T2 after administration at minT3 after administration at h T4 after administration at h T5 afteradministration at hbGroup E n was treated with nalbuphine hydrochloride diluted withsaline to mgLcGroup C n was administered tramadol hydrochloride diluted withsaline to mgLDifference is statistically significant compared with T1 difference isstatistically significant compared with T0pain which is the main cause of readmission after daysurgery []According to the Chinese Society of Anesthesiology[] systemic opioids given to patients undergoing ambulatory surgery with general anesthesia activate opioidreceptors and stimulate various ans This often results in nausea and vomiting pruritus urinary retentionexcessive sedation and respiratory inhibition Thusinprinciple systemic opioids are not used for postoperativepain relief after day surgery The analgesic and adversereactions of mixed agonistantagonist opioids such asnalbuphine and dezocine also exhibit a ceiling effectImplementation of multimodal analgesia using NSAIDscan significantly reduce the dose of opioid and adversereactions and can be used postoperatively to managemoderate pain after ambulatory surgeryNalbuphine a mixed agonistantagonist opioid is associated with milder μ receptorrelated side effects Itsplasma halflife is h and in clinical studies the durationof analgesic activity ranges from to h [] In ourstudy the VAS at rest of group E was less than pointsand the difference was statistically significant comparedwith the VAS at rest before administration This indicates that nalbuphine could effectively relieve pain afterambulatory surgery Similar results were also observedin animal studies that showed amelioration of somaticand visceral pain in mice after treatment with nalbuphine []In the present study the VAS at rest at timepoints T1to T4 of the nalbuphine group group E did not differfrom that of the control At T5 the VAS at rest of thenalbuphine group was significantly lower than that ofthe tramadol group This indicates that the duration ofnalbuphine for pain relief after ambulatory surgery waslonger than that of tramadol There were cases of adverse reactions in the nalbuphine group which wasnot significantly different from the cases in thetramadol groupThe incidence of adverse reactions associated with nalbuphine is relatively low compared with other opioidmedications A metaanalysis of randomized controlledtrials by Zeng [] showed that nalbuphine hassimilar analgesic effects compared to morphine and abetter drug safety profile with a low incidence of postoperative pruritusrespiratory inhibition nausea andvomiting In addition studies have reported that antagonism of the μ receptor by nalbuphine could reduce theadverse reactions of other opioids as seen in the combination of morphine and nalbuphine in patientcontrolled analgesia or patientcontrolled epidural analgesia [ ] and the rate of adverse effects such asurinary retention related to morphine pruritus and nausea was significantly less Nalbuphine with sufentanilused in patientcontrolled analgesia could reduce the 0cGuan BMC Anesthesiology Page of incidence of opioidrelated nausea and vomiting and improved patients™ satisfaction with analgesia [ ]In the present study the difference in respiratoryrates before and after administration in both the nalbuphine and tramadol groups was not statistically significant and no respiratory depression was observedMany studies have reported that respiratory depression caused by nalbuphine is small and has a ceilingeffect [ ] In one study a neonate was wronglyadministered a tenfold higher dose than required ofnalbuphine and it resulted in only prolonged sedationwith no respiratory failure []studyIn the presentStudies have shown that preanesthetic injections ofnalbuphine could reduce stress responses and fluctuations in blood pressure and heart rate during intubation [ ]the bloodpressures and heart rates of both groups after administration were significantly lower than at T1 althoughstill within normal ranges The blood pressure at T0Inception of the study was compared to the bloodpressure after surgery T1T5 the blood pressure atT1 was significantly higher than at T0 However atthe later timepoints T2 to T5 there were no statistical differences in the blood pressures compared toT0 The decrease in blood pressure after administration T2T5 may have been due to the alleviation ofpain If sothen the lowered blood pressure couldalso indicate the analgesic efficacy of nalbuphineThere are several limitations in this study First a limited number of parameters VAS score adverse eventsand change of vital signs were observed within the halflife of the medication Secondly the VAS scores were recorded at rest and not during movement Finally due toethical issues a placebo control group was not possibleTherefore we were not able to assess the effectiveness ofnalbuphine or tramadol at and h after administration Fortunately none of the patients dropped out during the or h after administration of medication forpain However the present results warrant further experiments to determine comprehensively the effectiveness and safety of nalbuphine for the treatment of painafter ambulatory surgeryConclusionThis study indicates that nalbuphine at a recommendeddose of mgkg is safe and effective for pain management after ambulatory surgeryAcknowledgementsNot applicableAuthors™ contributionsGYJ This author contributed to the data collection contributed to the dataanalysis and wrote the manuscript WL This author contributed to the datacollection and analysis LQ This author contributed to the data collectionand analysis FQW This author contributed to the data collection andanalysis HN This author contributed to the data collection and analysis HCFThis author contributed to the data collection and analysis MCH This authorcontributed to the data collection and analysis LCJ This author contributedto the data collection and analysis WHB This author contributed to the datacollection and analysis CH This author contributed to the data collectionand analysis GQL This author designed most of the research plancontributed to the data collection CZG This author designed most of theresearch plan contributed to the data collection All of the authors haveread and approved the manuscriptFundingNot applicableAvailability of data and materialsThe datasets generated and analyzed during the present study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was approved by the Ethics Committee of Xiangya Hospital ofCentral South University IRB Written informed consent wasobtained from all subjects participating in the trial The trial was registeredprior to patient enrollment at chictrcn ChiCTRIOR16010032 Principalinvestigator Qulian Guo Date of registration Consent for publicationAll data published here are under the consent for publication Writteninformed consent was obtained from all individual participants included inthe studyCompeting interestsThe authors declare that they have no conflict of interestAuthor details1Department of Anesthesiology Xiangya Hospital of Central South UniversityNo Xiangya Road Changsha Hunan China 2Department ofAnesthesiology Hunan Provincial People™s Hospital Changsha Hunan China3Department of Anesthesiology Third Xiangya Hospital of Central SouthUniversity Changsha Hunan China 4Department of Anesthesiology PainMedicine Critical Care Medicine Aviation General Hospital of ChinaMedical University Beijing Institute of Translational Medicine ChineseAcademy of Sciences Beijing China 5Department of Anesthesiology PekingUniversity Shougang Hospital Beijing China 6Department of AnesthesiologyShanxi Academy of Medical Sciences Shanxi Dayi Hospital Shanxi China7Department of Anesthesiology Fudan University Shanghai Cancer CenterShanghai China 8Department of Anesthesiology Third Affiliated Hospital ofSun YatSen University Guangzhou Guangdong China 9Department ofAnesthesiology First People™s Hospital of Foshan Foshan Guangdong China10Department of Anesthesiology Beijing Ditan Hospital Capital MedicalUniversity Beijing ChinaReceived March Accepted August AbbreviationsALT alanine aminotransferase ASA American Society of AnesthesiologistsAST Aspartic aminotransferase BIS Bispectral index BMI Body mass indexBUN Blood urea nitrogen C Control CONSORT Consolidated Standards ofReporting Trials Cr Creatinine DBP Diastolic pressure E ExperimentalMAP Mean arterial pressure SBP Systolic blood pressure VAS Visual analogscaleReferencesShirakami G Teratani Y Namba T Hirakata H TazukeNishimura M FukudaK Delayed discharge and acceptability of ambulatory surgery in adultoutpatients receiving general anesthesia J Anesth “ httpsdoi101007s0054000402976Tong D Chung F Postoperative pain control in ambulatory surgery SurgClin North Am “Rawal N Postoperative pain treatment for 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Mehta KD Effect of nalbuphine on haemodynamicresponse to orotracheal intubation J Anaesthesiol Clin Pharmacol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Melnyk M Casey RG Black P Koupparis AJ Enhanced recovery after surgeryERAS protocols Time to change practice Can Urol Assoc J “httpsdoi105489cuaj11002Lee JH Anesthesia for ambulatory surgery Korean J Anesthesiol “ httpsdoi104097kjae2017704398Acalovschi I Cristea T Margarit S Gavrus R Tramadol added to lidocaine forintravenous regional anesthesia Anesth Analg “Jankovic RJ Visnjic MM Milic DJ Stojanovic MP Djordjevic DR Pavlovic MSDoes the addition of ketorolac and dexamethasone to lidocaineintravenous regional anesthesia improve postoperative analgesia andtourniquet tolerance for ambulatory hand surgery Minerva Anestesiol“Kumar A Sharma D Datta B Addition of ketamine or dexmedetomidine tolignocaine in intravenous regional anesthesia A randomized controlledstudy J Anaesthesiol Clin Pharmacol “ httpsdoi104103AbdelGhaffar HS Kalefa MA Imbaby AS Efficacy of ketamine as an adjunctto lidocaine in intravenous regional anesthesia Reg Anesth Pain Med “ httpsdoi101097AAP0000000000000128 Youssef MIEN Lidocainenalbuphine versus lidocainetramadol forintravenous regional anesthesia AinShams J Anesthesiol “ Bakri MH Ismail EA AbdElshafy SK Analgesic effect of Nalbuphine whenadded to intravenous regional anesthesia a randomized control trial PainPhysician “ Vilsbøll T The effects of glucagonlike peptide1 on the beta cell DiabetesObes Metab Suppl “ httpsdoi101111j14631326200901073xShin D Kim S Kim CS Kim HS Postoperative pain management usingintravenous patientcontrolled analgesia for pediatric patients J CraniofacSurg “ Mukherjee A Pal A Agrawal J Mehrotra A Dawar N Intrathecal nalbuphineas an adjuvant to subarachnoid block What is the most effective doseAnesth Essays Res “ httpsdoi1041030259116294759 Hua X Chen LM Zhu Q Hu W Lin C Long ZQ Efficacy of controlledrelease oxycodone for reducing pain due to oral mucositis innasopharyngeal carcinoma patients treated with concurrentchemoradiotherapy a prospective clinical trial Support Care Cancer “ httpsdoi101007s0052001946435 Ali M Khan FA Comparison of analgesic effect of tramadol alone and acombination of tramadol and paracetamol in daycare laparoscopic surgeryEur J Anaesthesiol “ httpsdoi101097EJA0b013e328324b747 Chow SC Shao J Wang H Sample size calculations in clinical research 2nded Boca Raton Chapman HallCRC Apfelbaum JL Chen C Mehta SS Gan TJ Postoperative pain experienceresults from a national survey suggest postoperative pain continues to beundermanaged Anesth Analg “ table of contents Aubrun F Ecoffey C Benhamou D Jouffroy L Diemunsch P Skaare K et alPerioperative pain and postoperative nausea and vomiting PONVmanagement after daycase surgery The SFAROPERA national study AnaesthCrit Care Pain Med httpsdoi101016jaccpm201808004 Xu J Expert consen

Thyroid_Cancer

Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous 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 cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR “ p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR “ p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR “ p RMSSD 19ms vs�19ms vs adjusted HR “ p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors [“]HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [“] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients™ prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease [“]The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law ˜scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons™ in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients™ number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARB™s as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECG™s per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECG™s per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÞ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÞ2Pn 00 n 00 ð1Þð2ÞSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohn™sdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR “ all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR “To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECG™s older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR “ p and RMSSD19ms versus �19ms HR “ p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR “ p and RMSSD HR “ p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR “ p and RMSSD 19ms versus �19ms HR “ p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [“] [“] [“] [“] [“] [“] [“] [“] Mean BMI SD [“] [“] [“] [“]Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR “ p and RMSSD HR “p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR “ p and RMSSD HR “PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI “ “ reference “ “ “ reference “ “ “p reference reference “ “ reference reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “ reference “ “Postoperative complicationNoYes reference reference “ “101371journalpone0237244t003baseline HRV remained not significant SDNN HR “ p andRMSSD “ p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n “RMSSD 19ms n “SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference “ reference “RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference “ reference “SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [“] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [“] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [“] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n “SDNN �20ms n referenceAdjusted�SDNN 20ms n “SDNN �20ms n referenceUnadjustedRMSSD 19ms n “RMSSD �19ms n referenceAdjusted�RMSSD 19ms n “RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn “n “SDNN �20msn referencen referenceAdjusted�SDNN 20msn “SDNN �20msn referenceUnadjustedRMSSD 19msn “RMSSD �19msn referenceAdjusted�n “n referencen “n referenceRMSSD 19msn “n “RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform

Thyroid_Cancer

Hepatocellular carcinoma HCC is a high mortality disease the fifth most general cancer worldwide and the second leading to cancer‘related deaths with more than new patients diagnosed each year First the high expression of centromere M CENPM in mammary gland tissue of b‘catenin transformed mice was identifiedMaterials and methods In our study we evaluated the expression of CENPM in hepatocellular carcinoma based on data obtained from an online database Multivariate analysis showed that the expression of CENPM and M classifica‘tion was an independent prognostic factor for patients with hepatocellular carcinomaResults Survival analysis showed that patients with high CENPM had a worse prognosis than patients with low CENPM P A multivariate Cox regression hazard model showed that B cells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P Using the network the most frequently changed neighbor genes of CENPM were shown and the most common change was RAD21 Conclusion Our study found that the expression of CENPM was significantly increased in patients with hepatocellu‘lar carcinoma and it was related to a variety of clinical characteristics its correlation with the level of immune infiltra‘tion and poor prognosis so CENPM can be used as a useful prognosis for patients™ markers and HCCKeywords Hepatocellular carcinoma Centromere protein M Data mining PrognosisBackgroundHepatocellular carcinoma HCC a high mortality disease which is the fifth most general cancer in the world and the second most common lead to cancerrelated deaths with over new patients diagnosed each year [ ] Viral hepatitis and nonalcoholic steatohepatitis are the most common causes of cirrhosis and approximately of cases develop to HCC [] Due to the recurrence of HCC the prognosis of HCC remains discouraging and the 5year overall survival rate which Correspondence wawang123soutlookcomDepartment of Infectious Diseases Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Chinais only to [] Despite the rapid development of advanced medical technology there are still no useful curable strategies for HCC patients [] Byeno et a0al [] reported that based on longterm survival data serum OPN and DKK1 levels in patients with liver cancer can be deemed as novel biomarkers that show prognostic useful for liver cancer Other serum markers such as alphafetoprotein AFP and alkaline phosphatase ALP or AKP are proverbially used in clinical but they lack sufficient sensitivity and specificity [] Therefore finding useful biomarkers is indispensable for diagnosis and treatment for HCC patientsPosttranscriptional modifications are essential for tumorigenesis and development Centromere protein M CENPM otherwise called PANE1 CENPM and 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 0cWu a0and Yang Cancer Cell Int Page of C22orf18 which encodes a kinetic protein binds to spindle microtubules to regulate chromosomal separation during cell division [] Expression of the PANE1 gene was found preferentially in immune cells involving tumor tissues and tumor derived cell lines and leukemias and lymphomas [] Brickner et a0 al [] found highly expressed in B lineage chronic lymphocytic leukemia BCLL cells and resting CD19 B cells may be a potential therapeutic target for BCLL Bierie et a0al [] also demonstrated that human CENPM transcript cRNA was detected only in a0vivo or in a0vitro in activated B cells and T cells These studies suggested CENPM may play critical role in tumor immune response and may be deemed to therapeutic target for immunotherapy However the role of CENPM in HCC prognostic remains unclear In our study we evaluated the expression of CENPM in HCC based on data from an online database to further understand the biological pathway of CENPM related to the pathogenesis of HCC In addition we also analyzed the connection between CENPM expression and clinical features as well as the correlation of its expression with immune infiltration level in HCC comes an online tumor infiltrating immune cells analysis toolMaterials and a0methodsData collectionInformation on RNAsequencing data tissues workflow type HTSeqCounts and comparative clinical data patients data format BCR XML were identified and got from the level standardized FPKM of the TCGAHCC cohort Use boxplots to imagine expression differences for discrete variables [] The clinical factors included gender stage age grade Tphase Mphase Nphase survival status and number of days of survival Data analysis were checked by R version and R Bioconductor software packagesGSEA enrichmentGene Set Enrichment Analysis GSEA created a list of all gene permutations related to CENPM expression The samples were then divided into a high CENPM group and a low CENPM group as training sets to distinguish potential functions and use GSEA to clarify significant survival differences Genome replacement is performed multiple times with each exam The degree of expression of CENPM was used as a phenotypic marker Normalized enrichment scores NES and nominal Pvalues have been used to classify the pathways of enrichment in each phenotypeImmune infiltrates analysisTIMER [] is a comprehensive database for the systematic study of immune infiltration in various malignant tumor types The abundance of immune infiltrates CD8 T cells B cells CD4 T cells macrophages neutrophils and dendritic cells was evaluated by our statistical methods and has been estimated using pathology Methods evaluated it The network also enables users to explore the clinical relevance of one or more tumor immune subpopulations and has the flexibility to correct multiple covariates in a multivariate Cox proportional hazard model Meanwhile we contrast the differential level of CENPM between tumors and normal on all TCGA tumorsUALCAN and a0c‘BioPortal analysisUALCAN [] is a userfriendly intelligent network asset for analyzing discovering cancer data and indepth analysis of TCGA gene expression information One of the highlights of the portal is that it allows users to found between biomarkers or computer approval of potential genes of interest and to evaluate genes in different clinical subgroups such as gender age race tumor grade etc expression cBioPortal [] is an online free asset that can visualize analyze and download largescale cancer transcription datasets The portal included cancer studies The tab biological interaction network of CENPM and its coexpressed genes was got and neighboring genes with altered frequencies were containedTargetScan analysisTargetScan [] is a web for predicting potential biological targets of miRNAs TargetScanHuman deems that the match to human ²UTR and its orthologs is estimate by a UCSC genomewide adjustment As an alternative they are ranked according to their predicted conservative positioning possibilities FunRich [] is a tool designed to process varieties of geneprotein datasets in spite of the anism and used for functional enrichment analysis We used Funrich tools for miRNA enrichment analysis including analysis of biological pathways biological processes BP cellular components CC and molecular functions MFStatistical analysisScatter plots and paired plots visualize the differences between normal and tumor samples Use delete ways to handle disappeared data and if any individual value is disappeared the data will exclude the full sample The relationship between clinical factors and CENPM was used by logistic regression Wilcoxon rank sum test and Kruskal test Multivariate Cox analysis was used to assess the effect of CENPM expression on survival and other clinical factors such as age gender stage distant metastasis Benjamini“Hochberg™s means of converting P values to FDR 0cWu a0and Yang Cancer Cell Int Page of ResultsPatients™ characteristicsThe TCGA database contains patients The clinical and pathological properties of these samples are shown in Table a0 The middle age at diagnosis in TCGA was a0 years old range “ a0 years and median finally contact for subjects was a0 months range “ a0months Meanwhile followup for subjects conformed alive and death patients Our study cohort included female and Table TCGA hepatic carcinoma patient characteristicsClinical characteristicsAge at diagnosis yearFutime monthGender Female MaleStage I II III IV NAGrade G1 G2 G3 G4 NAT‘classification T1 T2 T3 T4 TX NAM‘classification M0 M1 MXN‘classification N0 N1 NX NAStatus Alive DeathData express as mean min“maxTotal “ “ male patients Stage I was located in patients stage II in stage III in and stage IV in Tumor stage was found T1 in patients T2 in T3 in and T4 in Node stage contained N0 in and N1 in of cases had distant metastases All the subjects were adenomas or adenocarcinomasCENPM expression and a0clinical factorsScatter plot showing difference in CENPM expression among normal and tumor samples P we then use paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P Fig a01a b The outcomes suggested that the expression of CENPM was significant difference The expression of CENPM correlated significantly with the patient grade P clinical stage P and Tclassification P Fig a01d“f Univariate analysis utilizing logistic regression uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors Table a0 CENPM expression in HCC as appreciably connected with grade OR CI “ G1 vs G3 stage OR CI “ I vs III and Tclassification OR CI “ T1 vs T3 indicated that patients with low CENPM expression are inclined to advance to a further advanced stage than those with high CENPM expressionSurvival and a0multivariate analysisSurvival analysis found that HCC with CENPMhigh had a worse outcome than that with CENPMlow P Fig a0 1c The univariate analysis suggested that CENPM linked essentially to stage HR CI “ P and Tclassification HR CI “ P Table a0 Multivariate analysis showed that the expression of CENPM HR P and M classification HR P were independent prognostic factors for patients with HCC Table a0GSEA analysisTo identify useful pathways that may be differentially initiated in liver cancer we performed a GSEA analysis between low and high CENPM expression datasets We chose the most abundant signaling pathway depending on the standardized enrichment score NES Table a0 The results showed that CENPM high expression differentially enriched cell cycle DNA replication RNA degradation certain cancers phagocytosis P53 signaling pathway and purine metabolism Fig a0 0cWu a0and Yang Cancer Cell Int Page of Fig CENPM expression and the association among clinicopathologic factors a The scatter plot showed the difference of CENPM expression between normal and tumor samples P b paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P c Survival analysis P d Grade e Stage f T‘stageTable CENPM expression associated with a0pathological characteristics logistic regressionclinical Clinical characteristicsTotal NAge vs ‰¤ Gender female vs maleGrade G1 vs G3Stage I vs IIIT‘stage T1 vs T3Odds ratio in a0CENPM expression “ “ “ “ “P‘valueCategorical dependent variable greater or less than the median expression levelImmune infiltrates related to a0CENPM in a0HCCThe correlation between CENPM liver cancer in expression and the abundance of immune infiltrates was statistically significant P Fig a0 3a A multivariate Cox proportional hazard model showed that Bcells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P indicating that these immune cells significantly affect the prognosis it is worth further research and exploration Table a0 At the same time the expression of CENPM was also statistically significant P Finally we compared CENPM expression between various tumors and normal tissues The results showed that CENPM was overexpressed in various cancers P Fig a03bAssociations survival Table a and a0 clinicopathologic characteristics in a0 TCGA patients using Cox regression b Multivariate survival model after a0variable selectionwith a0overall Clinicopathologic variableHR CIP‘valuea Age continuous Gender female vs male Stage IIIIIIIV Grade G1G2G3G4 T‘classification T1T2T3T4 Distant metastasis M0M1MX Lymph nodes N0N1NX CENPM expression high vs lowb Distant metastasis M0M1MX CENPM expression high vs low “ “ “ “ “ “ “ “ “ “UALCAN and a0c‘BioPortal analysis in a0HCCIn the age subgroup normal age “ a0years normal age “ a0years normal age “ a0years and normal age “ a0 years among patients with liver cancer CENPM has substantially higher transcription levels than healthy individuals Analysis in the weight subgroup gender subgroup ethnicity subgroup tumor grade subgroups analysis also showed significantly higher CENPM in HCC patients Fig a0 In order to determine the 0cWu a0and Yang Cancer Cell Int Page of Table Gene sets enriched in a0phenotype highGene set nameKEGG_CELL_CYCLEKEGG_DNA_REPLICATIONKEGG_RNA_DEGRADATIONKEGG_BLADDER_CANCERKEGG_NON_SMALL_CELL_LUNG_CANCERKEGG_THYROID_CANCERKEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSISKEGG_P53_SIGNALING_PATHWAY KEGG_PURINE_METABOLISMSizeNESNOM P‘valFDR q‘valNES normalized enrichment score NOM nominal FDR false discovery rate Gene sets with NOM Pval and FDR qval are considered as significantbiological interaction network of CENPM in liver cancer we used the network in the Network tab in cBioPortal showing the most frequently changed neighbor genes in CENPM and the most common change was RAD21 Fig a0 and Table a0miRNAs related to a0CENPMAccording to the online database the top of the miRNA families are hsamiR13075p hsamiR449b3p and hsamiR67785p related to the gene CENPM The conserved sites of the miRNA family that are widely conserved in vertebrates Fig a06a Using the Funrich database to explore the function of the identified miRNAs BP are significantly enriched in the regulation of nucleobases signal transduction cell communication transport regulation of gene expression and anogenesis CC are mainly concentrated in the nucleus cytoplasm Golgi apparatus endosome actin cytoskeleton and early endosome The MF are mainly transcription factor activity transcription regulation activity protein serine GTPase activity and ubiquitinspecific protease activity rich biological pathways in the ErbB receptor signaling network TRAIL signaling pathway Glypican pathway and syndecan1 mediated signaling events and signal transduction events mediated by hepatocyte growth factor receptor cMet Fig a06b“eDiscussionIn this work we performed a detailed assessment of CENPM expression in hepatocellular carcinoma based on the TCGA database and explored its relationship with clinicopathological features survival function immune infiltration and expression differences Understanding whether higher expression biomarkers in tumors are directly related to hepatocellular carcinoma can help us understand the mechanism of the observed clinical survival patterns In our findings the significant expression of CENPM suggests that CENPM may play an important CENPM is an role in regulating cancer progression This should draw attention to current views on the improvement of liver cancer and may reveal potential biomarkers or indicators to determine prognosisindispensable centromere protein involved in centromere assembly which regulates mitochondrial protein assembly and chromosome segregation [] Huang et a0 al [] cloned and identified the cDNA sequence of porcine PANE1 and found that porcine PANE1 gene was expressed differently in seven different tissues with the highest expression in lymph nodes and the lowest expression in kidney Until now the expression of CENPM and its potential prognostic effect on hepatocellular carcinoma has not yet been investigated our outcomes showed that the expression of CENPM in hepatocellular carcinoma was related to advanced clinical pathologic factors grade clinical stage Tclassification survival time and poor prognosis Univariate analysis uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors and Mclassification may play an indispensable role in the inclined to advance to a further advanced stage The univariate and multivariate analysis also suggested CENPM still remained freely connected with OS and recommended that CENPM may act as a potential prognostic biomarker of prognosis and therapeutic target in hepatocellular carcinoma but more researches needed to conduct for further study In addition we further analyzed various clinicopathological features of HCC samples using the UALCAN database and all of them showed high transcription of CENPMTo identify differential signaling pathways in liver cancer GSEA analysis results show that cell cycle DNA replication RNA degradation some cancers phagocytosis P53 signaling pathway and purine metabolism are differentially enriched in CENPM high expression phenotype CENPM may influence cell cycle DNA replication RNA degradation then controls the begins and development 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment plots from gene set enrichment analysis GSEAof cancer cells Kim et a0al [] was identified CENPM as a key gene that mediates the anticancer effect of garlic and cisplatin on bladder cancer and showed that patients with low CENPM expressed better progressionfree survival than patients without high expression Studies also found the CENPM genes encode a human minor histocompatibility antigen expressed by tumor cells [ ] Yu et a0al [] found CENPM could as AFPrelated diagnostic biomarkers in HCC and validate the results using quantitative realtime PCR Our study for the first time investigated the CENPM mRNA expression and its prognostic significance in hepatocellular carcinoma Chen et a0al [] demonstrated that LHX6 can inhibit the proliferation invasion and migration P53 signaling pathways during hepatocarcinogenesis Qin et a0 al [] found that P53stabilizing and activating RNA can strengthen the interaction between hnRNP K and P53 which ultimately leads to the accumulation and transactivation of P53 So CENPM may play a role via P53 signaling pathway and more researches needed to conduct in the future 0cWu a0and Yang Cancer Cell Int Page of Fig Immune infiltrates correlation with CENPM in HCC a Correlation between CENPM in HCC expression and abundance of immune infiltrates P b CENPM expression between various tumor and normal tissueTable Multivariate survival model analysis based on a0TIMER online toolClinicopathologic variableCoefHR CIP‘value SigAgeGender MaleRace BlackRace WhiteStage IIStage IIIStage IVPurityB cellsCD cellCD4 T cellsMacrophages “ˆ’ “ “ˆ’ “ “ “ “ “ˆ’ “ˆ’ “ˆ’ “ “NeutrphilsDendriticCENPMPvalue significant codes ‰¤ ‰¤ ‰¤ ‰¤ · ˆ’ “ “ “·Previous studies demonstrated that human CENPM transcript cRNA was only detected in activated B and Tcells either in a0vivo or in a0vitro These studies suggested CENPM may play important role in tumor immune response so we used an online tool to analysis immune infiltrates correlation with CENPM in HCC Multivariable Cox proportional hazard model showed that B cells CD8 T cells macrophages and dendritic cells of immune infiltrates statistically significant P in HCC indicating that these immune cells significantly affecting the prognosis A latest study showed CD8 CD68 and FoxP3 immune cells were associated with HCC particularly in the invasive margin [] Macrophages not only promote the proliferation colony formation and migration of HCC cells but also maintain tumor growth and metastasis by secreting hepatocyte growth factor HGF [] Pang et a0 al [] proposed that fusion of dendritic cells DC with tumor cells can effectively activate antitumor immunity in the body and affect tumor progression [] These studies indicate that CENPM may play an important role in tumor immune response and can be a good therapeutic target for immunotherapy 0cWu a0and Yang Cancer Cell Int Page of Fig Boxplot showing relative expression of CENPM in subgroups of patients with HCC UALCANTo determine the biological interaction network of CENPM in liver cancer we applied the most frequently changed neighbor genes of CENPM on the Network tab in cBioPortal and the most frequent change was RAD21 RAD21 is a nuclear phosphoprotein which becomes hyperphosphorylated in cell cycle M phase One study found that depletion of RAD21 resulted in reduced levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters resulting in enhanced selfrenewal of hematopoietic stem and progenitor cells HSPC [] Recent studies have shown that removing RAD21 in a background lacking Pds5 can rescue the phenotype observed only in the absence of Pds5 [] Our study may provide information on adhesion kinetics in replication fork studies in patients with liver cancer Our study also used the Targetscan online tool to distinguish CENPMrelated miRNAs To check the function of the identified miRNAs bioenrichment was performed through the Funrich database It is rich in ErbB receptor signaling network TRAIL signaling pathway Glypican pathway syndecan1 mediated signaling events and biological pathways of hepatocyte growth factor receptor cMet signaling events Studies have reported that selective cMet inhibitors have antitumor activity in HCC and have acceptable safety and tolerability in Child“Pugh A liver function patients [] A recent study found that abnormal HGFcMet upregulation and activation are often observed in bladder cancer [] Studies have also found that metastasis associated with colon cancer MACC1 regulates PDL1 expression and tumor immunity in gastric cancer GC cells through the cMetAKTmTOR pathway [] We hypothesized that CENPM may regulate the expression of cMet leading to the occurrence of HCC and more related research Fig The network for CENPM and the most frequently altered neighbor genesTable The type and a0frequency of a0CENPM neighbor gene alterations in a0HCC cBioPortalGene symbolRAD21RPS27AHCTF1NUF2PMF1Amplification Homozygous deletionMutation Total alteration 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment analysis of the miRNA altered in the CENPM in HCC Funrich and Targetscan a Conserved sites for miRNA families broadly conserved among vertebrates b Cellular components c KEGG pathway analysis d Biological processes e Molecular functionsis needed To date this study demonstrates for the first time the important role of CENPM in the prognosis of hepatocellular carcinoma However future clinical trials are needed to validate these results and promote the use of CENPM in the prognostic evaluation of hepatocellular carcinomaConclusionsOur study found that the expression of CENPM was significantly increased in patients with hepatocellular carcinoma and was related to a variety of clinical features its correlation with the level of immune infiltration and poor prognosis so CENPM may become a useful biomarker for the prognosis of patients with liver cancerKEGG Kyoto encyclopedia of genes and genomes BP Biological processes CC Cellular components MF Molecular functions OS Over survivalAcknowledgementsNot applicableAuthors™ contributionsWZH designed and analyzed the research study WZH wrote and revised the manuscript YDL and WZH collected the data and all authors contributed to final manuscript All authors read and approved the final manuscriptFundingThis work is not supported by grantsAvailability of data and materialsRNA‘seq data and corresponding clinical data were acquired from the data portal for TCGA https porta lgdccance rgovEthics approval and consent to participateNot applicableAbbreviationsHCC Hepatocellular carcinoma CENPM Centromere protein M GSEA Gene set enrichment analysis TCGA Cancer genome atlas GO Gene ontology Consent for publicationNot applicable 0cWu a0and Yang Cancer Cell Int Page of Competing interestsThe authors declare that they have no competing interestsReceived January Accepted August References Torre LA Bray F Siegel RL Ferlay J Lortet‘Tieulent J Jemal A Global cancer statistics CA Cancer J Clin “Tang Y Wang H Ma L et al Diffusion‘weighted imaging of hepatocellular carcinomas a retrospective analysis of correlation between appar‘ent diffusion coefficients and histological grade Abdominal Radiol “ Coskun M Hepatocellular carcinoma in the cirrhotic liver evaluation using computed tomography and magnetic resonance imaging Exp Clin Transplant 201715Suppl Lang H Sotiropoulos GC Brokalaki EI et al Survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers J Am Coll Surg “Jiao Y Fu Z Li Y Meng L Liu Y High EIF2B5 mRNA expression and its prognostic significance in liver cancer a study based on the TCGA and GEO database Cancer Manag Res “ Byeon H Lee SD Hong EK et al Long‘term prognostic impact of osteo‘ pontin and Dickkopf‘related protein in patients with hepatocellular carcinoma after hepatectomy Pathol Res Pract “Shen Y Bu L Li R et al Screening effective differential expression genes for hepatic carcinoma with metastasis in the peripheral blood mononu‘clear cells by RNA‘seq Oncotarget “ Renou JP Bierie B Miyoshi K Cui Y Djiane J Reichenstein M Shani M Hennighausen L Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated beta‘catenin Onco‘gene “ Bierie B Edwin M Melenhorst J et al The proliferation associated nuclear element PANE1 is conserved between mammals and fish and preferentially expressed in activated lymphoid cells Gene Expr Patterns “ Brickner AG The PANE1 gene encodes a novel human minor histocom‘patibility antigen that is selectively expressed in B‘lymphoid cells and B‘CLL Blood “ Kruppa J Jung K Automated multigroup outlier identification in molecu‘lar highthroughput data using bagplots and gemplots BMC Bioinf Li T Fan J Wang B et al TIMER a web server for comprehensive analysis of tumor‘infiltrating immune cells Cancer Res 20177721e108“e110110 https doi1011580008‘5472CAN‘‘ Chandrashekar DS Bashel B Balasubramanya SAH Creighton CJ Rodri‘guez IP Chakravarthi BVSK Varambally S UALCAN a portal for facilitat‘ing tumor subgroup gene expression and survival analyses Neoplasia “ https doi101016jneo201705002 Gao et al Sci Signal Cerami et al Cancer Discov when publishing results based on cBioPortal https doi1011582159‘ Agarwal V Bell GW Nam J Bartel DP Predicting effective microRNA target sites in mammalian mRNAs eLife 20154e05005 https doi107554eLife Pathan M Keerthikumar S Ang CS Gangoda L Quek CY Williamson NA Mouradov D Sieber OM Simpson RJ Salim A Bacic A FunRich an open access standalone functional enrichment and interaction network analysis tool Proteomics “ https doi101002pmic20140 Foltz DR Jansen LE Black BE Bailey AO Yates JR III Cleveland DW The human CENP‘A centromeric nucleosome‘associated complex Nat Cell Biol “ Huang H Deng H Yang Y et al Molecular characterization and associa‘tion analysis of porcine PANE1 gene Mol Biol Rep “ Kim WT Seo SP Byun YJ et al The anticancer effects of garlic extracts on bladder cancer compared to cisplatin a common mechanism of action via centromere protein M Am J Chin Med “ Yu Z Wang R Chen F et al Five novel oncogenic signatures could be uti‘lized as AFP‘related diagnostic biomarkers for hepatocellular carcinoma based on next‘generation sequencing Dig Dis Sci “ Chen HQ Zhao J Li Y et al Epigenetic inactivation of LHX6 mediated microcystin‘LR induced hepatocarcinogenesis via the Wntβ‘catenin and P53 signaling pathways Environ Pollut 2019252Pt A216“ Qin G Tu X Li H et al lncRNA PSTAR promotes p53 signaling by inhibit‘ing hnRNP K deSUMOylation and suppresses hepatocellular carcinoma Hepatology https doi101002hep30793 Ihling C Naughton B Zhang Y et al Observational study of PD‘L1 TGF‘β and immune cell infiltrates in hepatocellular carcinoma Front Med Laus‘anne Dong N Shi X Wang S et al M2 macrophages mediate sorafenib resistance by secreting HGF in a feed‘forward manner in hepatocellular carcinoma Br J Cancer “ Pang YB He J Cui BY et al a potential antitumor effect of dendritic cells fused with cancer stem cells in hepatocellular carcinoma Stem Cells Int Janco JMT Lamichhane P Karyampudi L Knutson KL Tumor‘infiltrating dendritic cells in cancer pathogenesis J Immunol “ Fisher JB Peterson J Reimer M et al The cohesin subunit Rad21 is a negative regulator of hematopoietic self‘renewal through epigenetic repression of HoxA7 and HoxA9 Leukemia Carvajal‘Maldonado D Byrum AK Jackson J et al Perturbing cohesin dynamics drives MRE11 nuclease‘dependent replication fork slowing Nucleic Acids Res “ Bouattour M Raymond E Qin S et al Recent developments of c‘met as a therapeutic target in hepatocellular carcinoma Hepatology “ Sim WJ Iyengar PV Lama D et al c‘Met activation leads to the establish‘ment of a TGFβ‘receptor regulatory network in bladder cancer progres‘sion Nat Commun Tong G Cheng B Li J et al MACC1 regulates PDL1 expression and tumor immunity through the c‘MetAKTmTOR pathway in gastric cancer cells Cancer Med “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

Thyroid_Cancer

"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

Moringa oleifera L from the Moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions M oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine With no reports of side effects in doses achievable by ingestion different parts of M oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders This plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women The seed and leaves powder has water purification properties through flocculation It also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries So M oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil According to the holistic or traditional medicine M oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients It is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life The high and specific immunological potential of M oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against Covid19 diseaseKeywords Moringa oleifera a0· Drumstick tree a0· Miracle tree a0· Medicinal plant a0· Cosmetics a0· Food supplementDiana Meireles and Jo£o Gomes authors contributed equally to this workdianameireleslivecompt Diana Meireles ICBAS Institute of a0Biomedical Sciences Abel Salazar University of a0Porto Rua de Je Viterbo Ferreira no a0Porto Portugal YIDAO Acupuncture and a0TCM Center Porto Portugal CIIMAR Interdisciplinary Centre of a0Marine and a0Environmental Research University of a0Porto Porto Portugal CBSin Center of a0BioSciences in a0Integrative Health Porto PortugalIntroductionMoringa oleifera L Moringa pterygosperma G wellknown as the œdrumstick or œhorseradish tree is native of Northwest India its main producer but can also be found in South Africa Northeast Africa Madagascar Tropical Asia Southwest Asia and Latin America The Moringa genus comprises species M arborea M longituba M borziana M pygmaea M hildebrandtii M drouhardii M longituba M peregrina M stenopetala M rivae M ruspoliana M Ovalifolia M Concanensis and M ole­fera Rani a0 From the Moringaceae family M oleifera is the most known studied and used species Anwar Olson with human and animal applications The various resources obtained from this plant”leaves flowers seeds pods bark and roots”can be used for cooking or in traditional medicine to treat several pathologies M oleifera has the capability to survive in humid or dry hot climates Vol01234567891 0c D a0Meireles et aland poor soils Anwar et a0al Mainenti M oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries Zongo ValdezSolana et a0al Gopalakrishnan Debajyoti et a0al M oleifera gained the title of œMiracle Tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry Tables a0 and In fact when compared to other plants from a0g of dry leafs of M oleifera we can obtain times more vitamin C than from oranges times more vitamin A than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach Oduro et a0al a0 a0Rockwood Saini et a0al Table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the United States Department of Agriculture USDA database although it is Table Nutritional values per a0g of the edible portion of M oleifera pods and leavesComponentsPer a0gRaw podsa“Raw leavesa““““Energy kcalWater gProtein gTotal lipid gCarbohydrate by difference gFibre total dietary gFatty acids total saturated gFatty acids total monounsaturated gFatty acids total polyunsaturated gFatty acids total trans gCholesterol mgVitamin A RAE µgVitamin D D2 D3 µgVitamin D IUThiamin mgRiboflavin mgNiacin mgPantothenic acid mgVitamin B6 mgVitamin B12 µgVitamin E mgVitamin C total ascorbic acid mgFolate total µgFolic acid µgSodium mgPotassium mgCalcium mgPhosphorus mgMagnesium mgIron mgZinc mgCopper mgManganese mgSelenium µga Information obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June b Average values and standard deviation published by Witt Only two values were found Witt Dried leavesb ± ± ± ± ± ““““““ ± ““““““ ± “ ± ± ± ± ± ± ± ± ““ 0cTable Amino acids and flavonols per a0g of the edible raw portion of M oleifera leavesPer a0gRaw leavesComponentsAmino acids a0Tryptophan g a0Threonine g a0Isoleucine g a0Leucine g a0Lysine g a0Methionine g a0Cystine g a0Phenylalanine g a0Tyrosine g a0Valine g a0Arginine g a0Histidine g a0Alanine g a0Aspartic acid g a0Glutamic acid g a0Glycine g a0Proline g a0Serine gFlavonols a0Isorhamnetin mg a0Kaempferol mg a0Myricetin mg a0Quercetin mgInformation obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June known that the nutrient content varies according to the plantation site Aslam and seasons Witt The nutritional value of dried leaves not existent in USDA database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by Witt From leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds Anwar et a0al Leone et a0al 2015a b Saini et a0al Fahey Debajyoti et a0al Divya et a0al M oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant Fahey Abdull Razis et a0al Divya et a0al Anwar et a0al published a table with various traditional medicinal uses of the different parts of M oleifera Anwar et a0al The attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans According the revision by Stohs and Hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of M oleifera leaves and leaf extracts achievable by oral ingestion Although there was not any report of major adverse side effects there are some important information that should be registered In fact there are some studies suggesting that M Oleifera cannot be used in combination with other modern medicines in humans A research by Gholap et a0al concluded that M oleifera has been noted to be a good regulator of insulin Thus according Sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using M oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications Another study suggests that when treating thyroids M oleifera compounds of the leaf may improve thyroid function Tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactionA research work concerning the œAcceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of M oleifera on children girls in Zambia Barichella et a0al With regards to safety concerns supplementation of a0g per day of M oleifera powder was deemed safe for children and adolescents both in the short and long term This research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of M oleifera daily showing to be still an inadequate and symptomatic dose in childrenOther studies suggest that M oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function Das et a0al Kelly Sileshi et a0al Despite the numerous positive health benefits associated with M oleifera phytochemicals there are suspicions that it contains harmful substances Fahey et a0al Annongu et a0al Maizuwo It contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects Maizuwo et a0al Some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin Fahey et a0al There are unconfirmed reports that M oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women Dutta It is also suspected that it can prevent implantation in women hence it must be avoided A review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c D a0Meireles et alTable Compilation of food supplements containing M oleifera tree parts or extracts in June Tree partsProduct informationBrandProductNaturingaMoringa capsulesLeavesMoringa teaMoringa kids multivitamin complexMoringa powderBioheraDried seeds extract Moringa capsulesLeavesMoringa syrupLeavesLeavesMiracle treeMoringa anic teaanic Moringa superfood supplements”capsulesMoringa Superfood PowderMoringa Superfood SticksIswariMoringa powder anicDrasanviLeaves dry extract Nutrabasics”MoringaRegulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditionDelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindStrengthens the immune system rich in vitamins and minerals stimulates natural defensesAdds nutritional value source of fiber protein vitamins and minerals improves physical conditionStrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoIt is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids Daily use of Moringa can help to restore your imbalances in your dietThe Moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundThe richness of its active ingredients helps maintain blood glucose levels Provides Flavonoids and Polyphenols by those attempting to conceive as it functioning as an abortifacient Nath et a0al Dutta Finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rat™s cells was verified at a0mgkg Asare et a0al However all mentioned side effects were verified with doses that far exceed the amounts used in food intake Asare et a0al So research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of M oleifera extracts and products on both human and animal models Adedapo et a0al Stohs et a0al Many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of M oleifera Fahey In fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as Ayurveda and Traditional Chinese Medicine TCM Karadi et a0al Kasote et a0al Debajyoti et a0al As an endemic source with highly digestible protein Ca Fe Vitamin C and carotenoids is considered as a suitable natural product to be used by undernourishment populations Dixit The resources obtained from a0M oleifera tree on a0a a0conventional approachLeaves and a0podsIn some countries leaves and fruits are commonly used in culinary as vegetables Leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption Moyo Olson et a0al In all ways of use and conservation M oleifera does not lose nutritional value Mahmood Leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants Table a0 Anwar Rebufa et a0al Phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids Table a0 M oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content Moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0cTable Compilation of cosmetics containing M oleifera tree parts or extracts as ingredients in June Cosmetic ingredientsProduct informationProduct brandnameSkinSecretAntiwrinkle face creamAntiaging moisturizer face creamHand creamBody milkLushAfrican paradise body conditionerQueen bee hair honeyMagical Moringa facial moisturizerCharity pot Hand and body lotionPassion fruit lip balmGo faster feet foot lotionTwinkle toes foot powderLush gardener cold pressed soapLaboratoires S©robiologiquesPURISOFT®Body shopMoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubMoringa eau de toilette Moringa body mistPurifying and protective action against environmental stress such as smoke and pollutionMoisturizing nourishingDeodorizingRemove dirt moisturizing nourishingSkin cleansingpurification protects skin against pollution heavy metals cigarette smokeSkin feels smooth and restoredDelicately scent your skin in a crisp floral aroma with MoringaLeavesM pterygosperma oilM pterygosperma leaf infusionoilM pterygosperma powderActive ingredient peptide from Moringa seedsM pterygosperma oilM oleifera leaf extractM pterygosperma seed extractM oleifera seeds and oilM oleifera leaf extractoilM pterygosperma seed extractM pterygosperma extractClarinsExtracomfort antipollution cleansing cream Eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionNeutralizes the effects of pollution and purifies the skin to restore its natural radiancePurifies and refines while preserving your skin™s natural moisture balance Neutralizes the harmful effects of pollutionOnestep facial cleanserExfoliating body scrubOnestep gentle exfoliating cleanserWater purifycomfort onestep cleanserDaily energizer cleansing gelNaturingaMoringa soap bioMoringa exfoliating face scrubMoringa O2Herbal moisturizing lotionfacial tonersoapherbal shampooconditionerShu UemuraAntiOxi pollutant and dullness clarifying cleansing oilUrban moisture hydronourishing shampooconditionerdouble serumdeep treatment masqueHigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissueMoringa seeds peel and exfoliate the skin while Moringa oil moisturizes and regenerates the skinRejuvenate nourish and protects skinRepairs strengthens reduces hair fallEnhanced power to remove micro impurities and stubborn makeup antipollution breakthroughHighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c Table continuedCosmetic ingredientsM oleifera seed extractProduct brandnameBioBeaut©Antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating gelDualphase waterproof eye makeup removerD a0Meireles et alProduct informationRemoves makeup pollution particles and excess sebum while leaving the skin well moisturized The Seed of Moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination This extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health Anwar et a0al Table a0 In dried M oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet In the leaves is found greater a variety and quantity of proteins when comparing to other tree parts Rebufa et a0al Wang et a0al Due to its nutritional rich values M oleifera can be a good enriching food additive to human diet and also an animal feed fortifier Moyo Adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition Bhargave Studies of acceptance by the consumer of enriched foodssnacks with M oleifera have been obtaining good results Ellis Jung M oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children Phytosterols from M oleifera increase estrogen production that enhance the activity of the mammary glands ducts Gopalakrishnan Doses of a0mgg of body weight given to mice result in increased milk production Also the pup weight augment with increasing doses of M oleifera leaf powder intake Titi et a0al Titi and Nurjanah Studies of toxicity in animals show that M oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes M oleifera may cause toxicity by accumulation of some elements a0Ali et a0al The amount of a0g of M oleifera dried leaf per day is the maximum recommended doseage AsieduGyekye et a0 al Table a0 compiles some food supplements based on M oleifera tree parts or extracts A hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice Table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis Bharali et a0al M oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer Gopalakrishnan et a0al carotene the major component reported from the drumsticks of the M oleifera plant as well as the presence of vitamin A and C suggest an action in the induction of antioxidant and antiinflammatory profiles Geervani and Devi Bharali et a0al Praengam et a0al It was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model Gupta et a0al Kraiphet et a0al Studies in rats showed that M oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function Kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases Sutalangka et a0al It was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a M oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days Kaur et a0al This effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitor”SSRI according to Sutalangka et a0al and Kaur et a0al 2015The influence of M oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function Vauzour et a0al Other studies with consumption of M oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects William reduction on post prandial blood glucose Ghiridhari increased insulin secretion in healthy subjects Anthanont et a0al decreased total plasma cholesterol and increased HDL Nambiar The presence of sitosterol in M oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces Jain Mbikay 0cnoitcudorpmubes niks ni noitcudeRledom laminAdna ortiv nI la a0te amreV a0 a0 pytivitca tnadixoitnAledom laminAAN yehaFnoitcefni la a0te ruaK la a0te akgnalatuSelfiorp dipil no tcapmi evitisoPledom laminA a0 a0 p raibmaN ledomnamuH a0 a0 pyattasiri yehaF la a0te lukaK la a0te ruozuaV la a0te roknoD la a0te la a0te eednoDnesodU hanajruNdna iti T la a0te itiT la a0te inor™aS la a0te nanhsirkalapoGledom laminA a0 a0 p ledomnamuH a0 a0 p la a0te tnonahtnA mailliW irahdirihGnilusni esaercnI doolb laidnarp tsop no noitcudeR ni slihportuen gnitalucric esaercnIseiduts ortiv nI a0 a0 pesoculgledom namuH a0 a0 p la a0te eurDledom laminAsserts etuca a0 a0 pledom laminA a0 a0 p lariv uehritna recnac ni romutitnAdna lairetcab itna msitamRSShti serpeditnA wnoitanibmoc ni tnas recnahne evitingoC tnatcetorporuen ralucsavorbereCairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaerTsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercnIselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo M tnereffid fo snoitca lacigolocamrahp fo elbat yrammuS elbaTarefielo M fo snoitca lacigolocamrahP ledomnamuH a0 a0 pmsidioryhtrepyh etalugeR ledomnamuH a0 a0 pamehtyre niks ni ecudeRseiduts ortiv nI a0 a0 pa ilA la a0te ilA la a0te dammahuMgnigaitna nikSstcartxe suoeuqa fognilaehdnuoW tsaerb tnadixoitna yrotammaflniitnAicrac noloc ni sisotpopa ecudnI la a0te ilarahB la a0te tehpiarK la a0te inailihaTledom laminA a0 a0 pnoitacfiixoted dnaledom laminA a0 a0 p ledomnamuHc ilA a0 a0 pamonytivitca laiborcimitnAledom laminA a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnAdna la a0te nanhsirkalapoG la a0te iramsAlAseiduts ortiv nIselknirwitnA a0 a0 pniap tnioj dna aehrraid taerT la a0te ilAledom namuH a0 a0 p la a0te nanhsirkalapoG a0 a0 pAN raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitnA la a0te lednoroTnoitardyh niks ni esaercnI ledomnamuH a0 a0 p ni laitnetop evitneverpomehCrecnac neelps dna citapeh nanhsirkalapoG la a0te ilarahBseiduts ortiv nI a0 a0 p ledomnamuHb ilA a0 a0 p dna tnadixoitna citebaiditnAseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonnikS tnadixoitna la a0te yebaR l Ednaledom laminA a0 a0 p ledomnamuHANammaflni gnul etuca fo esaerceD la a0te thginKcMledom laminA a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerceD ilarahB eDdna atpugsaD la a0teledom laminA a0 a0 p yebaR l Edna iklaMlAledom laminA a0 a0 pdik lla detaroilema dna desaercnIsretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihnIledom laminA a0 a0 pseiduts ortiv nI a0 a0 p azmaH la a0te areiV iklaMlAnajahaM narmI dna meedaNsevaeLsdoPsdeeSA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiuGledom laminA la a0te reugiuGledom laminA inamkcuRledom laminA hzimahtabnI la a0te nanhsirkalapoGseiduts ortiv nIANdipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorPstceffe evitaxalyticixototapeh decudni”lomatecarap no evitcetorpotapeH etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnArecnac eruc dna cimeloretselohcopyHsmelborp yraniruarefielo M fo snoitca lacigolocamrahPdeunitnoc elbaT eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecudeR la a0te idaraK dna ytivitca evitcetorpotycyrotercesitna recluitnA la a0te yrahduohCAN a0p a0evitcetorpotapeHipilitna dna cimeretselohcitnAledom laminA a0 a0 p amruKledom laminA a0 a0 pledom laminA a0 a0 p ahceneSledom laminA a0 a0 pcimed tcart yraniru fo tnemeganaMsmotpmys snoitcefni hgni Sdna ayruaM la a0te alkuhSledom laminA a0 a0 p ledomnamuH a0 a0 p serec¡Cledom laminAlairetcabitnA a0 a0 p la a0te reffaZseiduts ortiv nIANtceffe noitatnalpmiitnA a0p a0yrotammaflniitnA a0p a0yticixototapeHdecudnI ”lomatecaraPno evitcetorpotapeHaropsorueN tsniaga lagnufitnA inamkcuRledom laminA a0 a0 pseiduts ortiv nI ahJ a0 a0 pD a0Meireles et alSeveral M oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive Stohs and Hartman The antioxidant activity derives from the high amounts of polyphenols Leone et a0al 2015a b Verma et a0al Leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing Reactive Oxygen Species ROS production only in cancer cells which leads to cell apoptosis Gopalakrishnan The active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and G2M enrichment AlAsmari et a0al Some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model Krishnamurthy et a0al Table a0In traditional medicine a paste made of leaves is applied externally in wounds Siddhuraju and Becker Some scientific studies have shown that leave extracts have beneficial properties in skin Aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing Muhammad et a0al A M oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts Gothai et a0al A hydroalcoholic extract of M oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds Sunscreen and photo protective characteristics were studied very recently Baldisserotto et a0al When applying a cream with this extract it was also verified a reduction in sebum production Ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration Ali et a0al 2013a b c Wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with M oleifera leaf extract Ali et a0al The compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins A C and B Jadoon et a0al M oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema Ali et a0al 2013a b c Table a0 M oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed Torondel et a0al The efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarceA leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing ANyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutSsrewolFstooRkraB 0crate size and resistance on those plants and fruits Bhargave M oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that Moringa tea has adaptogenic capabilities in cases of stress Drue et a0al Table a0 Previous studies using dried Moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis McKnight et a0al An ethanolic extract of Moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of M oleifera leaves may have produced its anxiolytic effects via multiple mechanisms Bhat SeedsSeeds collected from pods can be eaten raw or cooked From M oleifera seeds a rich vegetable oil can be produced M oleifera seed oil or BehenBen oil is produced through the cold pressing of the M oleifera seeds M oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry Rashid et a0al The oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils Ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic Anwar It is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties It is also a good skin cleansing product Nadeem and Imran Table a0 details some cosmetic brands that use M oleifera leaves oil or active extracts as ingredients in the composition of their products This oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals Milled M oleifera seed shells can be used as a natural exfoliating agentMoringa oleifera seeds can also help diabetic patients Table a0 Some studies by AlMalki and El Rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of M oleifera seeds powderkg body weight during a0weeks At the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids Moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters In fact M Oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the CCl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity Hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities Hamza Treatment with M oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity Histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening From all above results and observations it can be concluded that the seeds possess promising antiarthritic property Mahajan et a0al These seeds have others appeals to the daily life and industry Seed powder showed capacity to purify water and remove heavy metals and anic compounds Sharma et a0al through low molecular weight cationic proteins mediated precipitation Kansal and Kumari There was a reduction of “ in the turbidity of the water and “ of bacterial reduction Bhargave Lea The remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value Lea Compounds such as pterygospermin moringine and benzyl isothiocya

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

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

"increasing number of studies have focused on the extragastrointestinal effects ofHelicobacter pylori H pylori including metabolic syndrome fatty liver and rheumatic and skin diseasesOsteoporosis is an asymptomatic disease that can eventually lead to fractures and has a significant impact on thequality of life of elderly individuals Sex is an influential factor that plays a crucial role in the development ofosteoporosis The aim of this study was to investigate the relationship between H pylori infection and osteoporosisand to identify potential influencing factorsMethods We conducted a crosssectional study of individuals older than years old who had undergone regularphysical examinations at the Beijing Shijitan Hospital Health Examination Center from July to October Weevaluated the associations of oste ia and osteoporosis with H pylori infection and related serum markers byusing multiple linear regression and logistic regression Then we analysed the correlation between sex andpotential serum biomarkersResults There were significant relationships between H pylori infection status and bone density in premenopausalfemales but not in males P according to Fisher™s exact test In females H pylori positivity OR P Body Mass Index BMI OR P and homocysteine HCY OR P wereassociated with osteoporosis Calcium had a trend but no statistically significant OR P relationshipwith osteoporosis Furthermore the waisttohip ratio OR P BMI OR P andtriglyceride levels OR P were significantly different by sex after adjusting for age as a confounderConclusion H pylori positivity BMI and HCY are associated with osteoporosis in premenopausal females Chronicinflammation may be involved in the relationship between H pylori and osteoporosisKeywords Female H Pylori infection Osteoporosis Premenopausal Chronic inflammationBackgroundHelicobacter pylori H pylori a gramnegative spiralshaped microaerophilic bacterium has been shown to bean important pathogen in gastrointestinal diseases []Approximately of the world population has been Correspondence maggie19950426163com3Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity Beijing Beijing ChinaFull list of author information is available at the end of the affected by H pylori and approximately millionChinese individuals are affected by this disease It maycause chronic inflammation of the gastric mucosa whichmay lead to chronic atrophic gastritis peptic ulcer diseases and gastric cancer [ ] Moreover the latest reports have described the investigation ofthe extragastrointestinal effects of H pylori including metabolicsyndrome [] fatty liver [] and rheumatic and skin diseases [] These parenteral diseases associated with H 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 Musculoskeletal Disorders Page of pylori infection seriously affect the patient™s general condition and cause a series of complicationsOsteoporosis is an asymptomatic disease characterizedby a decreased density of normally mineralized bone thatusually occurs in elderly persons It has been reportedthat approximately million men and million womenover the age of in the United States have been diagnosed with osteoporosis with an estimated millionsuffering from oste ia [] In the development ofosteoporosis there is often a long latent period beforethe appearance of the main clinical manifestation pathologic fractures Moreover the most prevalent sequelae ofosteoporosis are compression fractures of the vertebralbodies and fractures of the ribs proximal femurs humeri and distal radiuses which could have a significantimpact on the quality oflife of elderly individualsTherefore the prevention and early detection of osteoporosiselderlypopulationis particularlytheimportantforA majority of studies support the idea that at anygiven age women have a higher risk of fracture thanmen [] However men tend to have worse outcomesafter fracture than women they are twice as likely to dieafter a hip fracture than women [] Moreover sexrelated factors remain unclear Therefore exploring thedifferences in factors influencing osteoporosis in malesand females will be helpful to explore the pathogenesisof osteoporosis and early prevention of its occurrenceand developmentThere are some wellestablished risk factors for theemergence of osteoporosis such as age sex body massindex and alcohol consumption [] Recent shave focused on H pylori and osteoporosis Different researchers have proposed different theories including butnot limited to inflammation induced by H pylori infection [] and malabsorption of nutrients [ ] However there is still a lack of systematic data analyses onthe relationship between H pylori and osteoporosis Theassociation between osteoporosis and H pylori has beenstudied by many Japanese scientists and remains controversial In addition there are still some deficiencies inexisting research Few studies have focused on the correlation between H pylori and osteoporosis in Chinesepremenopausal females The sample sizes have been insufficient there is a lack of investigations on the influence of sex and there is a lack of sufficient serummarkers The aim of this study was to investigate the relationship between H pylori and osteoporosis and toidentify potential influencing factorsMethodsStudy populationBriefly men and women older than years old ienot including those aged years old who had regularphysical examinations at the Beijing Shijitan HospitalHealth Examination Center from July to October were included Our research used the following exclusion criteria for the data collection periods patientsusing the following drugs and having comorbidities thatmay cause secondary osteosis glucocorticoids thyroidparathyroid drugs psychotropic drugs anticonvulsantsselective estrogen receptor modulators SERMs vitaminD calcium and bisphosphonate patients who had ahistory of gastrectomy inflammatory bowel disease malignant diseases chronic kidney disease diabetes mellitushypohyperparathyroiddisorder acromegaly and rheumatoid arthritis includingcollagen disease Study participants who had been diagnosed with H pylori infection before or had potentiallyantiH pylori drugs in the past month were recruitedas wellhypohyperthyroidismWe also excluded postmenopausal women in thepresent study The criteria for determining menopausebased on the latest guidelines included any of the following prior bilateral oophorectomy age ‰¥ years old age years and amenorrhoeic for ormore months in the absence of chemotherapy tamoxifen or toremifene [] The female study participantswere not pregnant or lactatingData collectionAmong all the eligible individuals eligible study participants gave informed verbal consent and providedtheir basic informationincluding demographics agesex race smoking status and medicine use All ethicsapprovals were given by the Ethics Committee of BeijingShijitan Hospital affiliated with Capital Medical University and the study was performed in accordance withthe Declaration of Helsinki Blood measurements wereperformed with fresh serum obtained after a 12h fast tominimize the confounding effects of diurnal variation onhormone concentrations and included tests for glucosemetabolism liver function renal function lipid metabolism ions calcium iron tumour markers pepsinogenPG and progastrinreleasing peptide proGRP Anthropometric measurements including waist circumference cm blood pressure mmHg body weight kgand height cm were measured by trained nurses usinga standardized protocol Diastolic and systolic bloodpressure were measured in the morning Body massindex BMI was calculated by taking a person™s weightin kilograms divided by their height in metres squaredThe waisttohip ratio WHR is measured as waist circumference divided by hip circumference H pylori infection status was measured by a []C breathing test onthe same day with an empty stomach Tumour markerswere measured using enzymelinked immunosorbentassay methods 0cWang BMC Musculoskeletal Disorders Page of Diagnosis of osteoporosisThe bone mineral density BMD of lumbar vertebrae“ L2“ was measured by DXA using a DiscoveryDXA system Hologic Bedford Massachusetts The results provided BMD gcm2 and young adult meanbone mineral density The diagnosis of osteoporosis wasperformed in accordance with the World Healthanization diagnostic criteria from the World Healthanization WHO Collaborating Center for Metabolic Bone Diseases [] A value for BMD within onestandard deviation SD of the average BMD of normaladults was regarded as normal Oste ia is defined asa BMD that lies between and standard deviationsbelow the young adult mean value BMD more than SD below the young adult mean value was classified asosteoporosis []Statistical analysesWe used SPSS statistical software version for dataanalyses Continuous variables were reported as means ±standard deviations whereas categorical variables werepresented as percentages Study subjects were first classified into three groups according to BMD classificationnormal oste ia and osteoporosis The KolmogorovSmirnov test was used to verify whether the data fit anormal distribution and all continuous variables thatdid not conform to a normal distribution underwenttransformation for analysis Summary and grouping datafor baseline characteristics the laboratory examinationwere compared using a t test for continuous variablesand Fisher™s exact test for categorical variables in the Hpylori and H pylori groups Moreover we divided thestudy population by sex and used Fisher™s exact test toverify whether there were sex differences in the relationship between H pylori and osteoporosisMultivariateadjusted odds ratios ORs and confidence intervals CIs were calculated using logistic regression among the three subgroups To further analysethe relationship between H pylori infection status andosteoporosis we created a model using total cholesterolTC triglycerides TG uric acid UA BMI WHRlowdensity lipoprotein cholesterol LDLC Creactiveprotein CRP homocysteine HCY H pylori infectionstatus calcium Ca vitamin B12 and the BMD groupsthat analysed the different sexes separately The studypopulation™s highdensity lipoprotein cholesterol HDLC and glucose levels were all normal so we did not include them in the modelMeanwhile we analysed the differences in markers between males and females We separated the study population according to sex and further analysed the patients™basic data in the same way as we analysed the baselinecharacteristics We further analysed the relationship between sex and markers to find sex differences in therelationship between H pylori infection and osteoporosis The markers included TC LDLC UA TG glucoseGLU CA724 CEA BMI SP systolic blood pressureBP diastolic blood pressure WHR HCY CRP vitaminB12 Ca and Ghb Other serum biomarkers were entered into the model as factors using their normal valueas the grouping criterion All the models were adjustedfor age as a confounder A twotailed Pvalue wasconsidered to be statistically significantResultthe participants areThe baseline characteristics ofshown in Table The mean age was ± years were male and were femaleAmong the study population had osteoporosis had oste ia and hadnormal BMDs The mean ages ofthe osteoporosisoste ia and normal groups were ± ± and ± respectively There wereno significant differences in age among the participantsin the osteoporosis oste ia and normal BMD groupsFifty percent of the males had normal BMDs ofthe males had oste ia and of the males hadosteoporosis In addition of the females had normal BMDs of the females had oste ia and of the females had osteoporosis There was no significant difference in sex distribution among these threegroupsAmong all the study participants had H pyloriinfections and did not have H pylori infectionsTable shows that CA724 t P was significantly different between the study participants withH pylori infection and those without H pylori infectionand there were no significant differences between the Hpylori infection status and the BMD status groupsIn Table we separate the study population accordingto sex We found that there was a significant relationshipbetween H pylori infection status and bone density infemales P but not in males P As shown in Table there was a significant association of H pylori positivity OR CI“ P BMIOR CI“ P and HCY OR CI “ P with osteoporosis inpremenopausalOR CI“ P and TC OR CI“ P had a trend but neither wassignificantly associated with osteoporosisfemales CaMeanwhile we analysed the differences in markers between males and females which are shown in Table We found that age P SP P BP P P WHR P Hb P BMI platelets P UA P TC P TG P HDLC P GLU P 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients according to the H pylori infection statueFemaleMalenormaloste iaosteoporosisAgeSexBMDSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueB12pmolLWHR Waisttohip ratio TC Total cholesterol Ghb Glycosylated hemoglobin TG Triglyceride UA Uric acid AST Aspartate aminotransferase ALT Alanineaminotransferase Ca Calcium Cre Creatinine DP Diastolic blood pressure Hb Hemoglobin HDLC Highdensity lipoprotein cholesterol LDLC Lowdensitylipoprotein cholesterol BMD Bone mineral density OR Odds ratio proGRP Progastrinreleasing peptide WHR WaisttoHip Ratio SP Systolic blood pressure PGPepsinogen GLU Glucose CRP Creactive protein HCY HOMOCYSTEINEBold indicates statistically significant values ± ± ± Table the relationship between the H pylori infection andthe BMD in different genderFemaleH pylori infection ˆ’H pylori infection Normal BMDoste iaosteoporosisMaleH pylori infection H pylori infection ˆ’Normal BMDoste iaosteoporosisBMD Bone mineral densityBold indicates statistically significant valuesPvaluePvalue CEA P Ghb P iron P HCY P and B12 P demonstrated significant differences between males and femalesThe relationship between sex and markers is shown inTable We found that WHR OR CI“ P TG OR CI“ P and BMI OR CI“ P were significantly different bysex adjusting for age as a confounderDiscussionOsteoporosis is an important health and societal burdenin elderly people not only in females but also in malesThere are numerous osteoporosisrelated fracture riskfactors including age sex race lifestyle and concomitant medical conditions [] In men osteoporosis isunderrecognized and undertreated Only a few men are 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and BMDOR95CIPvalueTC mmolLQ1365“Q2521“BMI kgm2Q1179“Q2240“UA umolLQ1187“Q2358“TG mmolLQ10“Q2171“LDLC mmolLQ10“Q2313“C13Q1without H pylorQ2with H pylorWHRQ1079“Q2085“HCYQ10“ μmolLQ2 μmolLCRPQ1 “ mgLQ2 mgLCaQ1 “ mgLQ1 mgLB12Q10516pmolLQ2 pmolLAbbreviations as in Table OR Odds ratio C13 13C breathing test positiveBold indicates statistically significant valuesscreened for osteoporosis even after a fracture [] Thetreatment rate is much lower in males than in females[] Meanwhile more men than women die every yeardue to hip fractures [] Hence we also included menin the study population to determine the risk factors forosteoporosisSome studies about the influence of sex on osteoporosis remain controversial In our study there was a significant relationship between H pylori and osteoporosisin premenopausal females but not in males The reasonsfor the difference between males and females are asfollows First differences in clinical outcomes of osteoporosis in men and women may be rooted in the biologic properties of bone BarrettConnor E holds theview that there are sexspecific differences in the number of osteoprogenitor cells and in hormone responsesand regulation [ ] Second men have a greater bonesize trabecular BMD and bone area at the radius andtibia than women even after adjusting for weight andheight which may lead to a decrease in osteoporosis andfracture [] Third men undergo a slow decrease inBMD with increasing age while women experience aprofound period of rapid bone resorption especiallyafter entering menopause [] Last but not least somestudies support the idea that men are more likely to suffer from secondary diseasefor example rheumatoidarthritis alcoholism excessive smoking gonadal deficiencies and others [] which may lead to sustainablebone lossUnfortunately the relationship between osteoporosisand H pylori infection is still controversial Some studieshold the view that there is no difference between menand women in the relationship between H pylori andosteoporosis [ ] Some studies hold the view that Hpylori is related to osteoporosis only in women ShihChun Lin conducted a retrospective study including women and showed that H pylori is related to osteoporosis in females [] while others think that there is nocorrelation between them in females Daisuke Chindaconducted a study of healthy women and found thatH pylori is not a significant risk factor for oste ia[] In our study we analysed the relationship betweenH pylori infection and osteoporosis We found a significant relationship between H pylori infection status andbone density in premenopausal females but not in malesWe suspect this may be due to the difference in the aetiology of osteoporosis between males and females However we did not find any other studies on this and itrequires more systematic research for analysisAfter analysing the differences between males and females we found that there were significant differencesin BMI WHR and TG in the study population Thisstudy provides evidence for followup research on sexdifferences in the relationship between H pylori andosteoporosisMost studies hold the view that obesity is related toosteoporosis [] It is generally believed that obesitymay be a protective factor against bone loss and osteoporosis [] However the effect of obesity remains unclear On the one hand obesity has traditionally beenconsidered positive for bone because of the beneficial effect of mechanicalloading [] On the other handpeople hold the view that BMI may harm BMD Osteoblasts and adipocytes both stem from marrow mesenchymal stromal cells Osteoblasts and adipocytes are in a 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients in different genderAgeSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± B12pmolLAbbreviations as in Table OR Odds ratioBold indicates statistically significant values ± Female ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvaluecompetitive relationship and an increase in adipocyteswill inhibit osteoblasts [] In our study there was asignificant relationship between BMI and osteoporosisIncreased BMD levels in obese people may be associatedwith increased mechanical loading and strain this is acomplicated problem that cannot be generalizedIn our study we found that H pylori infection is associated with a decrease in bone density The possible reasons are as follows First H pylori infection may causesystemic inflammation and increase the production oftumour necrosis factorα interleukin1 and interleukin [] These cytokines are directly involved in the reduction of BMD We found that HCY is related toosteoporosis which supports this hypothesis Secondosteoporosis may be related to a decrease in vitamin B12levels [] Serin et al™s study examined patientswithout atrophy erosions or ulcers and they found thatthe histopathological scores for both antral and corpusH pylori density and inflammation were significantly inversely associated with serum vitamin B12 levels [] Inour study although we did not find a significant relationship between B12 and osteoporosis we still supportthe relevant theory The absence of our results may bedue to a lack of sufficient data and the influence of confounding factors Last but notleast most patientschronically infected with H pylori manifest pangastritiswith reduced acid secretion due to bacterial virulencefactors inflammatory cytokines and various degrees ofgastric atrophy [] Calcium is ionized in acidic conditions and absorbed in the small bowel Therefore in either hypochlorhydria or achlorhydric stomachs calciumabsorption is impaired [] Moreover the longtermuse of acid suppressants for example proton pump inhibitors may lead to osteoporosis or a decrease in BMDLimited experimental evidence indicates that PPI mayinfluence calcium absorption leading to compensatoryphysiologic responsesincluding secondary hyperparathyroidism which may cause an increase in the rate ofosteoclastic bone resorption [] The results showedthat calcium had a trend though it was not statisticallysignificant P with osteoporosis Our results donot support the theory that there is a correlation between Ca and osteoporosis but it may be that Helicobacter pyloriinfection may cause calcium absorptiondamage and affect BMD We did not analyse vitamin Dlevels which could affect both bone homeostasis and theinflammatory state [] Although H pyloriinfectioncausing a decrease in bone density is supported by mostresearchers the effect of early eradication therapy is stillinsufficient Replogle ML holds the view that early 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and genderWHRQ1079“Q2085“TC mmolLQ10“Q2 LDLC mmolLQ10“Q2313“UA umolLQ1187“Q2358“TG mmolLQ10“Q2171“GLU mmolLQ10“Q2611“CA724UmlQ10“Q2690“CEA ngmlQ10“Q2500“BMI kgm2Q1179“Q2240“SP mmHgQ190“Q2 BP mmHgQ160“Q2 HCY μmolLQ10“Q2 CRP mgLQ1 “Q2 B12pmolLQ10“Q2 Ca mgLQ1 “OR95CI Pvalue 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and gender ContinuedQ1 GhbQ10“Q261“OR95CIPvalueAbbreviations as in Table OR Odds ratioBold indicates statistically significant valuesMale and female have different normal value in UA and WHR UA 149416umolL in male89357umolL in female WHR in male in femaleeradication therapy may eliminate chronic inflammationfrom H pylori [] Some s have also reported animprovement in B12 levels after complete eradication[ ] which requires further investigationDespite its relevant findings our study had several limitations First because most patients cannot rememberthe time of HP infection accurately we were not able toobtain the time of HP infection so different infectiontimes may have had an impact on the results Secondwe did not collect vitamin D data the sample size of ourdata was not large enough and the study populationonly included participants from Beijing Shijitan Hospitalmeaning that there might have confounding factors because of differences in the distribution of hospital studypopulations Further largescale studies in the generalpopulation are needed to validate our results Third thestudy participants were all Chinese and the findingsmight not be generalizable to other ethnic populationsIn addition we only found some differences betweenmen and women but failed to further explore themConclusionsH pyloriis associated with osteoporosis in premenopausal females BMI and HCY are related to osteoporosis in premenopausal females Chronic inflammationmay be involved in the relationship between H pyloriand osteoporosisSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035867Additional file AbbreviationsDXA Lumbar dualenergy xray absorptiometry ORs Odds ratiosCIs Confidence intervals H pylori Helicobacter pylori SERMs Selectiveestrogen receptor modulators proGRP Progastrinreleasing peptideBMI Body mass index WHR Waisttohip ratio BMD Bone mineral densityWHO World health anization SD Standard deviation TC Totalcholesterol TG Triglycerides UA Uric acid LDLC Lowdensity lipoproteincholesterol Ghb Glycosylated haemoglobin HDLC Highdensity lipoproteincholesterol GLU Glucose PG Pepsinogen CA Calcium DP Diastolic bloodpressure Hb Haemoglobin SP Systolic blood pressure C13 13C breathingtest positiveAcknowledgementsNot applicableAuthors™ contributionsLH has made substantial contributions to the design of the work SSJ hasprovided the data obtained the consent of participants and analyzed thedata preliminarily ZLC has made contributions to the collection of data andparticipated in the drafting of the DFX has analyzed and interpretedof the data WJW has drafted the manuscriptSH has helped to revise themanuscript All authors have read and approved the manuscriptFundingThe study was supported by a program from the Beijing City Health Systemœ High Levels of Health Technical Personnel Training Aid and the CapitalClinical Characteristic Applied Research Project NoZ181100001718120Funds are used for the data collection portionAvailability of data and materialsThe datasets analyzed during the current study are not publicly availablebecause it includes the study population personal information which isillegal to but are available from the corresponding author onreasonable requestEthics approval and consent to participateAll the ethics approval has been given by the ethics committee of Beijingshijitan hospital affiliated to capital medical university and have beenperformed in accordance with the Declaration of Helsinki We used theparticipants data by anonymous All involved study populations were fromthe previous physical examination group and part of the population are notin Beijing All the participants received the informed consent by email Weread informed consent to patients or their immediate family members bytelephone and inform them of the purpose and significance of the studyand obtain their oral consent which is approved by the ethics committeeConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests in this sectionAuthor details1Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity No Tieyi Road Beijing China 2Department of Physicalexamination center Beijing Shijitan Hospital Capital Medical University No Tieyi Road Beijing China 3Department of Gastroenterology BeijingShijitan Hospital Capital Medical University Beijing Beijing ChinaReceived March Accepted August ReferencesGlaser DL Kaplan FS Osteoporosis Definition and clinical presentationSpine Phila Pa Suppl12s“6s Malfertheiner P Megraud F O'Morain CA Atherton J Axon AT Bazzoli F Management of Helicobacter pylori infectionthe Maastricht IVFlorence consensus report Gut “ 0cWang BMC Musculoskeletal Disorders Page of Matsuhisa T Aftab H Observation of gastric mucosa in Bangladesh the Pan BL Huang CF Chuah SK Chiang JC Loke SS Relationship betweenHelicobacter pylori infection and bone mineral density a retrospectivecrosssectional study BMC Gastroenterol GłogowskaSzeląg J Szeląg M Stolecki M Kudła M Obesity andosteoporosisconnections between adipose tissue and bone Wiad Lek cz “Fassio A Idolazzi L Rossini M Gatti D Adami G Giollo A The obesityparadox and osteoporosis Eat Weight Disord “Tucker KL Hannan MT Qiao N Jacques PF Selhub J Cupples LA Lowplasma vitamin B12 is associated with lower BMD the Framinghamosteoporosis study J Bone Miner Res “Smolka AJ Schubert ML Helicobacter pyloriinduced changes in gastric acidsecretion and upper gastrointestinal disease Curr Top Microbiol Immunol“ Wright MJ Proctor DD Insogna KL Kerstetter JE Proton pumpinhibitingdrugs calcium homeostasis and bone health Nutr Rev “ Bellavia D Costa V De Luca A Maglio M Pagani S Fini M Vitamin Dlevel between calciumphosphorus homeostasis and immune system newperspective in osteoporosis Curr Osteoporos Rep Shih HM Hsu TY Chen CY Lin CL Kao CH Chen CH et alAnalysis ofPatients with Helicobacter pylori Infection and the Subsequent Risk ofDeveloping Osteoporosis after Eradication Therapy A NationwidePopulationBased Cohort Study PLoS One 2016119e0162645 Published Sep httpsdoi101371journalpone0162645 Avcu N Avcu F Beyan C Ural AU Kaptan K Ozyurt M The relationshipbetween gastricoral helicobacter pylori and oral hygiene in patients withvitamin B12deficiency anemia Oral Surg Oral Med Oral Pathol Oral RadiolEndod “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationscountry with the lowest incidence of gastric cancer and Japan the countrywith the highest incidence Helicobacter “Upala S Jaruvongvanich V Riangwiwat T Jaruvongvanich S Sanguankeo AAssociation between helicobacter pylori infection and metabolic syndromea systematic review and metaanalysis J Dig Dis “Tang DM Kumar S The association between helicobacter pylori infectionand nonalcoholic fatty liver disease Curr Gastroenterol Rep Smyk DS Koutsoumpas AL Mytilinaiou MG Rigopoulou EI Sakkas LIBogdanos DP Helicobacter pylori and autoimmune disease cause orbystander World J Gastroenterol “Office of the Surgeon G Reports of the Surgeon General Bone Health andOsteoporosis A Report of the Surgeon General Rockville MD Office of theSurgeon General US Nguyen ND Ahlb HG Center JR Eisman JA Nguyen TV Residual lifetimerisk of fractures in women and men J Bone Miner Res “Haentjens P Magaziner J ColonEmeric CS Vanderschueren D Milisen KVelkeniers B Metaanalysis excess mortality after hip fracture amongolder women and men Ann Intern Med “ Yoshimura N Suzuki T Hosoi T Orimo H Epidemiology of hip fracture inJapan incidence and risk factors J Bone Miner Metab 200523Suppl78“ Chung YH Gwak JS Hong SW Hyeon JH Lee CM Oh SW et alHelicobacter pylori a possible risk factor for bone health Korean J FamMed “ Recker RR Calcium absorption and achlorhydria N Engl J Med “Serin E Gumurdulu Y Ozer B Kayaselcuk F Yilmaz U Kocak R Impact ofhelicobacter pylori on the development of vitamin B12 deficiency in theabsence of gastric atrophy Helicobacter “Tyagi NK DhesyThind S Clinical practice guidelines in breast cancer CurrOncol 201825Suppl 1S151“s60Kanis JA Melton LJ 3rd Christiansen C Johnston CC Khaltaev N Thediagnosis of osteoporosis J Bone Miner Res “ Dontas IA Yiannakopoulos CK Risk factors and prevention of osteoporosisrelated fractures J Musculoskelet Neuronal Interact “ Gennari L Bilezikian JP New and developing pharmacotherapy forosteoporosis in men Expert Opin Pharmacother “ Bougioukli S Κollia P Koromila T Varitimidis S Hantes M Karachalios T et alFailure in diagnosis and undertreatment of osteoporosis in elderly patientswith fragility fractures J Bone Miner Metab “ Alejandro P Constantinescu F A review of osteoporosis in the older adultan update Rheum Dis Clin N Am “ McMillan J FatehiSedeh S Sylvia VL Bingham V Zhong M Boyan BD et alSexspecific regulation of growth plate chondrocytes by estrogen is viamultiple MAP kinase signaling pathways Biochim Biophys Acta “Lenart BA Neviaser AS Lyman S Chang CC EdoborOsula F Steele B

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

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

Cabozantinib is an oral multikinase inhibitor whose targets include vascular endothelial growth factor receptors MET and the TAM family of kinases TYRO3 AXL MER Cabozantinib is approved for patients with advanced hepatocellular carcinoma who have been previously treated with sorafenib based on improved overall survival and progressionfree survival relative to placebo in the phase III CELESTIAL study During CELESTIAL the most common adverse events AEs experienced by patients receiving cabozantinib included palmarplantar erythrodysesthesia fatigue gastrointestinalrelated events and hypertension These AEs can significantly impact treatment tolerability and patient quality of life However AEs can be effectively managed with supportive care and dose modifications During CELESTIAL more than half of the patients receiving cabozantinib required a dose reduction while the rate of treatment discontinuation due to AEs was low Here we review the safety profile of cabozantinib and provide guidance on the prevention and management of the more common AEs based on current evidence from the literature as well as our clinical experience We consider the specific challenges faced by clinicians in treating this patient population and discuss factors that may affect exposure and tolerability to cabozantinib IntroductionThere has been a marked increase in liver cancer deaths in recent years In there were a0 new cases of liver cancer worldwide and liver cancer accounted for almost deaths making it the sixth most prevalent cancer worldwide [] The most common primary malignancy of the liver is hepatocellular carcinoma HCC [] The frequency burden and etiology of HCC vary across geographic regions and populations but are linked to prevalence of predisposing chronic hepatic conditions such as Electronic supplementary material The online version of this s1152 contains supplementary material which is available to authorized usersKey Points Cabozantinib represents a treatment option for patients with advanced hepatocellular carcinoma who progress after sorafenibAdverse events associated with cabozantinib may be effectively managed with supportive care and dose modifications thereby allowing patients to continue treatment at the appropriate dose with minimum interruptionStudies of cabozantinib in the firstline setting are ongoing by understanding the safety profile of this drug clinicians will be able to balance efficacy with tolerability for each patient Gabriel Schwartz GabrielSchwartzucsfedu Gastrointestinal Medical Oncology Clinic University of a0California San Francisco Fourth St Fourth Floor San a0Francisco CA a0 USAIndiana University Health Simon Cancer Center Indianapolis IN USA Department of a0Medicine University of a0California San Francisco San a0Francisco CA USAIRCCS Istituto Clinico Humanitas Rozzano Milan Italy viral hepatitis and nonalcoholic fatty liver disease NAFLD or nonalcoholic steatohepatitis NASH which generally develop in the setting of cirrhosis [ ] In recent years the incidence of nonviral HCC has increased while the proportion of HCC cases related to viral hepatitis has declined [ ] Additional risk factors for HCC include alcohol consumption smoking obesity and diabetes [] As the epidemiology of these conditions has evolved so too has the etiology of HCC []Vol0123456789 0c G a0Schwartz et alFor patients with advanced HCC the vascular endothelial growth factor receptor VEGFR“targeting tyrosine kinase inhibitor TKI sorafenib has been a standard of care [] however the treatment landscape has been transformed in recent years with the introduction of newer TKIs immunotherapies and monoclonal antibody therapies [] This provides clinicians and patients with a variety of treatment options based on mechanism of action and safety profileCabozantinib is a multikinase inhibitor that targets VEGFR “ MET the TAM family of kinases TYRO3 AXL MER RET ROS1 KIT TRKB FLT3 and TIE2 [ ] several of which are implicated in tumor growth angiogenesis and immune regulation [] VEGFR MET and AXL have been implicated in the pathogenesis of HCC [“] A capsule formulation of cabozantinib was first approved in for treatment of progressive metastatic medullary thyroid carcinoma MTC [] The tablet formulation not bioequivalent or interchangeable with the capsule [] was subsequently approved for patients with advanced renal cell carcinoma RCC [ ] and more recently for patients with advanced HCC who have received prior sorafenib [ ] The approval in HCC was based on outcomes from the pivotal phase III CELESTIAL trial which showed significantly improved overall survival OS and progressionfree survival PFS with cabozantinib relative to placebo in patients who received prior sorafenib [] The safety profile of cabozantinib was manageable nearly all patients receiving cabozantinib experienced an adverse event AE but these were effectively managed with dose modification and supportive care measuresClinicians treating patients with advanced HCC can face significant challenges as many patients present with cirrhosis and comorbidities that can impact treatment tolerability Adequate assessment of liver function and management of comorbidities are therefore essential before and during HCC treatment [] Here we provide guidance on the management of AEs associated with cabozantinib in patients with advanced HCC We briefly review outcomes from CELESTIAL and focus on managing some of the more common AEs experienced by patients based on current evidence from the literature as well as our own clinical experience Cabozantinib in a0Hepatocellular Carcinoma CELESTIALIn the phase III CELESTIAL study patients with advanced HCC were randomized to treatment with cabozantinib a0mg daily or placebo [] Patients were required to have had prior treatment with sorafenib and could have received up to two prior systemic regimens for HCC Eastern Cooperative Oncology Group ECOG performance status PS of or and ChildPugh class A liver function see Electronic Supplementary Table a0 for definition were also required At the second planned interim analysis patients had been randomized The study met its primary endpoint with significantly improved OS with cabozantinib relative to placebo median OS was versus months hazard ratio confidence interval [CI] “ p a0 a0 Cabozantinib also improved PFS with a median of versus months hazard ratio CI “ p a0 a0 as well the objective response rate per Response Evaluation Criteria In Solid Tumors RECIST v11 vs a0 p a0 a0 Safety and a0TolerabilityAllcause AE rates were generally higher in the cabozantinib arm than in the placebo arm some of the more common AEs experienced by patients in the cabozantinib a0 versus placebo arms included diarrhea vs decreased appetite vs palmarplantar erythrodysesthesia PPE vs fatigue vs nausea vs hypertension vs vomiting vs asthenia vs and increased aspartate aminotransferase AST vs Fig a0 The most common grade AEs in the cabozantinib versus placebo arms were PPE vs hypertension vs increased AST vs fatigue vs and diarrhea vs Overall the safety profile of cabozantinib was consistent with those from the phase III studies in RCC and MTC with gastrointestinal GI events PPE fatigue and hypertension being the most common AEs experienced by patients across studies [ ]In addition to supportive care measures protocolspecified dose modification including dose interruption and reduction was utilized to manage AEs [] Eightyfour percent of patients in the cabozantinib arm had an AE that led to dose interruption and had a dose interruption due to a grade AE [] Sixtytwo percent of patients had at least one dose reduction due to an AE [] and dose reduced due to a grade AE [] Thirtythree percent of patients had a second dose reduction [] Median time to first and second dose reduction in the cabozantinib arm was a0days and a0days respectively PPE was the event that most commonly led to dose interruption and dose reduction followed by diarrhea and and fatigue and [] Although most patients receiving cabozantinib required a dose interruption the rate of discontinuation due to treatmentrelated AEs was relatively low in the cabozantinib arm vs in the placebo arm indicating that the majority of AEs were adequately managed with dose modification and supportive care In the cabozantinib group AEs that led to treatment discontinuation in ‰¥ a0 of patients were PPE fatigue decreased appetite diarrhea and nausea In a subgroup analysis of patients 0cAE Any grade [Grade Grade ]Fatigue [ ]Hypertension [ ]Increased AST [ ]Increased ALT [ ]Asthenia [ ]Nausea [ ]Vomiting [ ]Decrease appetite [ ]Weight loss [ ]Diarrhea [ ]Constipation [ ]Abdominal pain [ ]PPE [ ]Fig Incidence rates for select AEs experienced by patients with HCC receiving cabozantinib during the CELESTIAL trial [] AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders AE adverse event ALT alanine aminotransferase AST aspartate aminotransferase HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesiawho received sorafenib as the only prior treatment for HCC duration of prior sorafenib did not appear to impact the types or rates of grade AEs []Generally the more common AEs emerged in the first weeks of treatment Fig a0 However clinicians should be aware of infrequent or serious events that can occur in the later phases of treatment Hemorrhagic events of grade or higher were reported in of patients in the cabozantinib arm including five patients with a grade event Bleeding complications are associated with antiangiogenic therapies and may arise as a result of reduced vascular integrity [] Median time to onset of hemorrhagic events was a0weeks in CELESTIAL Other grade or higher rare but serious AEs in patients receiving cabozantinib included fistulas of patients GI perforations and arterial and venous or mixed thrombotic events [] Median time to first occurrence was approximately a0weeks for GI perforations weeks for venous and arterial thromboembolisms and weeks for fistulas [] Two patients in the cabozantinib arm had developed ChildPugh C ie decompensated cirrhosis by the week assessment []Reversible posterior leukoencephalopathy syndrome RPLS a syndrome of subcortical vasogenic edema diagnosed by magnetic resonance imaging has been reported with cabozantinib and other TKIs [ ] Although there were no RPLS events during CELESTIAL [] clinicians should be aware of the symptoms which include headaches seizures confusion changes to vision or altered mental function [ ] Osteonecrosis of the jaw ONJ whereby necrotic jaw bone becomes exposed is another rare but serious AE associated with TKIs including cabozantinib [“] although again there were no ONJ events reported during this study [] The use of antiresorptive drugs in patients with bone metastases is also associated with development of ONJ []A post hoc analysis estimated the incremental qualityadjusted lifeyears accrued with cabozantinib compared with placebo using the fivedimension fivelevel EuroQol questionnaire [] Cabozantinib treatment was associated with an initial decline in mean total qualityadjusted lifeyears during the first “ a0months relative to placebo followed by longterm improvement that was significantly greater than that observed with placebo p a0 a0Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c Fig Rates and timing of select AEs in patients with HCC receiving cabozantinib during the CELESTIAL trial The size of the circle is proportional to the AE rate AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders black generalother AE adverse event ATE arterial thrombotic event GI gastrointestinal GR grade HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesia VTE venous thrombotic eventMedian time to first dosereduction to mg weeksG a0Schwartz et alMedian time to seconddose reduction to mg weeksFistulas Hemorrhage GR ATEs VTE Wound complication GI perforations Hepatic encephalopathy Diarrhea PPE Hypertension Median time to first occurrence weeks Factors Affecting Tolerability of a0Cabozantinib Co‘morbiditiesHCC emerges primarily in older adults [] In addition to the underlying HCC etiology older adults with HCC are likely to have additional comorbidities such as cardiovascular or pulmonary disease [] and it is not uncommon for patients with HCC to have multiple comorbidities [] Liver cirrhosis with compromised liver function and decreased hepatic reserve is a major risk factor for HCC development Other HCCrelated comorbidities include hepatitis B virushepatitis C virus infection alcoholic liver disease NASH and diabetes [] In addition metabolic syndrome characterized by hyperlipidemia and hypertension is linked to development of NAFLD which may progress to NASH cirrhosis and finally HCC [] For patients with HCC assessment of liver function is a key step in treatment decisionmaking [] Patients with moderate or severe hepatic impairment are predominantly excluded from clinical trials in HCC therefore treatment of these patients is complicated by a lack of prospective clinical data as well as competing comorbidities []Although the number of patients with HCC and prior an transplant is limited these patients are generally excluded from clinical trials and treatment is complicated by the need for immunosuppression TKIs may be used to treat posttransplant HCC recurrence although supporting data are limited The use of TKIs in these patients is complex so treatment decisions should involve collaboration between the oncology and transplant medicine care teams The use of sorafenib in patients receiving mammalian target of rapamycin inhibitorbased immunosuppression has been associated with an increased risk of fatal bleeding [ ] Immunotherapies are associated with an increased risk of an rejection in posttransplant patients [] Cabozantinib Clearance and a0ExposureTKIs are associated with high interpatient variability in clearance and exposure which may affect both efficacy and tolerability This variability may be due to a variety of factors including genetic background drug“drug interactions drugfood interactions and renal or hepatic impairment [] As evidenced by exposureresponse modeling patients with low clearance of cabozantinib may have higher exposure and an increased risk of developing certain AEs [ ] Awareness of these nuances may help clinicians to mitigate their effects thereby balancing efficacy with tolerability Hepatic and a0Renal ImpairmentAccording to pharmacokinetic analyses of patients with HCC and other tumor types mild hepatic impairment is predicted to have a minimal effect on cabozantinib exposure [] therefore adjustment of the recommended 60mg starting dose is not necessary for patients with Child“Pugh A 0cliver function [ ] Data on the pharmacokinetics of cabozantinib in patients with moderate ChildPugh B or severe Child“Pugh C hepatic impairment are limited [] As per the US Food and Drug Administration FDA prescribing information the starting dose of cabozantinib should be reduced to mg in patients with moderate hepatic impairment while cabozantinib is not recommended for patients with severe hepatic impairment [] Note that the European Summary of Product Characteristics SmPC does not recommend dose adjustments for moderate hepatic impairment owing to limited data [] For patients with HCC increased exposure due to hepatic impairment should be considered if intolerable AEs develop and dose modification undertaken as recommended Fig a0 [ ] Cabozantinib should be used with caution in patients with mild or moderate renal impairment owing to the potential for increased exposure although no dose adjustments are necessary Cabozantinib is not recommended for use in patients with severe renal impairment owing to lack of data on safety and efficacy in this population [ ] Drug“Drug and a0Drug“Food InteractionsGiven the range of comorbidities that may exist in patients with advanced HCC it is important to review all concomitant medications for potential interactions prior to initiation of treatment with cabozantinib Certain medications and foods have been shown to modulate the pharmacokinetics of cabozantinib which may in turn impact exposure levels efficacy and risk of AEs Cabozantinib is metabolized in the liver primarily by the enzyme cytochrome P450 3A4 CYP3A4 [] therefore CYP3A4 inhibitors or inducers may impact exposure examples of CYP3A4 inducersinhibitors are shown in Electronic Supplementary Table a0 Strong CYP3A4 inhibitorsinducers should be avoided in patients receiving cabozantinib If concomitant administration of a strong CYP3A4 inhibitor is necessary then the cabozantinib Recommended dose at initiation mg Except for¢ Patients with moderate hepatic impairment or coadministration of a strong CYP3A4 inhibitor initiate cabozantinib at mg ¢ Patients with coadministration of a strong CYP3A4 inducer initiate cabozantinib at mg Safety assessmentNo AEsGrade Grade AE or ONJSupportive caresee Tables “DosemodificationImprovementtolerableelbarelotnIlitnu esod dloHgrade ‰¤Continue at tolerated doseReduce dose by mg and restart mg †’ mg mg †’ mg mg †’ mg mg †’ mg or discontinueImmediate Discontinuation¢ Severe hemorrhage¢ Development of GI perforation or unmanageable fistula¢ Serious thromboembolic event eg myocardial infarction cerebral infarction¢ Hypertensive crisis or severe hypertension despite optimal medical management¢ Nephrotic syndrome¢ Reversible posterior leukoencephalopathy syndromeFig Cabozantinib dosing algorithm [ ] AE adverse event CYP3A4 cytochrome P450 3A4 GI gastrointestinal ONJ osteonecrosis of the jawManagement of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et aldose should be reduced by a0mg for example from to a0mg [] Conversely the cabozantinib dose should be increased by a0mg if strong CYP3A4 inducers need to be coadministered [ ]Cabozantinib should not be taken with any food as this may affect absorption [] The label recommends that cabozantinib be taken at least h before or at least h after eating [] Grapefruit and grapefruit juice are strong CYP3A4 inhibitors and should be avoided [ ]Cabozantinib may be used with caution in patients who are receiving concurrent antiarrhythmics or other QTprolonging agents [] This is based on a study of patients with MTC who received a daily 140mg capsule dose of cabozantinib recommended for this indication in which the mean deltadelta QT interval was increased by approximately “ a0ms with upper CIs not exceeding ms [] Such an increase is within the range considered to be acceptable for oncology drugs in this setting [] No patient in the aforementioned study or in CELESTIAL had a confirmed QTcF QT corrected using Fridericia™s method a0 ms [] which is considered clinically significant [] For patients receiving cabozantinib monitoring with periodic electrocardiogram and electrolyte measurements may be advisable particularly in patients with risk factors such as cardiac disease or a prior history of QT prolongation [] Concomitant use of proton pump inhibitors PPIs such as esomeprazole does not affect cabozantinib exposure levels [] However PPIs may cause hypomagnesemia which is linked to an increased risk of QT prolongation [] Therefore coadministration of PPIs and cabozantinib should be undertaken with caution following an individualized assessment of the patient™s baseline magnesium levels and concomitant medications that may also influence QT Pretreatment AssessmentsGiven the heterogeneity of the HCC patient population and the complexity associated with comorbidities and concomitant medications all patients should undergo a comprehensive assessment of medical history prior to initiation of treatment with cabozantinib Ideally the multidisciplinary care team should include an oncology pharmacist [] A œbrown bag medication review should be carried out prior to treatment initiation [] whereby the patient brings in all current medications including overthecounter medicines vitamins herbal remedies etc Therapeutic duplications should be eliminated for example concomitant PPIs and histamine H2 antagonists H2 blockers Switching and deprescribing should be considered where possible to minimize the risk of drugdrug interactions Adverse Event ManagementThe AE profile of cabozantinib is generally similar to that of other VEGFRtargeting TKIs with GIrelated AEs fatigue PPE and hypertension being the most common AEs [] Other AEs that occur less frequently can also have a significant impact on quality of life QoL and treatment adherence such as mucosal inflammation [] Hepatobiliary AEs such as elevated AST alanine aminotransferase ALT and bilirubin are particularly relevant in the context of advanced HCC and need to be carefully monitoredProphylactic and supportive care measures for the more common cabozantinibassociated AEs grade or tolerable grade are outlined in Tables a0 and discussed in the upcoming sections Symptom gradings are summarized in Electronic Supplementary Table a0 Dose interruption is recommended for management of intolerable grade AEs not resolved with supportive care measures or for any grade AEs Fig a0 [ ] Cabozantinib may be reinitiated at a reduced dose once the event resolves to grade ‰¤ a0 Palmar“Plantar ErythrodysesthesiaPPE is one of the more common events associated with anticancer therapies including VEGFRtargeting multikinase inhibitors [“] PPE is characterized by pain redness tingling and swelling of hands and feet [] Presentation may vary according to the etiologic agent PPE induced by TKIs is typically localized to pressurebearing areas in contrast to that caused by chemotherapy which has a more diffuse pattern It has been hypothesized that inhibition of multiple angiogenic pathways by TKIs may compromise repair of capillary microtrauma in areas exposed to mechanical stress such as the hands and feet [ ] Although not lifethreatening PPE can rapidly progress to a debilitating condition negatively impacting QoL [ ]Prophylaxis and prompt management of emerging symptoms may help to minimize the impact of PPE on QoL and adherence Table a0 Prophylactic measures predominantly involve skin care practices to remove hyperkeratotic areas and to minimize friction and damage prior to the start of treatment [ ] Recommendations include use of thick cotton gloves and socks padded insoles in shoes and avoidance of heat or friction on the hands and feet [ ] Patients with potentially predisposing comorbidities such as peripheral neuropathy [ ] as well as patients with persistent symptoms may benefit from involvement of a podiatrist andor dermatologist within their multidisciplinary care team [] Treatment strategies involve moisturization prevention of infection and analgesia [ ] Monitoring is crucial so that emerging symptoms can be proactively managed Patients should be assessed at baseline 0cTable Adverse event management strategies”palmar“plantar erythrodysesthesia PPE PPEProphylaxisProvide education on prophylactic skin care before starting treatment []Advise manicure and pedicure before and during treatment to remove hyperkeratotic areas [ ]Protect sensitive areas recommend sunscreen with SPF protection ‰¥ a0 thick cotton gloves and socks padded insoles and wellfitting shoes avoid heat sources and use cooling aids and avoid activities that may cause force or rubbing on the hands and feet eg heavy lifting dish washing [ ] delegate such tasks to caregiversAdvise on optimal hand cleaning avoid fragrancedfoaming soaps and hand sanitizers containing alcohol ensure hands are dried thoroughly after cleaning []Prophylactically administer keratolytic cream eg urea [ ]Monitor regularly in order to proactively manage skin toxicities evaluate at baseline monitor up to weekly for the first “ months and monthly thereafter [ ]Supportive careContinue prophylactic measures []Maintain moisture of skin using emollients [ ]Consider topical treatment with salicylic acid urea “ cream either alone or with tazarotene cream or fluorouracil cream andor clobetasol cream topical analgesics may be added for pain control [ ]Topical cortisone and clobetasol may also be used consider oral analgesics eg NSAIDs pregabalin cautious use of opioids [ ]Consult with a dermatologist to drain blisters and remove hyperkeratotic areas []To prevent infection of cracked skin soak in equal parts vinegar and water for min per day [] a0Antibiotics should be prescribed only if there is evidence of infection [] a0There is limited evidence for the use of pyridoxine vitamin B6 []NSAID nonsteroidal antiinflammatory drug SPF sun protection factorTable Adverse event management strategies”fatigue FatigueProphylaxisProvide patient education about fatigue management tools and available support []Establish baseline fatigue levels with a fatigue scale and remeasure regularly during patient visits []Ensure adequate fluid and nutritional intake []Advise behavioral modifications balancing rest with physical activity recommendations include relaxation massage yoga aerobic or resistance exercise programs and energy conservation strategies [“]Assess thyroid function prior to treatment and monitor during treatment [ ]Supportive careRule out alternative causes of fatigue eg anemia endocrine disorders such as hypothyroidism pain dehydration hypercalcemia or depressionanxiety [ ]Advise patient to increase activity consider referral to a physical therapist []Consider referral to nutritional counselor for nutritional therapy []Incorporate psychosocial measures including cognitive therapy social support biofeedback and sleep therapy []Incorporate management with psychostimulants eg methylphenidate [ ] or corticosteroids eg methylprednisolone []Owing to effects on CYP3A45 substrates including cabozantinib longterm use of modafinil should be avoided []CYP3A4 cytochrome P450 3A4 CYP2C19 cytochrome P450 2C19Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et alTable Adverse event management strategies”gastrointestinal GastrointestinalDiarrheaProphylaxisInstruct patients to monitor food and fluid intake [] a0Recommended water intake per day from all beverages and food [] L oz for women L oz for men a0Advise patients to keep a stool diary and to promptly report diarrhea to their healthcare provider [ ]Advise patients to avoid foods that may cause GI events such as lactosecontaining foods caffeine highfat or highfiber food eg nuts seeds legumes and raw fruit and vegetables [ ]Implement dehydration prevention management through oral rehydration with electrolytes []Supportive careAdminister loperamide at the first sign of diarrhea [ ] a0 mg orally followed by mg every h until “ h after last bowel movement maximum of mg in h a0For chronic diarrhea “ mg twice daily titrated as needed a0Alternatives to loperamide include diphenoxylate and tincture of opium []Implement supportive dietary modifications continuous oral hydration correction of fluid and electrolytes small frequent meals avoid lactosecontaining food and drink [ ] a0The BRAT bananas rice applesauce toast diet may help to alleviate mild diarrhea []If there are signs of severe dehydration administer IV fluid replacement isotonic saline or balanced salt solution []Rule out nontreatmentrelated causes eg infectious diarrhea []Decreased appetiteProphylaxisAdvise patients to monitor their appetite and weight []Encourage patients to consume highprotein calorierich food fruit and vegetables nutritional supplements that they may snack on throughout the day [ ]Advise patients to preprepare and freeze nutritional preferred food []Supportive careTreat underlying nausea []Consider involving a dietitian who may recommend scheduled eating times []Recommend a highcalorie diet []Provide dietary education alongside dietary modifications andor nutritionalvitamin supplements []Use a pharmacologic agent to stimulate appetite such as a CB1 receptor agonist dronabinol [ ] systemic corticosteroid methylprednisolone [ ] progestin megestrol acetate [ ] or mirtazapine [ ]NauseavomitingProphylaxisAssess risk factors for nauseavomiting prior to treatment []Metoclopramide may be administered prophylactically []Advise patients to avoid foods that are overly sweet greasy fried or spicy []Supportive careAntiemetic agents such as dopamine receptor antagonists eg metoclopramide prochlorperazine or 5HT3 receptor agonists eg ondansetron are recommended for management of nausea or vomiting [ ] a0Certain NK1 receptor agonists eg aprepitant and netupitant and dexamethasone are inducers inhibitors andor substrates of CYP3A4 and thus could alter cabozantinib exposure [ ] however the potential for ondansetron to prolong the QT interval must also be considered [] There is moderate evidence for olanzapine an antipsychotic drug that blocks multiple neurotransmitters as an antiemetic in this setting [] 0cTable continuedMucosal inflammationstomatitisProphylaxisA comprehensive dental examination should be conducted prior to treatment to identify potential complications []Mitigation of potential risk factors [ ] a0Modification of illfitting dentures a0Appropriate care for preexisting dental problems such as caries ulcers etcRegular oral assessments should be conducted throughout treatment [ ]Educate patients on good oral hygiene and oral care protocols including written instructions [] a0The oral cavity should be washed using salinecontaining mouthwash up to four times daily and dentures should be regularly cleaned []Painful stimuli eg smoking alcohol hot fooddrink sharp or spicy food should be avoided [ ]Supportive careTreat pain with doxepin mouthwash or viscous lidocaine [ ]Lactobacillus lozenges may be used to reduce inflammation []Obtain bacterialviral culture if oral infection is suspected and treat infection as clinically indicated []5HT3 5hydroxytryptamine CB1 cannabinoid CYP3A4 cytochrome P450 3A4 GI gastrointestinal IV intravenous NK neurokininTable Adverse event management strategies”hypertension HypertensionProphylaxisMonitor BP before initiation of cabozantinib using a minimum of two standardized BP measurements alongside patient history physical assessment directed laboratory evaluation and an instrument test to determine cardiovascular risk factors [ ]Educate patients on BP selfmonitoring and advise they keep a BP log []BP should be well controlled prior to initiating cabozantinib ensure patients who have already been prescribed antihypertensive therapy are adherent and that therapy has been titrated to effective doses [ ]Check for potential drugdrug interactions of existing antihypertensive agents with cabozantinib Supplementary Table a0Consider effects of concomitant medications on BP eg antiinflammatory drugs can increase BP opiates can lower BP []Monitor BP during cabozantinib treatment weekly during first cycle every ‰¥ a0“ weeks thereafter []Supportive careAdd antihypertensive medications or increase dose of existing medication as indicated [ ]Patients with portal hypertension should be treated with nonselective betablockers []The antihypertensive agent should be carefully considered owing to potential inhibition of CYP3A4 [ ] Supplementary Table a0 a0Thiazides angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may be used to treat hypertension and are not known CYP3A4 substrates [“ ] a0Thiazide diuretics should be prescribed with caution owing to the associated risk of diarrhea [] a0Diltiazem and verapamil are moderate inhibitors of CYP3A4 [] a0Amlodipine felodipine lercanidipine nisoldipine and nifedipine are not considered to be CYP3A4 inhibitors []BP blood pressure CYP3A4 cytochrome P450 3A4and monitored at least weekly for the first “ months of treatment and monthly thereafter [ ] Close monitoring in the early stages of treatment need not involve weekly visits”phone calls from a clinician nurse or pharmacist may facilitate monitoring in between scheduled appointments [] Patients should be encouraged to report early signs of PPE to their healthcare provider [] it may also be reassuring for patients to know that early reporting and management of AEs

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

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

Thyroid_Cancer

New diseaseCase reportAtypical manifestations of COVID19 in general practice a case of gastrointestinal a0symptomsSardam Faraidon Wahab1 Brian Bridal L¸gstrup2 SUMMARYDuring the previous months we have seen the rapid pandemic spread of SARS CoV2 Despite being considered a respiratory virus it has become clear that other clinical presentations are possible and some of these are quite frequent In this paper a case of a man in his late 70s showing atypical symptoms in general practice is presented Apart from fever the patient complained of diarrhoea borborygmus loss of appetite and nausea He developed no respiratory symptoms during his disease Due to his symptoms malignant disease was suspected and he was referred for further testing which revealed typical COVID19 findings on a chest CT scan The occurrence of atypical symptoms is discussed including the importance of recognising these in an ongoing pandemicBACKGROUNDIn December a cumulation of patients with pneumonia of unidentified cause was registered in Wuhan Hubei Province China Prior to this six strains of human pathogenic coronaviruses had been identified In February the WHO and the International Committee on Taxonomy of Viruses established the classification of the seventh human pathogenic virus SARS CoV2 as the disease causative agent of COVID191 While the name of the virus indicates respiratory disease the ongoing pandemic has shown that COVID19 is capable of causing symptoms from several an systems either concomitant with respiratory illness or as the only manifestation Recognising the clinical characteristics of COVID19 typical as well as atypical is of high importance in the prevention of further spread of the virus In this report an atypical case of COVID19 in general practice is presented Furthermore the significance of alternative manifestations is discussedCASE PRESENTATIONThe case of a man in his late 70s is presented The patient had a medical history of herpes zoster and stroke without sequelae He first contacted his general practitioner in mid March due to days of fever which was gradually decreasing Rectal temperature was °C The patient™s complaints were musculoskeletal body aches influenza like symptoms and headache These symptoms had subsided at the time of initial contact He also experienced rumbling of the stomach diarrhoea a general feeling of malaise and unease weight loss and night sweats He denied shortness of breath cough or other airway related symptomsAt the objective examination the patient appeared tired and exhausted but with normal awareness contact and cerebral condition There were no signs of respiratory distress and respiratory frequency was per minute Examination of the eyes and oral cavity as well as auscultation of the heart and lungs were normal There was no palpable lymphadenopathy in the head neck and periclavicular regions Abdominal inspection palpation percussion and auscultation were with normal findings Laboratory results are presented in table Blood pressure was mm Hg and pulse was estimated to be approximately beats per minute Urine dipstick showed trace of protein The condition was diagnosed as a viral infection and blood samples were collected for further testing He was given a control appointment days laterAt his control appointment the patient reported poor appetite and nausea and food intake was sparse Otherwise symptoms had remained the same with a continuous feeling of malaise There was no stomach pain but he had abdominal discomfort His headache had diminishedThe objective examination was with unchanged general condition The patient still appeared exhausted but otherwise his respiratory condition and appearance were natural Digital rectal examination revealed an enlarged and hard prostate with normal lateral boundaries No faeces or blood was seen on the examination glove and there was no pain on examination There was an increase in C reactive protein CRP to with a normal white and red blood cell count Due to continuous diffuse symptoms primarily abdominal discomfort and weight loss the patient was sent for further investigations This included occult cancer screening with thorough blood work and a CT scan of the throat chest abdomen and pelvisTwo weeks after the onset of symptoms the CT scan showed bilateral ground glass opacities GGO in the lungs raising the suspicion of infectious or postinfectious foci As a result of these findings combined with the short history fever and an increase in CRP COVID19 was thought of as a possible diagnosis The patient was tested for SARS CoV2 with materials obtained from tracheal suctioning The test came out positive for SARS CoV2 and negative for influenza virus and respiratory syncytial virus In the mean time the patient™s condition had deteriorated and he 1General Medicine Hospitalsenhed Midt Vib Denmark2Cardiology Aarhus University Hospital Aarhus N DenmarkCorrespondence toSardam Faraidon Wahab sardam w hotmail comAccepted August BMJ Publishing Group Limited No commercial re use See rights and permissions Published by BMJTo cite Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profileTestCRP mgLAlanine aminotransferase ULMonocytes —109LLeucocytes —109LNeutrophils —109LEosinophils —109LBasophils —109LLymphocytes —109LThrombocytes —109LHaemoglobin gLErythrocyte sedimentation rate mmhourReticulocytes —1012LHaptoglobin gLFerritin µgLTransferrin µmolLPlasma iron µmolLBlood glucose mmolLPlasma creatinine µmolLeGFR mLminPlasma kappa chain Ig free mgLPlasma lambda chain Ig free mgLPlasma kappa chain to lambda chain ratio Ig freeIgM gLHigh density lipoprotein mmolLValue day day day Reference range““““““““““““““““““Day indicates first contact to general practice CRP development is denoted and the rest are results from day The following tests were normal HbA1c INR potassium sodium alkaline phosphatase total bilirubin TSH antinuclear antibodies IgA urate alpha fetoprotein HBsAg anti HCV HAV IgM folate amylase M component IgG cobalamin B12 PSA albumin and human choriogonadotropinCRP C reactive protein eGFR estimated glomerular filtration rate HAV hepatitis A virus HbA1c Haemoglobin A1c HBsAg hepatitis B surface antigen HCV hepatitis C virus INR international normalized ratio PSA prostate specific antigen TSH thyroid stimulating hormonefelt unwell Therefore the doctor admitted the patient to the hospital at the COVID19 isolation floorAt admission oxygen saturation was without oxygen therapy Furthermore he had a respiratory frequency of per minute temperature of °C blood pressure of mm Hg and otherwise stable vital parameters During the first days of admission he developed an oxygen demand of up to Lmin to uphold an oxygen saturation of “ The patient was hospitalised for a total of days Throughout the course of the disease he did not complain of regular sore throat stuffy nose sneezing cough dyspnoea or other airway related symptomsINVESTIGATIONSThe suspicion of COVID19 in our patient was raised by the radiology department The chest CT scan was performed with arterial phase contrast It mainly showed peripheral but also central peribronchovascular GGO Streaky consolidations were seen in the right upper lobe Multiple swollen lymph nodes were detected in the mediastinum and both lung hila The key findings are presented in figures “ There were insignificant laminar subsegmental atelectasis in the posterobasal areas of both lower Figure CT scan in axial plane showing streaky consolidations in the right upper lobe arrowslobes No pericardial or pleural effusions were seen Apart from minimal mucosal thickening and polyp like changes in the right maxillary sinus there was no other pathology detected in the airwaysOUTCOME AND FOLLOWUPThe patient was seen for a follow up appointment months after discharge He reported complete recovery without sequelae On discharge he felt fatigue which gradually subsided in the following “ weeks There were no complications during Figure CT scan in axial plane showing ground glass opacities arrowsWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profile at follow up weeks after dischargeTestReference rangeValueFigure CT scan in axial plane showing ground glass opacities extending to the pleura arrowshospitalisation He particularly mentioned a pronounced positive effect of oxygen therapy which increased his appetite and general condition within hours He was slowly regaining the kg of weight that he had lost CRP alanine aminotransferase and blood monocytes had all normalised weeks after initial contact Follow up blood tests are shown in table DISCUSSIONSARS CoV and the Middle East respiratory syndrome coronavirus are among the previously identified human pathogenic coronaviruses Both are known to cause respiratory and enteric illness Gastrointestinal GI symptoms appear to be less common C reactive protein mgLAlanine aminotransferase ULMonocytes —109LLeucocytes —109LNeutrophils —109LLymphocytes —109LThrombocytes —109LHaemoglobin gLTotal cholesterol mmolLTriglycerides mmolLCreatine kinase ULLow density lipoprotein mmolLHigh density lipoprotein mmolL““““““““in SARS CoV2 compared with SARS CoV3 but studies are continually published Since COVID19 is still a novel disease it might be too early to conclude which symptoms are typical and which are not A recent comprehensive systematic review and meta analysis reported a pooled prevalence of digestive symptoms of The three most common GI manifestations were loss of appetite diarrhoea and nausea or vomiting While fever was the most frequent manifestation and relatively constant across the studies shown in table diarrhoea ranged from to In the meta analysis by Mao et al4 the frequency of diarrhoea ranged from to It is also important to note that symptoms from different an systems may present at different times in the course of the disease For instance diarrhoea and cough may occur after fever has abated5 These circumstances which may complicate the diagnostic process are important to recogniseThe cause of these different presentations is an interesting matter Factors such as genetics age immunological response and viral properties could be relevant explanations As a result of a synonymous mutation variations in viral components responsible for antigenicity and immunogenicity were found in a recent study This indicates the possible existence of strains with either increased or decreased virulence6 The founder effect must be taken into consideration though Further studies are needed to validate the significance of these findings and contribute to the understanding of the evolutionary trends of SARS CoV2 Studies of the already known coronaviruses have shown that the virions resemble a wreath or a crown due to the conformation of the surface glycoproteins This explains the Latin name corona In contrast to most enveloped viruses these glycoproteins enable the virus to withstand the conditions of the GI tract and thus spread faeco orally7 Xiao et al8 found that of patients had SARS CoV2 RNA in stool Among these still tested positive in faecal samples after converting to negative in respiratory samples Similar to SARS CoV virological studies Table Prevalence of symptoms Sore throatDyspnoeaFeverCough Diarrhoea MyalgiaFigure CT scan in coronal plane showing mediastinal lymph node enlargement arrowZhang et al21Liu et al22Xu et al23Chen et al24Myalgia or fatigue““Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseasehave pointed towards the ACE2 receptor as the site of cell entry of SARS CoV29 Previous studies have shown that ACE2 is an enzyme which physiologically counters the activity of the renin angiotensin aldosterone system and thus decreases blood pressure10 It is a membrane bound receptor which is expressed in the vascular endothelia cardiovascular and renal tissue epithelium of the small GI tract and testes ACE2 is abundantly present in the alveolar epithelium of the lungs and the small intestinal tract including the duodenum jejunum and ileum11 The mechanism by which GI manifestations occur is presumably multifactorial Positive immunofluorescent staining of ACE2 and intracellular viral capsid protein in GI tissue has been reported8 Combined with the presence of viral RNA in faecal samples this suggests secretion of infectious virions in faecal matter These findings indicate that direct virus mediated tissue damage may be a possible pathophysiological explanation The endothelial expression of ACE2 facilitates the involvement of vascular beds across several ans Histopathological examination of the small intestine has shown endotheliitis of the submucosal vessels with apoptotic bodies and accumulation of inflammatory cells This may cause endothelial dysfunction vasoconstriction and mesenteric ischaemia13 Evidence of interstitial oedema with infiltration of plasma cells and lymphocytes in GI tissue has also been reported8 These findings contribute to the understanding of GI manifestations of COVID19It is of high importance that healthcare workers are familiar with typical as well as atypical presentations and the course of this novel disease Due to the focus on respiratory symptoms in the early stages of the pandemic our patient was not initially suspected for COVID19 and thus he was sent for further testing without isolation In our case the main suspicion was influenza disease or occult cancer Among the different blood tests in table the elevated ferritin level particularly stands out It is known that ferritin levels may increase during inflammatory infectious and malignant disease among others Zhou et al14 found that ferritin levels were clearly elevated in cases of COVID19 with fatal outcome compared with survivors Another recent study found significantly elevated ferritin levels in patients with severe COVID19 and it was the last blood parameter to normalise A decrease in ferritin levels was not seen along with patient improvement Therefore it was suggested that it is a sensitive marker of severe COVID19 but it cannot be used for disease assessment15 Moreover it has been found that elevated ferritin erythrocyte sedimentation rate CRP fibrinogen and procalcitonin were higher in patients with thrombotic complications16 The present knowledge thus suggests that elevated ferritin level is a marker of severe COVID19 and is associated with thrombotic complicationsAfter the CT scan the main differential diagnoses were pneumonia eosinophilic pneumonia and COVID19 A systematic review of chest CT results in patients showed that GGO was the most frequent finding and was present in of the patients while had bilateral lung involvement17 Radiological findings of GGO have also been reported in asymptomatic patients18 This demonstrates the importance of guidelines being continually updated and communicated to clinicians As already mentioned a significant number of patients are tested positive for virus RNA in stool samples Although it is still not clear to what extent SARS CoV2 spreads faeco orally testing of stool should be further investigated in order to optimise disease controlAs an additional note related to atypical symptoms a recent study found that patients might present with conjunctivitis as the only symptom of COVID1919 Finally some patients experience Patient™s perspectiveIt all began when I came home from tennis and felt slightly cold with a mild fever In the following days the fever continued and fluctuated between °C and °C and my appetite was decreasing At first I could not eat anything sweet Later this came to include ordinary food as well I developed a feeling of unease in my body and I was prescribed paracetamol for my fever and general condition I experienced severe sweating every time I took paracetamol My wife is a former nurse and we were both convinced that I was having influenza The Danish Health Authorities had encouraged citizens to stay home in the presence of cough fever shortness of breath or musculoskeletal aches Approximately a week after onset I went to my physician and my blood tests showed signs of mild inflammation After three days I came back for a check up appointment which showed a further increase in the inflammatory count I had no appetite and felt very ill tired and disheartened I experienced severe unease and slept very poorly After a thorough examination the doctor said œAt this time I don™t know the cause of your condition I would like to refer you for further examination in order to rule out severe illness and malignant disease and to find the underlying cause I was given an appointment the following day for further blood tests and imaging The imaging of my lungs showed signs of possible coronavirus infection and I was admitted to hospital in isolation I was tested with a throat swab and some fluid was obtained from my airways through a tube in my nasal cavity I was then discharged and told that we would be informed about the test results Late in the evening close to midnight we were contacted with the results I had coronavirus Half an hour later an ambulance arrived and I was admitted to an isolated COVID19 unit at another hospital nearby Further tests were conducted and I was given nasal oxygen therapy because of a low oxygen level in my blood Shortly after I felt significantly better The only therapy I received during my eight days at the hospital was oxygen therapy For every day that passed I felt better and my appetite returned When I was discharged I was told that I had to be symptom free for at least hours before I could consider myself recovered Did I have any remnants of the disease I was very tired but this receded in the following three to four weeks While I am writing this it has been seven weeks since I was discharged I have recovered completely and my condition is now as good as it was before I fell sick with coronavirus I now play tennis three times a week again and I am looking forward to the reopening of the gym Throughout my illness I have received fantastic treatment by all parts of the health care service and I am deeply gratefulLearning points –º Our understanding of typical and atypical symptoms of COVID19 is still under development –º The existing evidence suggests that gastrointestinal manifestations are present –º This calls for increased focus on atypical non respiratory symptoms of COVID19 for optimal disease control –º It is important to recognise typical as well as atypical presentations of COVID19 in general practice and other places with initial contactWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0colfactory and gustatory disturbances such as hyposmia anosmia and dysgeusia in the absence of other symptoms20Acknowledgements Maria Tudb was actively involved in clinical decision making as senior consultant in general practiceContributors SFW was the lead author of the manuscript and was involved in initial care and management case identification manuscript write up and drafting editing and literature review BBL was senior supervisor and was involved in acquisition of data and imaging and performed critical revision and editing of the manuscript for important intellectual content SFW and BBL were equally involved in the design and interpretation of dataFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewedThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedREFERENCES Has¶ks¼z M Kili§ S Sara§ F Coronaviruses and SARS COV2 Turk J Med Sci “ Jin Y Yang H Ji W et a0al Virology epidemiology pathogenesis and control of COVID19 Viruses Cipriano M Ruberti E Giacalone A Gastrointestinal infection could be new focus for coronavirus diagnosis Cureus 202012e7422 Mao R Qiu Y He J S et a0al Manifestations and prognosis of gastrointestinal and liver involvement in patients with COVID19 a systematic review and meta analysis Lancet Gastroenterol Hepatol “ Kobayashi K I Kaki T Mizuno S et a0al Clinical characteristics of patients with coronavirus disease in Japan a single center case series J Infect Dis “ Kim S J Nguyen V G Park Y H et a0al A novel synonymous mutation of SARS CoV2 is this possible to affect their antigenicity and immunogenicity Vaccines 103390vaccines8020220 [Epub ahead of print May ]New disease Patrick R Murray KSR Michael A Pfaller medical microbiology ELSEVIER Xiao F Tang M Zheng X et a0al Evidence for gastrointestinal infection of SARS CoV2 Gastroenterology “ Wan Y Shang J Graham R et a0al Receptor recognition by the novel coronavirus from Wuhan an analysis based on decade long structural studies of SARS coronavirus J Virol 101128JVI0012720 [Epub ahead of print Mar ] Vaduganathan M Vardeny O Michel T et a0al Renin Angiotensin Aldosterone system inhibitors in patients with Covid19 N Engl J Med “ Donoghue M Hsieh F Baronas E et a0al A novel angiotensin converting enzyme related carboxypeptidase ACE2 converts angiotensin I to angiotensin Circ Res 200087E1“ Jia HP Look DC Shi L et a0al Ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia J Virol “ Varga Z Flammer AJ Steiger P et a0al Endothelial cell infection and endotheliitis in COVID19 Lancet “ Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Li Y Hu Y Yu J et a0al Retrospective analysis of laboratory testing in patients with severe or critical type novel coronavirus pneumonia Lab Invest “ Al Samkari H Karp Leaf RS Dzik WH et a0al COVID19 and coagulation bleeding and thrombotic manifestations of SARS CoV2 infection Blood “ Salehi S Abedi A Balakrishnan S et a0al Coronavirus disease COVID19 a systematic review of imaging findings in patients AJR Am J Roentgenol “ Pan Y Yu X Du X et a0al Epidemiological and clinical characteristics of asymptomatic SARS CoV2 carriers J Infect Dis Scalinci SZ Trovato Battagliola E Conjunctivitis can be the only presenting sign and symptom of COVID19 IDCases 202020e00774 Pellegrino R Cooper KW Di Pizio A et a0al Corona viruses and the chemical senses past present and future Chem Senses 2020101093chemsebjaa031 [Epub ahead of print May ] Zhang H Shang W Liu Q et a0al Clinical characteristics of cases of COVID19 in Huanggang and Taian China Infection 101007s15010020014405 [Epub ahead of print May ] Liu K Fang Y Y Deng Y et a0al Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province Chin Med J “ Xu X Yu C Qu J et a0al Imaging and clinical features of patients with novel coronavirus SARS CoV2 Eur J Nucl Med Mol Imaging “ Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “Copyright BMJ Publishing Group All rights reserved For permission to reuse any of this content visithttpswwwbmjcomcompanyproductsservicesrightsandlicensingpermissionsBMJ Case Report Fellows may reuse this article for personal use and teaching without any further permissionBecome a Fellow of BMJ Case Reports today and you can –º Submit as many cases as you like –º Enjoy fast sympathetic peer review and rapid publication of accepted articles –º Access all the published articles –º Reuse any of the published material for personal use and teaching without further permissionCustomer ServiceIf you have any further queries about your subscription please contact our customer services team on or via email at supportbmjcomVisit casereportsbmjcom for more articles like this and to become a FellowWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0c'

Thyroid_Cancer

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

Thyroid_Cancer

"Pancreatic cancer PC is one of the most aggressive cancers and has an extremely poor prognosisworldwide Long noncoding RNA lncRNA has been reported to be a potential prognostic biomarker in theinitiation and prognosis of PC Nevertheless the biological functions and the detailed molecular mechanism ofLINC00514 in PC remain unclearMethods We measured the expression level of LINC00514 in PC tissues and cell lines by quantitative realtime PCRGain and lossoffunction experiments were performed to explore the bioeffects of LINC00514 on PC developmentboth in vitro and in vivo Subcellular fractionation luciferase reporter assay RNA immunoprecipitation assay pulldown assay and western blotting were performed to investigate the oncogenic molecular mechanisms ofLINC00514Results In this study LINC00514 was shown to be upregulated in PC tissues and cell lines Increased LINC00514expression was significantly associated with the clinical progression and prognosis of PC patients In additionsilencing LINC00514 inhibited PC cell proliferation migration and invasion while LINC00514 overexpressionpromoted these processes Moreover LINC00514 knockdown remarkably inhibited PC development and metastasisin vivo Deeper investigations indicated that LINC00514 acted as a sponge for microRNA285p miR285p in PCand that Rap1b was a downstream target of miR285p Furthermore the positive correlation of LINC00514 andRap1b and the negative correlation between miR285p and LINC00514 or Rap1b were revealed Based on therescue assays Rap1b inhibition partially suppressed the oncogenic effect of LINC00514 overexpression on PC cellproliferation migration and invasionConclusions This study is the first to characterize the oncogenic function of the long noncoding RNA LINC00514in pancreatic cancer progression by acting as a competing endogenous RNA ceRNA of miR285p to upregulateRap1b expression Understanding this molecular mechanism might contribute to further discoveries of betterdiagnostic and therapeutic options for pancreatic cancerKeywords LINC00514 Pancreatic cancer Proliferation Invasion miR285p Rap1b Correspondence songzhiwangtongjieducnDepartment of Oncology the First Affiliated Hospital of Nanchang University Yongwaizheng Street Nanchang Jiangxi People™s Republic ofChina The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHan Journal of Experimental Clinical Cancer Research Page of Table Primers involved in the studyGeneLINC00514Rap1bMiR285pGAPDHForward primerGAGGCAGGAGAATCGCTTGAACCACAGCAATGAGGGATTTATACTGGTGTCGTGGGTCGACGCTCTCTGCTCCTCCTGTTCU6Abbreviation LINC00514 long intergenic nonprotein coding RNA GAPDH glyceraldehyde 3phosphate dehydrogenaseCTCGCTTCGGCAGCACAReverse primerGAGGCAGGAGAATCGCTTGAACCTGACCTTGTTCCTTCCCTACCTCGCTTCGGCAGCACAATCCGTTGACTCCGACCTTCACAACGCTTVACGAATTTGCGTFig LINC00514 was upregulated in PC and predicted a poor prognosis a LINC00514 expression was detected in PC tissue and adjacentnormal tissue by qRTPCR bd Associations between LINC00514 expression and tumor size Lymph node metastasis or clinical stage weredetected by qRTPCR e qRTPCR was applied to confirm the expression level of LINC00514 in PC cell lines and normal pancreatic epithelial cellline f KaplanMeier analysis was used to assess the relation between LINC00514 expression level and overall survival in PC patients p05p01 p001 All experiments were repeated at least for three times and mean ± SD was used to represent the final result PC pancreaticcancer qRTPCR quantitative realtime polymerase chain reaction SD standard deviation 0cHan Journal of Experimental Clinical Cancer Research Page of BackgroundAs an extremely aggressive cancer worldwide pancreatic cancer PC has shown an increasing incidencerate in recent years [] Due to its high level of malignancy PC has become the fourth leading cause ofdeath from malignanttumors with poor prognosisand the 5year survival rate is less than [ ]Early diagnosis of PC has been a considerable challenge due to its complicated pathological process andintricate molecular mechanism While some advancesin imaging and clinical treatment have improved diagnosis and therapy [] the outcome of PC patients remains unsatisfactory Hence a better understandingofthe underlying molecular mechanism is essentialfor seeking a novel therapeutic target for PChavestudiesfunctions HoweverLong noncoding RNA lncRNA is a ribonucleotidechain with a coding length of more than nucleotides[] In the past it was thought that since lncRNAs didnot have the ability to encode proteins they lacked biologicalin recent years scientistshave found that lncRNAs execute their biological effectsin epigenetics [] at the histone modification [] tranlevels [] Accumuscriptional and posttranscriptionallatedelucidatedtheextraordinarysignificance of lncRNAs in the progression of a widerange of diseases such as cardiovascular diseases []diabetes [] neurodegenerative diseases [] and human cancers For instancelncRNA SNHG1 whichcan be positively regulated by miR21 activates theAKT pathway to promote sorafenib resistance in hepatocellular carcinoma cells [] lncRNA EPB41L4AAS1 suppresses the Warburg effect and plays a significant role in metabolic reprogramming of cancer[]intestinalstem cells and promote tumorigenesis of colorectalcancer []lncGata6 could maintain stemness ofLINC00514 is a newly identified lncRNA and very fewreports about it are found in the literature Research byLi [] proved that LINC00514 could be an inhibitor of malignant behaviors of papillary thyroid cancer Inaddition another study has also shown a relationship between LINC00514 and neuroendocrine prostate cancer[] In our study we explored the function and mechanism of LINC00514 in PC We discovered thatLINC00514 expression was increased in PC tissue andPC celllines and that the upregulated expression ofLINC00514 was associated with PC cell proliferationmigration and invasion in vitro and tumor growth andmetastasis in vivo Mechanistically LINC00514 accelerated pancreatic cancer progression via the miR285pRap1b axis AllthatLINC00514 might act as a potential prognostic biomarker of PC occurrence and provide a novel target forPC therapythe evidence above suggestsMethodsClinical samplesPC tissue and adjacent normal tissue were collectedfrom the First Affiliated Hospital of Nanchang University with the informed content of the enrolled patientsin this research Patients received neither chemotherapynor radiotherapy before surgery Our study was approved by the Human Research Ethics Committee ofNanchang UniversityQuantitative realtime PCRRNA was extracted by TRIzol reagent Invitrogen fromtissue samples and cells Extracted RNA was later reverse transcribed into complementary DNA cDNA byPrimeScript RT Reagent Takara Japan A SYBR GreenKit Takara Japan was utilized to perform RTPCRGAPDH and actin were used as internal controlsGene expression levels were calculated by the ˆ’ΔΔCtmethod The primer sequences are shown in Table Cell lines and cell cultureThe normal pancreatic epithelial cell line HPDE andPC celllines BxPC3 SW1990 PANC1 AsPC1Capan2 and MIAPaCa2 were purchased from ATCCCells were cultured in Dulbecco™s modified Eagle™sTable Correlation between LINC00514 expression level andclinical featuresCharacteristicsN2P valueAllAge years ‰¥ GenderMaleFemaleTumor size cm ‰¥ DifferentiationPoorModerateWellLymph node metastasisAbsentPresentClinical stage AJCCIIIIIIIVLINC00514 expressionHighLowAbbreviation LINC00514 long intergenic nonprotein coding RNA p was considered statistically significant 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted PC cell proliferation migration and invasion ab Transfection efficiency of LINC00514 overexpression plasmids andshRNA in BxPC3 and SW1990 cells were evaluated by qRTPCR cd CCK8 assay was performed to detect cell BxPC3 SW1990 proliferationability with LINC00514 overexpression and LINC00514 silencing e Colony formation assay was carried out to further detect cell proliferationcapacity fg Transwell migration and invasion assay were carried out to detect cell migration and invasion under LINC00514 overexpression andknockdown h Western blot was conducted to evaluate the impact of LINC00514 on EMT progression p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8 EMT epithelialmesenchymal transitionmedium DMEM containing fetal bovine serumFBS at °C with CO2 in humidified airCell transfectionLINC00514 overexpression plasmid and shRNAs againstLINC00514 and Rap1b were purchased from GenePharma Shanghai China with scramble plasmid andshRNA used as negative controls MiR285p mimics andmiR285p inhibitors were acquired from Gene Pharma aswell All of the above reagents were transfected into cellsvia TF3000 Transfection Reagent Invitrogen accordingto the manufacturer™s recommendationsColony formation assayCells × cells per well were seeded in 6well platesand then incubated for days After being washed withPBS three times colonies were stained with hematoxylinand countedViability assayTo evaluate cell viability a CCK8 assay was carried outCells × cells per well were plated in 96well platesfor h h h and h The cell growth rate was analyzed by Cell Counting Kit8 Solarbio China reagentaccording to the manufacturer™s instructions The opticaldensity value was measured by a microplate reader at nmMigration and invasion assaysA transwell chamber Corning Tewksbury MA wasused to detect cell migration and invasion capacitiesCells × were seeded on the upper chamber covered with Matrigel Corning Tewksbury MA whileDMEM with FBS was placed on the lower chamberAfter h of transfection cells that passed from theupper chamber onto the lower chamber were fixed withmethanol stained with crystal violet and imaged under alight microscopeIn vivo analysisFiveweekold female nude mice were purchased fromthe National Laboratory Animal Center Beijing Chinaand maintained under specific pathogenfree conditionsSubsequently the mice were randomly separated intotwoLINC00514groups Cells × oftheoverexpression group and NC group were subcutaneously injected into the right axillary of nude miceTumor volume was measured every days and weightwas measured at the end of the experimentTo further evaluate the effects of LINC00514 we carried out pulmonary metastasis analysis PC cells × were injected into nude mice via the caudal vein After days the mice were euthanized The lungs of micewere removed to observe tumor metastasis All experiments were approved by the Animal Research EthicsCommittee of Nanchang UniversitySubcellular fractionation assayThe PARIS Kit Life Technologies was used to isolatenuclear and cytoplasmic RNAs according to the manufacturer™s protocol Reverse transcription of extractedRNAs and RTPCR were conducted as described beforeLuciferase reporter assayThe online software StarBase30 httpstarbasesysueducnwas used to predict the binding sites of LINC00514to miRNA285p Wildtype LINC00514 and mutantLINC00514 of the putative binding sites were clonedinto a luciferase vector Promega and cotransfected withmiR285p mimics into PC cells via LF3000 transfectionreagent After h cells were harvested for luciferase activity analysisPulldown assayWtmiR285p and NCmiRNA were labeled with biotinand transfected into BxPC3 and SW1990 cells The celllysates were incubated with streptavidin magnetic beads at °C for h After that the beads were rinsed with precooled lysis buffer and salt buffer The pulldown RNAswere extracted to detect LINC00514 levelsRNA immunoprecipitation assayThe Magna RIP RNABinding Protein ImmunoprecipitationKit Millipore MA was used to conduct the RIP assay according to the manufacturer™s protocol The cells were lysedand incubated with Ago2 and IgG Then cell lysates weremixed with antiAgo2 and antiIgG in RIP buffer MilliporePrecipitated RNAs were collected for RTPCR analysis 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted tumor growth and pulmonary metastasis in vivo ab The images of subcutaneous tumors were obtained on Day cf The tumor volumes and weights of shLINC00514 group compared with NC group and LINC00514 overexpression group compared withempty group were quantified Tumor volumes were analyzed by ANOVA gh qRTPCR was used to assess the transfection efficiency ij Theimage of pulmonary metastasis was photographed at the endpoint kl Pulmonary metastasis of LINC00514 silencing and LINC00514overexpression compared with their control groups were evaluated p05 p01 p001 The mean±SD was used to represent the finalresults of experiments repeated at least three times PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction SDstandard deviation NC negative control ANOVA analysis of variancePVDF membranesWestern blotProtein was extracted from cells and transferred topolyvinylidene difluorideafter sodium dodecyl sulfate polyacrylamide gel electrophoresis After that membranes were blocked with nonfat milk and incubated overnight at °C withprimary antibodies After rinsing the membranes threetimes with PBS a secondary antibody labeled withhorseradish peroxidase was used to incubate membranesroom temperature Antibodiesagainst ECadherin NCadherin and Vimentin wereall purchased from CST company and actin andRap1b antibodies were purchased from Abcam Thedilution ratio was determined accordingto theinstructionsfor h atStatistics analysisAll data are presented as the mean ± standard deviationAll experiments were repeated at least three times Student™s t test ANOVA Spearman™s rank correlation testand χ2 test were used for statistical analysis A value ofp was considered statistically significantResultsLINC00514 was upregulated in PC and predicted a poorprognosisFirst we analyzed the LINC00514 profile in PC Wefound that LINC00514 was remarkably increased in PCtissues compared with the corresponding normal tissuesFig 1a The upregulated expression of LINC00514 wassignificantly associated with the LINC00514 level andtumor sizelymph node metastasis and clinical stageFig 1bd while no significant correlation was foundbetween LINC00514 expression and age gender ortumor differentiation Table Additionally LINC00514expression was increased in PC cell lines compared withthe normal pancreatic epithelial cell line Fig 1e Furthermore Kaplan“Meier survival curves revealed thathigh LINC00514 expression was related to a lower overallthe lowLINC00514 level group Fig 1f Overall LINC00514was increased in PC and might be associated with clinical progression and a poor prognosis of PC patientsrate compared with that ofsurvivalcellandpromotedLINC00514 promoted cell proliferation migration andinvasionTo investigate whether LINC00514 is involved in cellproliferation migration and invasion we carried outgain and lossoffunction assays The LINC00514 overexpression plasmid and LINC00514 shRNA were stablytransfected into BxPC3 and SW1990 cells with ascramble plasmid and shRNA used as negative controlsFig 2ab According to the results of CCK8 and colony formation assays LINC00514 overexpression significantlySW1990proliferation capacity while suppression of LINC00514remarkably inhibited these processes Fig 2ce Moreovertranswell assays were utilized to prove thatLINC00514 increased cell migration and invasion capabilities Fig 2fg For further confirmation westernblotting was performed to measure the expression ofEMT markers in both BxPC3 and SW1990 cells As expected Ecadherin was observed to be strikingly downregulated by LINC00514 overexpression whereas Ncadherin and Vimentin were obviously upregulated andthe shLINC00514 group showed the opposite resultsFig 2h In summary LINC00514 promoted PC cellproliferative migratory and invasive capacitiesBxPC3LINC00514 knockdown inhibited tumor growth andpulmonary metastasis in vivoTo further identify the bioeffects of LINC00514 ontumor growth we constructed a subcutaneous xenografttumor model BxPC3 cells transfected with LINC00514shRNA compared with NC shRNA or transfected withthe LINC00514 overexpression plasmid and comparedwith the empty plasmid were subcutaneously injectedinto nude mice The tumors were measured every daysafter injection After euthanizing the mice we obtainedimages of the tumors Fig 3ab Compared with thoseof the NC group the volume and weight of tumors inthe LINC00514 shRNA group were significantly reducedwhileresults were observed in theLINC00514 overexpression group Fig 3cf QRTPCRwas used to assess the transfection efficiency Fig 3ghThen we further investigated the role of LINC00514 inPC metastasis in vivo Nude mice were injected withBxPC3 cells transfected with LINC00514 shRNA compared with NCLINC00514the oppositeshRNA orthe 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 was a sponge for miR285p ab Subcellular fractionation assay was used to determine the subcellular localization ofLINC00514 a BxPC3 cells b SW1990 cells C Sequence of WTLINC00514 MutLINC00514 and miR285p were conducted dg Luciferasereporter assay and RIP assay was performed to demonstrate that miR285p was a downstream target of LINC00514 h Pulldown assay wasconducted to detect the reaction between miR285p and WTLINC00514 or MutLINC00514 ij Relative miR185p expression level in BxPC3and SW1990 were determined by qRTPCR K The expression of miR285p in PC tissue and normal tissue were detected by qRTPCR LSpearman™s rank correlation test was utilized to analyze the correlation between the levels of LINC00514 and miR285p MN The miR285pexpression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo op CCK8 assay was performed to detectproliferation of cells transfected with LINC00514 shRNA and cells cotransfected with LINC00514 and miR285p inhibitor qr Transwell migrationand invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experiments were repeated atleast for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chainreaction RIP RNA immunoprecipitation SD standard deviation WTlINC00514 wild type LINC00514 MutLINC00514 mutant LINC00514 CCK8cell counting kitoverexpression plasmid compared with the empty plasmid into the tail vein Images of pulmonary metastasiswere acquired at the endpoint Fig 3ij There was anobviously lower incidence of pulmonary metastasis and asmaller number of metastatic tumors per lung in the shLINC00514 group compared with the NC groupwhereas the LINC00514 overexpression group showedthe opposite results Fig 3klin the celldetected the expression level of miR285p in the tumorswe collected before and the results showed that miR285p expression was higherlines withLINC00514 knockdown while miR285p was lower inthe cells with LINC00514 overexpression Fig 4mnLINC00514 promoted cell proliferation migration andinvasion at least partially by sponging miR285p Fig4or In summary LINC00514 accelerates PC progression by sponging miR285pLINC00514 acted as a sponge for miR285pTo explore the underlying molecular mechanism of theoncogenic effects of LINC00514 on PC we determinedthe subcellular localization of LINC00514 The resultsshowed that LINC00514 was mostly distributed in thecytoplasm Fig 4ab which suggested that LINC00514might exert its biological function by sponging miRNAStarBase30 was utilized to identify a candidate microRNA miR285p and predict the potential downstreamtargets of LINC00514 Fig 4c The luciferase reporterassay results confirmed that the luciferase activity ofWTLINC00514 was clearly decreased by miR285pmimics while the luciferase activity of MutLINC00514did not change significantly Fig 4de In addition theRIP assay further revealed that LINC00514 and miR285p were enriched in beads conjugated to Ago2 comparedwith the IgG group Fig 4fg Furthermore overexpression of WTLINC00514 but not MutLINC00514 decreased miR285p expression in BxPC3 and SW1990cells Fig 4h Additionally overexpressing LINC00514dramatically decreased miR285p levels in both BxPC3and SW1990 cells while silencing LINC00514 increasedmiR285p levels with NC shRNA used as an internalreference Fig 4ij Then we further detected miR285p expression in tumor tissue The results revealed alower level of miR285p in PC tissue than in normal tissue and a negative correlation between LINC00514 expression and miR285p levels Fig 4kl To obtainmore evidence in vivo experiments were performed WeRap1b was a downstream target of miR285p in PCThe posttranscriptional function of miRNAs is usually toinhibit protein synthesis by base pairing with the ²untranslated region [] Next to ascertain the detailed regulatory mechanism of LINC00514 in PC we searchedStarBase30 and observed that Rap1b was predicted to bea downstream target of miR285p Fig 5a MutRap1bor WTRap1b and miR285p or NCmiRNA were transfected into BxPC3 and SW1990 cells A luciferase reporter assay and RIP assay were used to confirm thehypothesis that Rap1b is a direct target of miR285p Fig5be Then we found that Rap1b expression was downregulated by LINC00514 silencing while cotransfectingmiR285p and shLINC00514 inhibited the effect ofLINC00514 knockdown on Rap1b at both the transcriptional and translational levels Fig 5fgThen we detected Rap1b levels in tumor tissue Rap1bwas obviously increased in PC tissue compared with adjacent normal tissue and there was a positive relationshipbetween Rap1b expression and LINC00514 levels while anegative correlation was observed between Rap1b expression and miR285p levels Fig 5hj In addition we alsodetected the expression of Rap1b in vivo and the transfection efficiency was examined previously As expected theexpression of Rap1b was clearly decreased in BxPC3 cellsby LINC00514 knockdown while increased expressionwas observed in LINC00514overexpressing cells Fig 5kl Thus far we have proven that Rap1b is a direct target 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig Rap1b was a downstream target of miR285p in PC a The sequence of WTRap1b MutRab1b and miR285p were conducted efLuciferase reporter assay and RIP assay were performed to determine the association between miR285p and Rap1b fg QRTPCR and westernblot were used to detect Rap1b expression in cells of LINC00514 silencing and cells of cotransferring miR285p and LINC00514 shRNA attranscription and translation level h Relative Rap1b expression in tumor tissue and normal tissue were detected by qRTPCR ig Thecorrelation between Rap1b and LINC00514 as well as the correlation between Rap1b and miR285p were analyzed by Spearman™s rankcorrelation test kl The Rab1b expression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo mn CCK8assay was performed to detect proliferation of cells transfected with miR285p inhibitor and cells cotransfected with miR285p inhibitor andRap1b shRNA OP Transwell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01p001 All experiments were repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction RIP RNA immunoprecipitation SD standard deviationof miR285p Thereafter functional experiments werecarried out to investigate the bioeffects of Rap1b The resultsdemonstrated that Rap1b silencing remarkably suppressedthe promoting effects of the miR285p inhibitor on cell proliferation migration and invasion capacities Fig 5mppatients which indicated that LINC00514 might be involved in PC progression In addition it was determinedthat LINC00514 facilitated PC cell proliferation migration and invasion in vitro and tumor growth and metastasis in vivo The underlying molecules however havenot yet been revealedRap1b inhibition inhibited the tumorigenesis effects ofLINC00514Finally we explored the role of LINC00514 mediatedby Rap1b in promoting tumor growth We knockeddown Rap1b in BxPC3 and SW1990 cells to determine whether Rap1b inhibition can reverse the oncogenic effects of LINC00514 Based on the rescueassays Rap1b inhibition partially inhibited the effectof LINC00514 overexpression on cell proliferationmigration and invasion Fig 6adIn conclusionthesethatLINC00514 acted as a key tumor promotor of PC bycompetitively binding to miR285p and then upregulating the expression of Rap1bdemonstratedcollectivelyresultsthrough the miR1883pBRD4 axisDiscussionIn recent years PC has received increasing attention dueto its high incidence and extremely poor prognosis []Accumulated studies have shown that lncRNAs play animportant role in the initiation and development of cancers including PC [] For instance LINC00346 accelerated PC progression and gemcitabineresistancepartially[]lncRNA GLSAS mediated the feedback loop of Mycand GLS and provided a potential therapeutic strategyfor metabolic reprogramming in PC [] AFAP1AS1was shown to exert inhibitory effects on the stemness ofPC cells and ultimately PC tumorigenicity in vivo via themiR384ACVR1 axis [] LINC00514 has been previously reported in papillary thyroid cancer [] and neuroendocrine prostate cancer [] butthere are noreports in PC Our study revealed that LINC00514 expression was markedly elevated in PC tissues and PC celllines and that increased expression of LINC00514 wasassociated with the progression and prognosis of PCIn recent years increasing evidence has proven the hypothesis that lncRNAs exert their biological impact by acting as competitive endogenous RNAs ceRNAs to affectthe development of cancers [] There has been considerable progress in the study of ceRNAs in PC For examplelncRNAPVT1 promotes PC cell proliferation and migration by sponging miR448 [] cucurbitacin B inhibits PCcell proliferation both in vitro and in vivo throughlncRNAAFAP1AS1 binding with miR146b5p [] andPXNAS1 acts as a ceRNA of miR3064 which upregulates PIP4K2B expression and suppresses the progressionof pancreatic cancer [] In our research subcellular fractionation assays indicated that LINC00514 was mostly located in the cytoplasm which provided a basis forLINC00514 to act as a ceRNA in the initiation and progression of PC Then the online software StarBase30 wasutilized to predict the possible downstream target miR285p for LINC00514 Luciferase reporter assay RIPassay and pulldown assay were used to confirm the interaction between LINC00514 and miR285p Overexpression of LINC00514suppressed miR285p whileLINC00514 silencing upregulated miR285p expressionFurther investigation was carried out to demonstrate themigration and invasion promoting effect of miR285p inthe initiation and development of PC which suggested atumorpromotingeffectthat wasdependent on miR285pLINC00514ofAccording to the ceRNA hypothesis mRNA expression is upregulated due to lncRNA competitively bindingto miRNA Rap1b was first reported in the study of Chajut [] and was found to be related to various cancers such as thyroid cancer [] breast cancer []gastric cancer [] and colorectal cancer [] Howeverthere is only a limited number of reports about Rap1b inPC [] In our current study Rap1b was predicted to be 0cHan Journal of Experimental Clinical Cancer Research Page of Fig Rap1b inhibition restrained the tumorigenesis effects of LINC00514 ab CCK8 assay was performed to detect proliferation of cellstransfected with LINC00514 overexpression plasmids and cells cotransfected with LINC00514 overexpression plasmids and Rap1b shRNA cdTranswell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8a direct target of miR285p by StarBase30 Luciferase reporter assay and RIP assay confirmed thedirect binding of Rap1b with miR285p Rap1bacting as a cancerpromoting gene had a positivecorrelation with LINC00514 while there was anegative relationship between Rap1b and miR285p Moreover silencing Rap1b partially abolishedthe tumorigenic effects of LINC00514 based on therescue assayIn conclusion ourstudy provides evidence thatLINC00514 promotes PC development by spongingmiR285p and increasing Rap1b expression Thishighlights the LINC00514miR285pRap1b axis as anovel diagnostic and therapeutic strategy for PCpatientsresults highlighted the significantConclusionsOurtheLINC00514miR285pRap1b axis in PC progressionsuggesting that LINC00514 may serve as a potential biomarker and therapeutic target in PCrole ofAbbreviationsPC Pancreatic cancer LncRNA Long noncoding RNA LINC00514 Longnoncoding RNA00514 MiR285p MicroRNA285p CeRNA Competingendogenous RNA EMT Epithelialmesenchymal transition QRTPCR Quantitative realtime PCR RIP RNA immunoprecipitation 0cHan Journal of Experimental Clinical Cancer Research Page of AcknowledgmentsNot applicableAuthors™ contributionsQH and ZWS designed the study QH JHL and JPX collated the data carriedout data analyses and produced the initial draft of the manuscript QH andZWS contributed to drafting the manuscript All authors have read andapproved the final submitted manuscriptFundingThis study was funded by Jiangxi Provincial Education Fund Project youth Science Foundation of Jiangxi Province20202BAB216027Availability of data and materialsAll the data and materials supporting the conclusions were included in themain paperEthics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinkiprinciples It was approved by the Ethics Committee of the First AffiliatedHospital of Nanchang UniversityConsent for publicationNot applicableCompeting interestsThe authors declare no competing interestsReceived May Accepted July ReferencesPei X Song F Wang Z Emerging incidence trends and application ofcurative treatments of pancreatic cancer in the USA Medicine e17175Ansari D Tingstedt B Andersson B Holmquist F Sturesson C Williamsson CSasor A B D Bauden M Andersson R Pancreatic cancer yesterdaytoday and tomorrow Future Oncol “Shin SJ Park H Sung YN Yoo C Hwang DW Park JH Kim KP Lee SS RyooBY Seo DW Prognosis of pancreatic Cancer patients with synchronousor Metachronous malignancies from other ans is better than those withpancreatic Cancer only Cancer Res Treat “Halbrook CJ Lyssiotis CA Employing metabolism to improve the diagnosisand treatment of pancreatic Cancer Cancer Cell “Beermann J Piccoli MT Viereck J Thum T Noncoding RNAs indevelopment and disease background mechanisms and therapeuticapproaches Physiol Rev “ Wei

Thyroid_Cancer

"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately “ of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory [“]According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts— HPF Grade G1 Ki67 ‰ mitoses Grade G2 Ki67 ‰ ‰ mitoses‰ Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [“] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients™ status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kim™sfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kim™s research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kim™s While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kim™s study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm‰¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm‰¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CI“Multivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size‰¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value“““ ““““““““““ “ ““““““““tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Park™s study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[“] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Van™s results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors™ contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital “ Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients™ written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “ Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer “Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer “La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol “Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg Pathol“Rayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc

Thyroid_Cancer

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

Thyroid_Cancer

"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

"protein protein interaction screen carried out to maphuman cellsurface receptorligandinteractions between proteins belongingto the immunoglobulin domainsuperfamily IgSF begins to unravel thecomplex network of cellsurfaceinteractions that allows cells to recognizeand respond to one another and theirdynamically changing environmentHighlightsd Human IgSF interactome reveals complex network of cellsurface protein interactionsd Phylogenetic homology analysis predicts proteinproteininteractionsd 18 previously unknown proteinprotein interactionsidentifiedd Deorphanization of receptors and new binding partners forwellstudied receptorsWojtowicz Cell “August ª Elsevier Inc101016jcell202007025ll 0cllResourceA Human IgSF CellSurface Interactome Reveals aComplex Network of ProteinProtein InteractionsWoj M Wojtowicz16 Jost Vielmetter26 Ricardo A Fernandes17 Dirk H Siepe137 Catharine L Eastman17Gregory B Chisholm2 Sarah Cox4 Heath Klock4 Paul W Anderson4 Sarah M Rue4 Jessica J Miller4 Scott M Glaser4Melisa L Bragstad4 Julie Vance4 Annie W Lam2 Scott A Lesley4 Kai Zinn2 and K Christopher Garcia13581Department of Molecular and Cellular Physiology Stanford University School of Medicine Stanford CA USA2Division of Biology and Biological Engineering California Institute of Technology Pasadena CA USA3Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA USA4The Genomics Institute of the Novartis Research Foundation San Diego CA USA5Department of Structural Biology Stanford University School of Medicine Stanford CA USA6These authors contributed equally7These authors contributed equally8Lead ContactCorrespondence wojstanfordedu WMW kcgarciastanfordedu KCG101016jcell202007025SUMMARYCellsurface proteinprotein interactions PPIs mediate cellcell communication recognition and responses We executed an interactome screen of human cellsurface and secreted proteins most ofwhich are immunoglobulin superfamily IgSF proteins using a highthroughput automated ELISAbasedscreening platform employing a pooledprotein strategy to test all PPI combinations Screen resultsaugmented by phylogenetic homology analysis revealed 18 previously unreported PPIs We validated asubset using surface plasmon resonance and cell binding assays Observed PPIs reveal a large and complexnetwork of interactions both within and across biological systems We identified new PPIs for receptors withwellcharacterized ligands and binding partners for ˜˜orphan™™ receptors New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous systemdevelopment and function differentiationproliferation metabolism vascularization and reproductionThese PPIs provide a resource for further biological investigation into their functional relevance and may offernew therapeutic drug targetsINTRODUCTIONProteinprotein interactions PPIs at the cell surface allowcells to respond to one another and their environment in ahighly dynamic contextdependent and spatiotemporalmanner Wood and Wright The essential role playedby cellsurface PPIs is exemplified by estimates that 18 of genes in the human genome encode cellsurfaceproteins and 18 encode secreted proteins Fonseca At present a comprehensive human cellsurface interactomeis lacking Mapping extracellular PPIs has proved challengingbecause most cellsurface proteins are refractory to classicbiochemical screening techniques and cellsurface PPIs are underrepresented in affinity purificationmass spectrometrybaseddatasets Huttlin Additionally cell surfacePPIs often have fast binding kinetics spanning a broad rangeof affinities low nM to hundreds of mM van der Merwe and Barclay rendering them difficult to detect using standardbiochemical assaysIn recent years several assays have been developed thatallow detection of lowaffinity cellsurface PPIs by impartingavidity through clustering of secreted proteins and the extracellular domains ECDs of single transmembrane STM cellsurface proteins Clustering is achieved using multimerization domains and can increase binding signal up to 250fold Bushell Experimental platforms that utilize clusteringinclude several variations of ELISAbased binding assaysWojtowicz Bushell O¨ zkan BioPlex beads Li protein microarrays Sun cellsignaling assays Barrow cellsurface staining microarrays Turner and beadbasedassays Husain Multiple groups have shown thatELISAbased binding assays have a remarkably low falsepositive rate Wojtowicz Bushell So¨ llnerand Wright Martin Crosnier O¨ zkan Visser Ranaivoson Previously we conducted a screen for all 18 Drosophilacellsurface and secreted proteins containing three types of domains immunoglobulin Ig and Iglike fibronectin type III FN3Cell “ August ª Elsevier Inc 0clland leucinerich repeats LRRs O¨ zkan This screenreported over new PPIs including a previously unknownimmunoglobulin superfamily IgSF PPI network between members of the Dpr and DIP subfamilies Since we reported theDprDIP network functional studies have revealed that thisnetwork mediates neuronal wiring decisions in the fly brain andneuromuscular system for review see Honig and Shapiro Sanes and Zipursky In humans there are an estimated 18 secreted and STMproteins totaling 18 M putative PPIs Screening this vast number requires a highthroughput assay Here we developed ascreening platform that combines a highthroughput version ofthe ELISAbased extracellular interactome assay ECIA O¨ zkan with an automated pooledprotein strategy apECIA We performed a screen of human IgSF secreted andSTM cellsurface proteins excluding antibodies and T cell receptors along with other select proteins of interest The IgSFis the largest and most functionally diverse family in the cellsurface proteome Members include receptor tyrosine kinasesphosphatases costimulatory or coinhibitory immune receptors growth factor and adhesion receptors among many othersand are present in most if not all cell typesWe produced proteins and screened every possible PPI We observed PPIs of which are previously unreported New PPIs form a complexnetwork and include PPIs between phylogenetically related proteins within a subfamily different subfamilies and distantlyrelated proteins Screen results were combined with phylogenetic homology analysis PHA to predict additional PPIs between subfamily members using a nearestneighbor approachWe confirmed a subset of both screen and PHA predictedPPIs using surface plasmon resonance SPR and cell bindingassaysRESULTSSelection and Production of Proteins for PPI ScreeningTo identify human IgSF proteins we utilized the HUGO GeneNomenclature Committee HGNC Yates HumanProtein Atlas Uhle´ n and UniProt UniProt Consortium databases ECDs and secreted proteins for IgSF and nonIgSF proteins of interest were produced with˜˜bait™™ and ˜˜prey™™ multimerization domains into cell supernatantsFigures 1A and 1B Data S1 and S2 and expression wasconfirmed by western blot Data S3 Westerns revealed detectable levels of protein for of baits and of preys We andothers have observed that PPIs can be detected in the ECIA andother ELISAbased binding assays even when proteins are present at levels below the limit of detection by western O¨ zkan Visser Ranaivoson Assuch all bait and prey were included in the screen regardlessof whether protein was detectedDevelopment of an Automated PooledPrey ECIAPlatform apECIAECIA and other ELISAbased assays allow bait and preyproteins to be tested for binding directly from conditionedmedia B These assays test one baitprey pair per Cell “ August Resourcewell To increase throughput we pooled three preys andfollowing screening deconvoluted positive wells to identifyPPIs B Pooling experiments with a panel of knownPPIs showed binding for all 3fold diluted prey Figures 1CS1C and data not shown As baitprey pairs are testedin both orientations a falsenegative resulting from poolingcan be ˜˜rescued™™ by a positive resultin the converseorientation We reasoned thatthe advantages of poolingwhich reduces the number of binding reactions outweighedthe potentialincrease in false negatives To further improvethroughput we optimized a 384well format and developedan automated workflow using liquid handling robots TheapECIA platform allows testing of baitprey combinations per weekPPI Screen Reveals a Complex Network of PPIsFollowing screening deconvolution of positive wells R2 foldoverbackground was performed by retesting each preyindividually B Nine prey gave rise to large numbers ofwells with positive signals and were excluded as nonspecificbinders Figure S2 Following removal of nonspecific PPIs deconvolution revealed positive wells comprising uniquePPIs Data S4 In each case only one of the three deconvolutedprey yielded a positive signal To confirm binding the positiveprey was retested againstin triplicateEightyone percent of PPIs are between IgSF proteinsThe remaining include PPIs between IgSF and otherproteins present in the screen Figure S3its cognate baitAlmost half of the proteins tested were involved in a PPI Proteins not involved in PPIs may be misfolded have binding partners notincluded in the screenrequire coreceptors or fall outside the dynamic range of theassay false negatives which is determined by PPI affinityand bait and prey concentrations Figure S4F Confirming thesensitivity of the assay many bait or prey proteins expressedat very low levels still engaged in one or two PPIs FiguresS4A and S4B A small number of PPIs were observed withbait or prey proteins exhibiting undetectable levels in conditioned media Figures S4C“S4E To interrogate the dynamicrange of the assay we plotted prey AP levels for PPIs with reported affinities Figure S4F These data suggest thatforvery poorly expressed prey proteins Figures S1A S4A andS4B PPIs with KD mM are likely to be missed false negatives Figure S4FOf the PPIs are previously unreported basedon literature and PPI databases Data S4 The majority of PPIsform one large network comprising proteinsD Only proteins involving PPIs are not connectedto the network Different regions of the network are shown inFigure Ninetyeight proteins had one PPI had two to five PPIs and had PPIs EBecause of proteins exhibit binding we calculated theexpected frequency with which each protein will bind at least˜˜x™™ number of proteins up to the maximally observed PPIsFigure S2B and compared the expected and observed frequencies F The observed number of binding partnersis significantly greater than predicted by random chance ofPPIs for a network of this size 0cResourcellABCEDFFigure apECIA Screen Details and Overview of ResultsA Schematic depiction of a subset of proteins in library Pie graph of library distribution Full protein domain names at martemblde Sun B Left flow chart of screen HT highthroughput Right example plate from screen Schematic of ECIA and pooledprey strategy Illustration of screen matrixC ECIA of undiluted prey single prey versus 3fold diluted prey pooled prey Background subtracted data are represented as ±SD Bkgd backgroundD Network of PPIs observed in screen Inset the PPIs that are not connected to the network Node size is proportional to number of PPIs Siglec subfamilynodes are highlighted in colorE Pie graph of distribution of the number of binding partners observed in screen overlaid on the network in a degreesorted circular layoutF Observed versus expected frequency with which each protein will bind at least ˜˜x™™ number of proteins up to the maximally observed PPIs assuming randomchance of interactions p KolmogorovSmirnov [KS] testSee also Figures S1 S2 S3 and S4 and Data S1 S2 S3 and S4Cell “ August 0cllResource Cell “ August legend on next page 0cResourcellPhylogenetic Homology Analysis PHA to Predict PPIsPPIs often occur between phylogenetically related proteins bothwithin and between subfamilies We performed multiplesequence alignments to identify subfamily members within ourlibrary and generated phylogenetic trees Using a combinedapproach which we call apECIAPHA we analyzed screendata alongside the phylogenetic trees to predict additionalPPIs between subfamily members that may have been missedin the screenPPI Validation by Surface Plasmon ResonanceWe selected a subset of screen and PHA predicted PPIs to validate by SPR Bona fide PPIs are expected to display distinct association and dissociation which can be observed with highsensitivity by SPR To increase avidity and therefore sensitivityFc dimerized ECD analytes and ligands were used in most experiments Figure S5 This increase in sensitivity prevented us fromdetermining monomeric KD constants Binding profiles characteristic of PPIs exhibiting clear resonance signals above background negative control ligand and receptor and concentrationdependent binding curves were deemed indicative of aspecific ligandanalyte PPI Nonspecific PPIs generally exhibitdeviations from this behavior such as high background andnonlinear concentration responsesTwentyfour newly identified PPIs observed in our screen weretested by SPR Of these we observed specific ligandanalyteinteractions We additionally tested PHA predicted PPIs andobserved PPIs for Three additional PPIs were observed between homologous proteins in mouse and crossspecies humanmouse In total we SPR validated newly identifiedPPIs Table Data S5Combined apECIAPHA Approach Reveals Multiple DCCSubfamily PPIsThe netrin1 receptor DCC has wellcharacterized functions inthe nervous system Finci DCC is a dependence receptor and is implicated in colorectal and other cancers but itsroles in these cancers are not well understood Goldschneiderand Mehlen We observed DCC binding to insulinlikegrowth factorbinding proteinlike IGFBPL1 but not to insulinlike growth factorbinding protein IGFBP7 Figures 2I and3A Our phylogenetic tree revealed IGFBPL1 and IGFBP7 residein a cluster and share the highest amino acid sequence similarity among subfamily members As such we examined binding of DCC to both IGFBPL1 and IGFBP7 by SPR and observedbinding for both Figure 3CPHA pointed us to four proteins that cluster with DCC PUNCPUNC e11 neogenin NEO1 and protogenin PRTG Figure 3ATogetherthese proteins play roles in diverse processesincluding nervous system development myogenesis and angiogenesis inflammation and tissue regeneration leukocyte migration neural tube and mammary gland formation development ofbone and connective tissues and stem cell differentiation Salbaum Wilson and Key Takahashi Schievenbusch DakouaneGiudicelli PUNC is an ˜˜orphan™™ receptor expressed in the developing nervous system Salbaum PUNC e11 and PRTG bound intercellular adhesion molecule ICAM5Figure 3A a proteinexclusively expressed in the brain that functions in synapse formation stabilization and refinement Gahmberg Cleaved ICAM5 ECD exhibits immunosuppressive functionsthrough cytokine regulation and may play important roles inregulation of brain inflammation We confirmed binding ofPUNC e11 and PRTG to ICAM5 by SPR Figure 3C AlthoughPPIs with ICAM5 were not detected in the screen for the remaining DCC subfamily members we tested them by SPR andobserved binding for all three Figure 3CIn our screen one or more DCC subfamily members alsobound to WFIKKN2 a secreted protein that binds transforming growth factorbeta subfamily members and modulates theirpresentation to cells Monestier and Blanquet lactotransferrin LTF an ironbinding protein with antimicrobial activity Hao interleukin6 receptor subunit alpha IL6Ra a cytokine receptor Schaper and RoseJohn and ISLR2LINX which functions in nervous system developmentMandai Panza Abudureyimu We confirmed binding of all DCC subfamily members tothese proteins by SPR and to other proteins observed in ourscreen Figures 3C 3D and 3F These results demonstratethe value of using a combined apECIAPHA approach to identifyadditional PPIs beyond screen data resulting in the elucidationof a more complete network Figure 3FLARPTPR Subfamily PPIs with SALMsThe LARfamily of type IIA protein tyrosine phosphatase receptors LARPTPRs comprises PTPRF also known as LARFigure Select Regions of the Complex PPI NetworkA Select PPIs including four proteins not connected to the network CD146 CNTN1 NFASC and NrCAMB Region largely comprised of immune system proteinsC Region comprising PPIs both within and across biological systemsD Two regions highlighting subfamilyspecific type IIA and type IIB PTPR PPIs Type IIA PPIs with SALMs and IL1APs include PPIs observed in screen and PHApredicted PPIs validated by SPR Figures and S6E Region highlighting PPIs for ˜˜orphan™™ receptors ILDR1 and PUNCF Region showing a subset of LILR subfamily PPIsG Region showing a subset of Siglec PPIs with nonSiglecs CD33Siglec3H Region showing SiglecSiglec PPIs CD33Siglec3 MAGSiglec4a SNSiglec1I Region highlighting PPIs between nervous system proteins and with proteins in immune and reproductive systems Within this region multiple additional PHApredicted PPIs were validated by SPR Figure Table Because a network is composed of interconnected nodes some linkage proteins are present in more than one panel Colored nodes denote proteins and PPIsvalidated by additional experiments Green line previously reported PPI gray line previously unreported PPISee also Data S4Cell “ August 0cllResourceTable SPRValidated apECIA Screen and PHA Predicted PPIsTable ContinuedLigandFcAnalyteFcapECIATIE1hISLR2mIslr2SALM1SALM2SALM3SALM4SALM5IL1RAPL1IL1RAPL2DSCAMCTDSCAML1CTKIR2DL5IL6RaPSG7PSG9ICAM5WFIKKN2PDL1PDL2LTFTrkAhLEPamLepahLEPamLepaPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRSPTPRDPTPRFPTPRSPTPRSFGFR3PTPRSPTPRMPTPRTPTPRMPTPRTPVRISLR2DCCDSCAMDSCAMDCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2PRTGPUNCPUNC e11PDL1PDL1DCCIL6RaNANANANANANAknownknownknownNANAknownknownknownknownNANANANANANANANANAknownNANAPHANANANANANANANANANANANANANANANANANANANANANANANANANANANANANAContinued on next page Cell “ August LigandFcAnalyteFcapECIAPHAIGFBPL1IGFBP7ISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11NANANANANANANANANANANANANANANANANANANAECDFc ligand and ECDFc analyte PPIs observed by SPR apECIA denotes PPI was observed in screen PHA denotes PPI was predicted byphylogenetic homology analysis SPR conditions are included in DataS5 See also Figure S6ahLEP and mLEP monomer proteinPTPRD and PTPRS Figures 4A and 4B LARPTPRs playimportant roles in synaptic anization and function Nam Presynaptic LARPTPRs mediate transsynapticadhesion through PPIs with multiple postsynaptic ligandsMouse mutants in LARPTPRs and their ligands exhibit defectsin synapse structure and function Lie Several PPIs are known between specific LARPTPRs andSALM235 Li Choi GotoIto Figure 4A We observed these as well as additionalPPIs in our screen Figures 2D 4A and 4C No LARPTPR binding to SALM1 or SALM4 has been reported and we did notobserve binding in our screen PHA led us to test binding of allLARPTPRs to all SALMs by SPR With the exception ofPTPRFSALM4 we observed binding of all LARPTPRs to allSALMs Figures 4E and S6A PTPRSALM pairs exhibited differences in maximum response units RU a relative comparison ofbinding strength suggesting a spectrum of binding affinities mayexist among LARPTPRs and SALMs Figure 4FLARPTPR Subfamily PPIs with Interleukin1 ReceptorAccessory Proteins IL1APsTranssynaptic LARPTPR interactions with IL1RAP and IL1RAPL1 induce bidirectional pre and postsynaptic differentiation Yoshida IL1RAP is known to interactwith all three LARPTPRs and IL1RAPL1 with PTPRD Weobserved these PPIs and additionally binding of IL1RAPL1 toPTPRS Figure 2D IL1RAPL1 shares sequence similarity 0cllBResourceACDEFlegend on next pageCell “ August 0cllResourcewith IL1RAPL2 an orphan receptor expressed in the brain withno known biological function Boraschi In ourscreen we detected binding of IL1RAPL2 to PTPRD and PTPRSFigure 2D We tested both known and previously unknown PPIsby SPR and observed binding for all Figure S6B IL1RAPL2additionally interacted with fibroblast growth factor receptor FGFR3 in the screen and by SPR Figure S6BType IIB PTPR Subfamily PPIs with DSCAMsType IIB PTPRs are expressed in most tissues and regulatediverse processes including cell growth migration and differentiation immune cell development endothelial cell adhesion andmigration neuronal development and synapse formation andoncogenic transformation Lee Stoker The typeIIB subfamily is composed of PTPRK PTPRM and PTPRT Figures 4A and 4B and multiple binding partners are known Weobserved binding of all type IIB PTPRs with Down syndromecell adhesion molecule DSCAM and Down syndrome cell adhesion molecule like1 DSCAML1 Figures 4A and 4C proteinsthat play various roles in nervous system development Sanesand Zipursky The ECD of DSCAMs contains nine Iglike domains followedby four FN3 domains one Iglike domain and two FN3 domainsFigure 4B The Nterminal region engages in homophilic bindingand mediates binding to secreted chemoattractant and chemorepellent ligands netrin1 and slit1 respectively Wojtowicz Ly Alavi No binding partnerhas been reported for the Cterminal ECD region In our screenwe observed binding between the Cterminal regions of bothDSCAMs DSCAMCT and DSCAML1CT and all three typeIIA PTPRs Figure 4C We confirmed binding of DSCAMCTand DSCAML1CT to PTPRT and PTPRM by SPR PTPRK wasnot expressed at sufficient levels Figure 4D vascular endothelial growth factorNew PPIs between Immune System ProteinsNew PPIs were observed for wellstudied immune systemproteins as well as orphan receptors Figure 2B Two PPIswere detected for the orphan granulocyte receptor CEACAM4receptor VEGFR3 and programmed cell death ligand PDL2 CEACAM4 is a member of the carcinoembryonic antigenrelated cell adhesion molecule subfamily expressed inthe immune system epithelial and endothelial cells and brainWakabayashiNakao Zinn and O¨ zkan Inthe immune system CEACAMs play roles in immunity anddevelopment We also observed binding between CEACAM1and the immune checkpoint receptor programmed cell deathprotein PD1Interactions of PD1 with its ligands PDL1 and PDL2 inhibitT cell proliferation cytokine production and cytotoxic activityBardhan PDL2 competes with PDL1 for bindingto PD1 and can be expressed on tumor cells where it mayplay a role in tumor evasion Ghiotto Cheng Bardhan Using SPR we confirmed the knownPPI between PDL1 and PDL2 Lee and observedhomophilic binding of PDL1 a previously unknown PPI Figure S6C By comparison with PDL1 binding to PD1 which approaches saturation at 18 nM we infer that the affinity of PDL1 homophilic binding is lower than binding to PD1 This mayexplain why this PPI within the wellstudied PD1PDL1 axishas not been identified previously and highlights the value of using multimerized proteins to detect lowaffinity PPIsHomophilic and Heterophilic Siglec Subfamily PPIsEight of the proteins with the highest number of PPIs in ourscreen are members of the Siglec subfamily members proteins highly restricted to the immune system that have immunemodulatory effects on Tolllike receptor TLR signaling andplay important roles in self versus nonself discrimination Macauley Siglecs display differential expression on cellsand exhibit a broad spectrum of Siglecspecific recognition ofsialylated glycan profiles present on healthy cells inflamed ormalignant cells and pathogens We observed a network of homophilic and heterophilic PPIs among Siglecs Figure 2H DataS4 as well as PPIs with distantly related proteins a subset areshown in Figure 2G Data S4PPIs between Immune and Nervous System ProteinsThe signaling lymphocytic activation molecule subfamily SLAM members is expressed on most immune cells Dragovich andMor SLAMs function as both costimulatory and coinhibitory molecules in innate and adaptive immunity and playan integral role in autoimmune disorders SLAMF9 is an orphanreceptorWe observed SLAMF9 binding to bactericidal permeabilityincreasing protein BPI and IGSF10 Figure 2C BPI is a neutrophilderived antibiotic protein that participates in bacteria killingthrough its highly cationic Nterminal region Bu¨ low The Cterminal region of BPI exhibits no bactericidal activity andis believed to interact with other proteins and function in differentprocesses IgSF10 is an orphan receptor involved in the migration of gonadotropinreleasing hormone expressing GnRH neurons Howard IgSF10 has no known function in theFigure SPR Validation of DCC Subfamily PPIs Identified by apECIAPHA ApproachA Dendrograms of a subset of DCC subfamily PPIs showing the number of SPRvalidated PPIs observed in the apECIA screen turquoise and predicted by PHAmagentaB Dendrogram of a subset of PSG PPIsC SPR sensrams for DCC subfamily IL6Ra and ISLR2 analytes 2fold dilutions “ nM nM for PRTGWFIKKN2 due to incomplete chipregeneration at higher concentrations binding to various ligands TrkA and TIE1 negative controlsD SPR sensram for IL6Ra analyte 2fold dilutions “ nM binding to ISLR2 ligand PUNC negative controlE SPR sensrams for DSCAM and DCC analytes 2fold dilutions “ nM binding to PSG ligands TIE1 negative controlF Subnetwork of SPRvalidated PPIsRU resonance unitsSee also Figure S5 and Data S5 Cell “ August 0cResourcellABCEDFlegend on next pageCell “ August 0cllResourceimmune system and SLAMF9 function in the nervous system isunknown revealing a PPI between two orphan receptors fromdifferent biological systemsFigure 2F These PPIs present MOG and MPZ PPIs with LILRsas new candidates for neuronal regeneration studiesPPIs with PregnancySpecific Glycoproteins PSGsDuring pregnancy PSGs members are the most abundanttrophoblastic proteins in maternal blood and serve as markersfor trophoblast quality and embryo viability Moore and Dveksler The mechanisms of PSG action in pregnancy are not wellunderstood Studies suggest PSGs also have immunoregulatory proangiogenic and antiplatelet aggregation functionsWe observed several PSG binding partners includingplateletderived growth factor receptor alpha PDGFRAfibroblast growth factor receptor FGFR4 Ctype lectindomain family member A CLEC4A DCC and DSCAMFigures 2I and 3BObserved PSG interactions occur selectively and differentiallyto these binding partners Figure 2I Data S4 Using SPR weexamined binding of DCC and DSCAM to PSG7 and PSG9 Figure 3E We observed binding for DSCAM with PSG9 and bothDCC and DSCAM with PSG7 Interestingly DCC is expressedin human placenta DakouaneGiudicelli ThesePPIs present new candidate receptors for studying the role ofPSGs in pregnancy immunoregulation and angiogenesisLILR Subfamily PPIs with BTNL8 and MyelinationProteinsLeukocyte immunoglobulinlike receptors LILRs membersare a subfamily of activating LILRA and inhibitory LILRB receptors that exhibit immunomodulatory activity and function ininflammation regulation tolerance and differentiation Burshtynand Morcos We observed binding of multiple activatingand inhibitory LILRs to butyrophilin8 BTNL8 Figure 2F BTNLsare members of the extended B7 family of molecules and function as costimulatory or coinhibitory signals for T cell activationRhodes LILRs are also expressed on neurons and function in the regulation of development synaptic plasticity and axonal regeneration Hirayasu and Arase Myelin the protective insulatinglayer around axons inhibits neuronal regeneration following spinal injury Monje Three myelin proteins Nogo MAG andOMgp are known to interact with PirB in mouse one of only twomouse LILRB orthologs Atwal We observed bindingof multiple LILRs to two additional myelin proteins myelinoligodendrocyte glycoprotein MOG and myelin protein P0 MPZPVR Selectively Interacts with a KillerCellImmunoglobulinlike Receptor KIRThe polygenic Killercellimmunoglobulinlike receptors KIRsare a highly polymorphic subfamily of activating and inhibitoryproteins expressed on natural killer NK cells that regulate development maturation and activation Pende NK cellsinitially express a stochastic combination of KIRs that is refinedduring maturation to tune killing response threshold and ensureoptimal discrimination of target cells from healthy cells Weobserved binding of KIR2DL5 to poliovirus receptor PVR Figures 2C and 5A which validates a recently reported PPI Husain We confirmed KIR2DL5 binding to PVR by ECIAtitration analysis and SPR Figures 5C and 5DTo examine binding at the cell surface fluorescent tetramersof PVRFc and KIR2DL5Fc ECDFcSA647 were incubatedwith fulllength KIR2DL5 and PVRtransfected cells respectively Flow cytometry analysis revealed concentrationdependent binding of both ligands to cells expressing cognate fulllength receptor but not control cells Figure 5E PVRFc tetramers did not bind cells transfected with fulllength KIR2DL4and KIR2DL1 which share and sequence similaritywith KIR2DL5 respectively Figures 5B and 5F To furtherexamine the specificity of PVR for KIR2DL5 ECIA was performed using KIRs PVR bound specifically to KIR2DL5 Figure 5G Because KIR2DL5 has been associated with increasedvirus susceptibility and reduced antiviral response to therapythis specificity may have implications for the role of KIR2DL5 inimmunityTrkA Selectively Interacts with TIE1Highaffinity nerve growth factor receptor NTRK1 also knownas TrkA has multiple wellstudied functions in the nervous system Amatu In the immune system where its function is not well understood TrkA is expressed on monocytesmacrophages dendritic cells resting and activated B cells andneutrophils and erythroblasts Minnone In ourscreen we observed TrkA binding to tyrosineprotein kinase receptor TIE1 but not to TIE2 Figures 2A and 6A TIE1 is expressed on endothelial cells immature hematopoietic cells andplatelets and functions in complex with the angiopoietinTIE2pathway to inhibit angiogenesis Eklund We performed SPR and observed TrkA binding to TIE1 but not toFigure SPR Validation of Type IIA and Type IIB PTPRs with SALMs and DSCAMsA Left dendrogram highlighting phylogenetic clustering of SALMs DSCAMs type IIA PTPRs PTPRFSD and type IIB PTPRs PTPRTMK Right SPRvalidated PPIs green line previously known PPI turquoise line previously unknown PPI observed in screen magenta line previously unknown PHA predictedPPI Gray line screen PPI not SPR testedB Protein domain structures Ig immunoglobulinlike domain FN fibronectin type III domain MAM meprin A5 protein domain LRR leucinerich repeat TMtransmembrane NT Nterminal region of ECD CT Cterminal region of ECDC Screen data showing PPI specificity of PTPRFSD and PTPRTMK subfamilies Background subtracted OD nm data are represented as mean oftriplicate wellsD SPR sensrams for DSCAML1CT and DSCAMCT analytes 2fold dilutions “ nM binding to PTPRM and PTPRT ligandsE SPR sensrams for PTPRS analyte 2fold dilutions “ nM binding to SALMs and DSCAMCT ligandsF SPR max RU values for every pairwise combination of PTPRFSD analytes with SALM ligandsRU resonance unitsSee also Figures S5 and S6 and Data S5 Cell “ August 0cResourcellABCEDFGlegend on next pageCell “ August 0cllResourceTIE2 Figures 6B and S7A As a positive control for TIE2 weconfirmed binding to monomer angiopoietin1 Figure S7A Davis We next investigated whether TrkA can bind NGF and TIE1simultaneously We preincubated TrkA with NGF and comparedbinding of T

Thyroid_Cancer

Signaling pathway analysis methods are commonly used to explainbiological behaviors of disease cells Effector genes typically decide functionalattributes associated with biological behaviors of disease cells by abnormal signalsthey received The signals that the effector genes receive can be quite different innormal vs disease conditions However most of current signaling pathway analysismethods do not take these signal variations into considerationMethods In this study we developed a novel signaling pathway analysis methodcalled signaling pathway functional attributes analysis SPFA method This methodanalyzes the signal variations that effector genes received between two conditionsnormal and disease in different signaling pathwaysResults We compared the SPFA method to seven other methods across GeneExpression Omnibus datasets using three measurements the median rank of targetpathways the median pvalue of target pathways and the percentages of significantpathways The results confirmed that SPFA was the topranking method in termsof median rank of target pathways and the fourth best method in terms of medianpvalue of target pathways SPFA™s percentage of significant pathways was modestindicating a good false positive rate and false negative rate Overall SPFA wascomparable to the other methods Our results also suggested that the signal variationscalculated by SPFA could help identify abnormal functional attributes and parts ofpathways The SPFA R code and functions can be accessed at githubcomZhenshenBaoSPFASubjects Bioinformatics Cell Biology Computational Biology Computational ScienceKeywords Signaling pathway analysis Functional attributes Cell behaviors SPFA Effector genesINTRODUCTIONRecently developed highthroughput functional genomics technologies have generatedlarge amounts of experimental disease data and detected new biological informationChallenge for biologists is understanding the biological behaviors of disease cells usingboth newly generated disease data and existing biological knowledge Signaling pathwayanalysis methods are used to better understand the biological behaviors of disease cellsHow to cite this Bao Z Zhang B Li L Ge Q Gu W Bai Y Identifying diseaseassociated signaling pathways through a noveleffector gene analysis PeerJ 8e9695 107717peerj9695Submitted February Accepted July Published August Corresponding authorYunfei Bai whitecfseueducnAcademic editorJun ChenAdditional Information andDeclarations can be found onpage 107717peerj9695Copyright Bao et alDistributed underCreative Commons CCBY 0cThe understanding of biological behaviors of disease cells benefits to understand thepathological scenery and treatment Overrepresentation analysis ORA based methodswere initially presented as signaling pathway analysis methods to help biologists identifyoverrepresented pathways from a list of relevant genes produced from experimentaldata ORAbased methods merely count the number of differentially expressed genes inspecific functional category gene sets such as the Gene Ontology GO Blake the Kyoto Encyclopedia of Genes and Genomes KEGG Kanehisa BioCarta Nishimura and Reactome Joshitope Then they determinesignificance of the overlaps via statistical tests such as Fisher™s exact test Many tools arebased on this method including OntoExpress Draghici Khatri Fisher Khatri Sirota Butte and the Gene Ontology Enrichment AnalysisSoftware Toolkit GOEAST Zheng Wang However ORAbased methods onlytake into account large changes in individual genes that significantly affect pathwaysand they do not account for smaller changes in sets of functionallyrelated genesie pathways capable of significant effects Functional class scoring FCS basedmethods have been used to avoid this limitation of ORAbased methods FCSbasedmethods take into account the coordinated gene expression changes in pathways such asgene set enrichment analysis GSEA Subramanian gene set analysis GSAEfron Tibshirani and meanrank gene set enrichment tests MRGSE Liu However ORAbased and FCSbased methods are both limited because theydo not take into account the complex interactions between genes or the complextopology of pathways To overcome this limitation pathwaytopologybased methodswere proposed Pathwaytopologybased methods integrate the complex interactionsbetween genes using pathway topology information specifically KEGG signalingpathway informationSignaling pathway impact analysis SPIA one of the most widelyused pathwaytopologybased methods considers both the number of differentially expressed genesDEGs in a given pathway and the topology information of that pathway Tarca Many improved methods based on SPIA have been proposed Li developed a method called subSPIA which used a minimum spanning tree way toprune signaling pathways and used the SPIA method to identify significant signalingsubpathways Li Bao developed two SPIAbased methodscalled PSPIA and MSPIA These two methods replaced or ˆ’ interaction strength inSPIA with the interaction strength of the Pearson correlation coefficients and mutualinformation respectively Bao There are different pathwaytopology methodsthat make use of the topological information of signaling pathways For instance GeneGraph Enrichment Analysis GGEA uses prior knowledge derived from directed generegulatory networks Geistlinger Liu Xu Bao used a subgraphmethod to take advantage of pathway topological information Liu Xu Bao ROntoTools introduced a term of perturbation factor by considering the type ofinteractions to take the pathway topology into consideration Tarca Bao PeerJ 107717peerj9695 0cVoichita Donato Draghici SebastianLeon developed a method usingtopology to detect liner subpathways in a signaling pathway SebastianLeon These methods still have their disadvantages Pathwaytopologybased methods donot consider the importance of genes in pathways Geneweightbased methods havebeen proposed to overcome this limitation Pathway analysis with downweighting ofoverlapping genes PADOG uses the frequency of a present gene in the analyzedpathways to improve gene set analysis Tarca Functional link enrichment ofgene ontology or gene sets LEGO measures gene weights in a gene set according toits relative association with genes inside and outside the gene set in a functional associationnetwork Dong Fang proposed an improved SPIA method calledSPIAIS that measured and assigned the importance as the average output degree ofthe gene in the pathwayA signaling pathway is a cascade of molecular reactions that bring out the functionalattributes eg cell proliferation apoptosis associated with the biological behaviors ofdisease cells using effector genes Effector genes receive signals without outputting signalsto other genes in an individual signaling pathway SebastianLeon Diseasesare always related to the abnormal signal that the effector genes receive Thereforethe signal that the effector genes receive can be very different under disease andnormal conditions The limitation of the previously mentioned methods includinggeneweightbased methods is that they do not consider the signal variations betweendisease and normal conditionsAdditionally the functional attributes in the same signaling pathway may be verydifferent from one another and can sometimes be opposites For example there are twoopposite functional attributes on the axon guidance pathway axon repulsion and axonattraction see the hsa04360 pathway in the KEGG dataset We cannot determinewhich functional attributes contribute more to the disease using most current pathwayanalysis methods Furthermore some pathways consist of several parts each with verydifferent contributions For example the Wnt signaling pathway is significant acrossdifferent diseases and can be divided into three parts Most existing pathway analysismethods cannot determine which part of the Wnt signaling pathway most significantlycontributes to a specific diseaseWe propose a new method that considers the signal variations between normal anddisease conditions that effector genes received in pathways the signaling pathwayfunctional attributes analysis SPFA method SPFA calculates the gene expression changesin a given pathway using an ORA method and then combines the ORA method resultswith the signal variation results under two conditions normal vs disease The signalvariations can help identify functional attributes and abnormal pathways We tested thecapabilities of the proposed signaling pathway analysis method on a series of real datasetsusing three parameters We also showed that the two types of probabilities consideredin this method were indeed independent Ultimately we verified the usefulness of thesignal variations the effector genes received under two different conditions using theSPFA methodBao PeerJ 107717peerj9695 0cMATERIALS AND METHODSData sources and preprocessingSignaling pathway analysis methods require two types of input a collection of pathwaysand a list of genes or gene products with accompanying expression values across differentsamples between the compared phenotypes We used the KEGG signaling pathway asit is the most common manuallycurated signaling pathway used for pathway analysisWe downloaded signaling pathways from the KEGG PATHWAY datasetWe acquired disease gene expression datasets from the KEGGdzPathwaysGEORpackage and KEGGandMetacoreDzPathwaysGEO Rpackages Table TarcaBhatti Romero Tarca Each disease gene expression dataset wasmatched with a corresponding disease KEGG pathway For example a colorectal cancerdataset was associated with the colorectal cancer pathway Tarca The corresponding disease KEGG pathways were called target pathways Three rules wereused to select the gene expression datasets The dataset™s DEGs were available If no DEGs were selected other comparable methodswould return null results The results of these datasets could be analyzed Pathway analysis result pvalues of could not be analyzed The target pathways of these datasets were KEGG pathways since we used KEGGpathways as examplesDEGs were selected if they contained more than genes with FDR adjustedpvalues Otherwise we selected more than genes with original pvalues and absolute log fold change If DEGs still less than genes we selected the top of genes ranked by pvalues as DEGs 0c 0cdse 0c 0cSPFA algorithm designTo assess the signal variations between two conditions normal vs disease that the effectorgenes received from upstream genes we calculated the sum of signal variations from allupstream genes to effector genes Given an effector gene ge and an upstream gene gsthe signal variation from the gene gs to the effector gene ge can be defined asese ¼ cordiseaseðgsgeÞ 00 cornormalðgsgeÞwhere cordiseaseðgsgeÞ and cornormalðgsgeÞ refer to the Pearson correlation coefficientbetween the gene expression data of gene gs and gene ge in the disease and normal statesrespectively dse is the network distance between gene gs and gene ge The Pearsoncorrelation coefficient is always used in gene coexpression networks to represent thestrength of interactions between two genes The Pearson correlation coefficient can also beused to represent the strength of an interaction between two gene expression valuesStudies have shown that the genetic regulatory patterns in signaling pathways betweenBao PeerJ 107717peerj9695 0cTable Data sets used for assessing the proposed method and compared methodsIDTarget pathwayGEO IDReferencesColorectal cancerColorectal cancerColorectal cancerColorectal cancerColorectal cancerNonsmall cell lung cancerPancreatic cancerPancreatic cancerPancreatic cancerThyroid cancerThyroid cancerAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseAlzheimer™s diseaseChronic myeloid leukemiaChronic myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaDilated cardiomyopathyDilated cardiomyopathyEndometrial cancerGliomaGliomaHuntington™s diseaseParkinson™s diseaseParkinson™s diseaseProstate cancerProstate cancerRenal cell carcinomaRenal cell carcinomaGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762Hong Galamb and Gyorffy SabatesBellver Hong Uddin SanchezPalencia Badea Pei Donahue He “Liang Liang Liang Blalock Juan Affer Affer Le Dieu Le Dieu Stirewalt “Barth Hever Liu Liu Runne Zhang Zheng Wallace Wallace Lenburg Wang genes are different under normal and disease conditions Jung If the geneticregulatory pattern between the two genes changes the signal transmitted between thetwo genes will be very different Thus we used the Pearson correlation coefficient tocalculate the signal variations that the effector genes received from their upstream genesHowever if an upstream gene does not directly transmit a signal the signal may beattenuated Therefore we used the network distance dse between gene gs and gene ge as apenalty coefficientBao PeerJ 107717peerj9695 0cFor each effector gene gi in a given pathway the accumulated signal variations betweeneijXsj¼1normal and disease conditions that the upstream genes received total s genes in theupstream of the gene gi were calculated using the formulaASVðgiÞ ¼The accumulated signal variation ASVðgiÞ of the effector gene gi in a pathway can helpus distinguish among the functional disease attributes Effector genes with high ASVðgiÞdemonstrate that these functional attributes significantly contribute to their correspondingdiseasesFor a given signaling pathway the total accumulated signal variation ASV can bedefined asASV ¼Xki¼1ASVðgiÞwhere k is the total number of effector genes in the given pathwayUltimately the probability Psd used to measure the signal variations between twoconditions normal vs disease that those effector genes received from genes upstream in agiven signaling pathway Px is based on the pathway™s ASVðPxÞ The same number of genesas the one observed on the given signaling pathway are randomly selected from allgenes random gene IDs and have any possible expression data in all samples in the rangeof the experimenter Therefore the observed signal variations were obtained by permutingthe gene IDs times ASVperðPxÞwas the total accumulated signal variation of thegiven pathway Px obtained in the perth time The probability PsdðPxÞ of the given pathwaywas calculated asPsdðPxÞ ¼IðASVperðPxÞ 15 ASVðPxÞÞPwhere I is a function that returns when the argument is true and when the argument isfalseThe probability Psd does not measure the gene differential expression in a givenpathway Thus we had to combine the probability Psd with the probability Pde which canmeasure the total gene differential expression in a given signaling pathway The probabilityPde of a given pathway Px can be calculated through the following hypergeometric test 13 12 13tr 12m 00 t 12 13n 00 rmnPdeðPxÞ ¼ 00where the whole genome contains a total of m genes n genes are the number of DEGs inthe m genes and the given pathway contains t genes and r DEGsBao PeerJ 107717peerj9695 0cThe probability Psd uses the Pearson correlation coefficient of the two genes™ expressiondata but the probability Pde uses the number of DEGs in a pathway Thus the twoprobabilities are independent of each other The significance of the given pathway wascalculated following the SPIA method which combines the probabilities Psd and Pde Tarca The formulas areP ¼ c 00 c 01 lnðcÞc ¼ Psd 02 Pdewhere c is a product of Pde and Psd P is the combined probability of the signalingpathwaySignificantly enriched pathway analysis using SPFAThe SPFA procedure identifies significantly enriched pathways in two steps Fig The first step measures the total gene differential expression in the signaling pathwaysDEGs need to first be identified from the gene expression datasets Then the DEGs aremapped onto the signaling pathways Finally the signaling pathway pvalues are calculatedusing a hypergeometric testThe second step is to measure the signal variations between the two conditions normalvs disease that effector genes received from upstream genes in the signaling pathwaysThis is completed by Finding all effector genes in each signaling pathway Ascertaining all paths from the upstream genes to the effector genes in each signalingpathway If a path exists between the upstream genes and effector genes an interactionmust exist between them The path™s network distances are used to weight thecorresponding interactions Using the Pearson correlation coefficient absolute difference values between the diseaseand normal samples to calculate the signal variations of the corresponding interactions Using the network distance of each interaction to decrease their signal variations Calculating the accumulation of the signal variations between the effector genes andupstream genes for each effector gene Calculating the sum of the accumulations of all effector genes in each signaling pathway Evaluating the statistical significance of each pathway based on their scoreUltimately the results of the two steps are combined into one pvalue We used the FDRadjust method on the combined pvalue to determine the significance of each signalingpathway The pathways with the adjusted combined pvalues smaller than a thresholdvalue were considered as significant pathwaysThe distribution of effector genes in the signaling pathwaysStudying the signal variations between two conditions normal vs disease that the effectorgenes received leads to a deeper understanding of the biological behaviors of disease cellsEffector genes are widely scattered throughout the signaling pathways If a gene has noBao PeerJ 107717peerj9695 0cFigure The workflow of SPFA method The step by step to identify significant signaling pathways using SPFAFullsize \ue90d 107717peerj9695fig1signal inputs in an individual signaling pathway the gene is not considered an effectorgene The distribution of effector genes in each signaling pathway can be seen in Fig One hundred and ninetyfive of the signaling pathways contained effector genesComparison methods and measuresWe compared seven methods to SPFA including Fisher Khatri Sirota Butte GSA Efron Tibshirani GSEA Subramanian MRGSE Liu SPIA Ullah ROnoTools Tarca Voichita Donato Draghici and PADOG Tarca We selected these methods for their stability andprevalence they can be compared using the same R environment as SPFAThere is no universally accepted technique for the validation of the results of pathwayanalysis methods Current pathway analysis methods use the results of a very smallnumber of datasets based on searching corresponding published life literature Thisapproach has its limitations First the number of datasets used is small Second authorsoften search their own leading to biased results Third complex biological phenomenaalways directly or indirectly correspond to multiple signaling pathwaysTarca compiled an objective and reproducible approach based on multipledatasets Tarca This approach avoided a biased literature search and requiredtesting on a large number of different datasets at least In this work we followedBao PeerJ 107717peerj9695 0cseneg rotceffe fo rebmuNPathwaysFigure The distribution of the effector genes™ number in each signaling pathway A total of ofFullsize \ue90d 107717peerj9695fig2 signaling pathways contain the effector genesTarca validation approach Two measurements were compared in thisvalidation approach The first measurement was the median pvalue of the targetpathways of the disease datasets Smaller median pvalues meant higher sensitivityThe second measurement was the median rank of the disease target pathwaysThe higher ranked methods were more accurate To further validate the different pathwayanalysis method results we used a third measurement the ratio of significant pathwaysusing a significance threshold of of the adjusted pvalue in the datasets Thismeasured the method™s ability to control false positive and false negative ratesRESULTSThe independence between the two probabilitiesThe two probabilities Pde and Psd are theoretically independent under the null hypothesisWe verified their independence by calculating the squared correlation coefficient betweenthe two probabilities using the gene expression datasets Table Our results showed thatthe average squared correlation coefficient of the datasets was R2 ¼ Only four ofthe squared correlation coefficients were slightly higher than R2 ¼ These resultsindicated essentially no correlation between the two probabilitiesSPFA method performanceWe compared SPFA with the other seven methods using three measurements the medianpvalue of the target pathways the median rank of the target pathways and the ratioof significant pathways The signaling pathways with adjusted pvalues ‰¤ weresignificantWhen comparing the median rank of the target pathways SPFA ranked first Fig When comparing the median pvalue of the target pathways SPFA ranked fourthBao PeerJ 107717peerj9695 0cTable The squared correlation coefficients between the two probabilities using the geneexpression datasets The four squared correlation coefficients which are slightly more than areshown in boldGEO IDGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762AverageSquared correlation between the probabilities Pde and Psd661523Eˆ’748134E”Fig Notably the methods with the highest ranking in one measurement did notnecessarily rank the highest in the others This is because different measurements analyzedifferent abilities For example MRGSE was first in median pvalue but was sixth inmedian rank Fisher was second in median pvalue but ranked fourth in median rankTo better compare SPFA™s performance against the other methods we added the ranks ofBao PeerJ 107717peerj9695 0csyawhtap tegrat fo sknaRSPFAPADOGSPIAFisherGSAMRGSEGSEA ROntoToolsFigure The distribution of the target pathways ranks of the eight methods using datasets SPFAperforms the 1st among eight methods in terms of the median ranks of the target pathwaysFullsize \ue90d 107717peerj9695fig3the median pvalue and median rank values from each method together We found that thecombined value of SPFA and PADOG was the smallest Table To further assess the performance of the eight methods we collected the results fromother general pathways typically associated with cancer using the out of datasetswith a form of cancer in Table Apoptosis and Pathways in cancer When using theApoptosis pathway and Pathway in cancer pathway instead of target pathways SPFA™smedian ranks were both first and the median pvalues of MRGSE were also both rankedfirst These results were in alignment with the target pathway results However when usingBao PeerJ 107717peerj9695 0ctsyawhap tegrat fo seuavplMRGSEFisherPADOGSPFASPIAGSEAGSA ROntoToolsFigure The distribution of the target pathways pvalues of the eight methods using datasetsSPFA performs the 4th among eight methods in terms of the median pvalues of detecting the tarFullsize \ue90d 107717peerj9695fig4get pathwaysthe Apoptosis pathway and Pathway in cancer pathway instead of the target pathwaysPADOG™s median pvalues were both ranked fifth When using the Apoptosis pathwaySPFA™s median pvalue ranked third When using the Pathway in cancer pathway SPFA™smedian pvalue ranked fourth All these results suggest that SPFA had the best accuracyand a good sensitivity when compared with the other seven methodsAdditionally our results showed that SPFA™s ratio of significant pathways wasmoderate Fig compared to the others MRGSE™s ratio of significant pathways wasalmost and it could be questioned whether a such number of pathways was realisticBao PeerJ 107717peerj9695 0cTable The combined rank values of the ranks in terms of the median pvalues and the medianranks of target pathways of eight methodsMethodsSPFAPADOGFisherMRGSESPIAGSAGSEAROnoToolsRanks of the median pvaluesRanks of the median ranksSumTable The results of other general pathways apoptosis and pathway in cancer typically associatedwith cancer using the out of datasets with a form of cancer For each pathway the values for thetype of methods with the smallest median pvalues and ranks strongest association with the phenotypeare shown in boldPathway statisticApoptosisSPFAFisherSPIAGSAGSEAMRGSERontoToolsPADOGpValues medianRanks medianPathway in cancerpValues median794Eˆ’225Eˆ’162Eˆ’27Eˆ’Ranks medianGSA™s ratio of significant pathways was lower than and it reflected that the GSAmethod had a high false negative rate The methods had a modest ratio of significantpathways indicated that the method had a modest false positive rate and a modest falsenegative rate Thus the discriminative ability of SPFA was good when compared with theother seven methods In conclusion our results strongly supported that SPFA waswellsuited for signaling pathway analysis and confirmed previously reported results inDong Sources of improvement for SPFAThe main source of improvement in SPFA is that it uses signal variations that effectorgenes received under normal and disease conditions SPFA is compared to the simplerORAbased method used to calculate the probability Pde without accounting for signalvariations Fig As shown in Fig the ORAbased method has a higher worse rankand pvalue than SPFA for the target pathwaysBao PeerJ 107717peerj9695 0csyawhtap tnacifings tion dna tnacfings fio oitar ehTsignificancenot significantsignificantSPFAFisherSPIAROntoToolsGSAMRGSEGSEAPADOGmethodsFigure Average percentage of the pathways detected as significant and not significant by eachmethod using the threshold of pvalues ‰¤ Fullsize \ue90d 107717peerj9695fig5Validating the correlation between diseases and the signal variationsthat effector genes received under two different conditionsTo validate the correlation between diseases and the signal variations that effector genesreceived under two different conditions normal vs disease we analyzed a colorectalcancer dataset GSE4183 and an Alzheimer™s disease dataset GSE16759 The colorectalcancer microarray GSE4183 Affymetrix array HGU133 Plus20 included colorectalcancer samples and normal samples Galamb Gyorffy Bao PeerJ 107717peerj9695 0cABttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa ttt tttt tfff foooo sss seeekuuunaaaavvvRppplllttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa t tttt tttf fffoooo s sssekkkunnnaaaavRRRplSPFASPFASPFASPFAORAORAORAORASPFASPFASPFASPFAORAORAORAORAFigure Determining the contribution of signal variations received by effector genes between twodifferent conditions normal vs disease in SPFA performance The boxplots show the distribution ofFullsize \ue90d 107717peerj9695fig6the target pathways ranks A and pvalues BThe Alzheimer™s disease dataset GSE16759 included four disease samples and four normalsamples Juan The Wnt signaling pathway was altered in of the colorectal cancer samplesGalamb We assessed the signal variations that effector genes received in theWnt signaling pathway using the GSE4183 dataset Fig The results of Galamb coincided with our signal variation results Galamb reported thatoverexpression of TNS1 could induce the activation of JNK ENTREZID and The signal variation that the effector gene ENTREZID received ranked first inour results Galamb detected that RBMS1 is another overexpressed geneand modulator of cmyc ENTREZID cmyc can regulate cell cycles and cause cellsto transform pathways The signal variation that the effector gene ENTREZID received ranked second in our results Galamb also identified that TCF4 is anoverexpressed gene that can participate in the transcriptional regulation of genesassociated with colon carcinogenesis These colon carcinogenesis associated genes includecmyc ENTREZID cyclin D1 ENTREZID PPARδ ENTREZID and MMP7 ENTREZID The signal variations that these effector genes receivedranked second fourth fifth and sixth respectivelyBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoitairav liangSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig7Many pathways can be studied in colorectal cancer datasets For example the PI3KAktsignaling pathway plays a critical role in the growth and progression of colorectal cancerJohnson The effector genes ENTREZID596 ENTREZID842 andENTREZID1027 have the highest signal variations and are linked to cell cycle progressionand cell survival Fig The GSE4183 dataset results further confirmed the role of thispathway in colorectal cancer developmentThe Wnt signaling pathway is also closely related to the occurrence and development ofAlzheimer™s disease Inestrosa The signal variations that different effectorgenes received calculating based on the Alzheimer™s disease dataset GSE16759 in the Wntsignaling pathway were shown in Fig The signal variations that the effector genesENTREZID and received were considerably higher than the other effector genesin the Wnt signaling pathway This result validated evidence of crosstalk between theAlzheimer™s disease signaling pathway and the two effector genes™ upstream genes in theWnt signaling pathwayAll these results indicated the high correlation between diseases and the signalvariations calculated using the SPFA methodBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoiitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the PI3KAktsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695fig8The other usages of the signal variations that effector genes receivedunder two different conditionsThe signal variations that effector genes received under two different conditions canshow the different contributions of different functional attributes contributed to theircorresponding diseases We can also identify which parts of the pathway contribute to theircorresponding diseases through the signal variations that effector genes receivedWhen looking at the Wnt signaling pathway results of GSE4183 Fig first we knowthe functional attributes participating in the cell cycle have abnormal signal variationsbecause most effector genes with high signal variations participate in the pathway cell cycleincluding cmyc ENTREZID cyclin D1 ENTREZID and PPARδENTREZID and MMP7 ENTREZID Second we can know that theabnormal state of the first and second parts of the Wnt signaling pathway may contributemore to colorectal cancer because that the effector genes with high signal variations areall in the two parts If we were only to observe DEG distribution in the Wnt signalingpathway using the GSE4183 dataset we would not know which abnormal part contributedto the disease Fig Through the result of the Wnt signaling pathway in GSE16759Fig on one hand according to this result we can know that the functional attributeslinked with the effector genes ENTREZID and which had the highest signalBao PeerJ 107717peerj9695 0cseneg rotceff eehi t ybdevecer snoitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using Alzheimer™s disease datasets GSE16759Fullsize \ue90d 107717peerj9695fig9variations were abnormal in Alzheimer™s disease On the other hand this may dominatethat the first part of the Wnt signaling pathway may be more related to t

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

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

Thyroid_Cancer

Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly citedCutaneous metastases are unusual presenting symptoms of lung cancer erefore they are prone to be misdiagnosed and missede report describes a case of a fortynineyearold female with painful zosteriform rashes showing multiple vesiclelike papuleslocalized on the left breast for days e patient had been diagnosed as lung adenocarcinoma at the department of oncology oneyear ago Skin biopsy revealed blue nodular lesions in the dermis composed of clustered heterogeneous tumor cells with glandularformation Immunohistochemical stains confirmed the diagnosis of metastatic lung adenocarcinoma IntroductionLung cancer can metastasize to almost all ans butmore often invades the hilar nodes liver adrenal glandsbones and brain [] e incidence of lung cancer withmetastases to the skin varies between “ [] A lungcancer metastasis is usually classified only as adenocarcinoma squamous cell carcinoma SCC or undiï¬erentiated carcinoma Until the 1980s SCC was reported asthe most common type of lung cancer However adenocarcinoma has replaced SCC as the most common lungcancer subtype especially in women and in neversmokers Sun reported that the type of adenocarcinoma was times more frequent than that of SCC []Skin metastases can appear on any cutaneous surface andthe most common sites are the chest abdomen head andneck [ ] Cutaneous metastases have various manifestations such as single papulesnodules or multiplelesions on anywhere of the skin while other rare formsmay show plaquelike lesions erysipelaslike papuleszosteriform lesions and scars [ ] Case PresentationA fortynineyearold nonsmoker female was admitted toour department with multiple painful papules localizedon the left breast ey appeared eruptively for about days and initially diagnosed as herpes zoster in anotherhospital e patient had been diagnosed as lung adenocarcinoma at the department of oncology one year agoShe was given oxitinib mesylate a targeted drug for thetreatment of nonsmallcell lung cancer In addition thepatient exhibited symptom of pain signs of weight lossanorexia and fatiguePhysical examination showed zosteriform vesiclelikepapules measuring “ cm on the left breast elesions were pink or red firm and tender Figure Excisional biopsy was performed revealing blue nodularlesions ltrating in the dermis composed of clusteredheterogeneous tumor cells with glandular formationSome tumor cells were detected within vessels or lymphatic vessels Some cells were transparent Mitosis wassignificant Figures 2a“2c In immunohistochemistumor cells were positive for cytokeratin CKtrycytokeratin7 CK7thyroid transcription factor1TTF1 and EMA and negative for cytokeratin20CK20 carcinoembryonic antigen CEA and grosscysticGCDFP15Figures 3a“3c Proliferative index as measured byKi67 was approximately “ oftumor cellsAccording to the clinical and pathological features cutaneous metastatic lung adenocarcinoma was madeprotein15diseasefluid 0cCase Reports in Dermatological MedicineFigure Zosteriform vesiclelike papules measuring “ cm on the left breast Pink or red firm and tenderabcFigure Skin biopsy revealed a blue nodular lesions ltrating in the dermis composed of clustered heterogeneous tumor cells withglandular formation HE magnification — b some tumor cells were detected within vessels or lymphatic vessels HE magnification — c some cells were blue and transparent and mitosis was significant HE magnification —abcFigure Immunohistochemical stain highlighting the tumor cells showing a CK7 b EMA and c TTF1 positive DiscussionSkin metastases suggest the progression of primary cancerand portend a poor clinical prognosis Skin metastases fromlung cancer are rare e percentage of patients with lungcancer that develops cutaneous metastases ranges from to percent [] It is seen more often in men than in women[] It does not show any specific presentation It is oftenpainless and less likely to be noticed making it more difficultto be diagnosed correctly which may delay treatment Although described cases show that metastatic nodules arepainless our patient showed severe pain e presence ofzosteriform painful vesiclelike lesions really mimics herpeszoster clinically in our casee mechanisms determining the metastasis of lungcancer in skin remain unknown Pathogenesis is suggested tobe by lymphovascular invasion with poor diï¬erentiationand upper lobe tumors increasing the risk [] Usually skinmetastasis develops after initial diagnosis of the primarymalignancy and late in the course of the disease Occasionally skin lesions that arise from lung cancer may develop before the primary tumor is recognized In our case 0cCase Reports in Dermatological Medicine[] R Koca Y Ustundag E Kargi G Numanoglu andH C Altinyazar œA case with widespread cutaneous metastases of unknown primary origin grave prognostic findingin cancer Dermatology Online Journal vol no p [] N A Babacan S Kilickap S Sene œA case of multifocalskin metastases from lung cancer presenting with vasculitictype cutaneous nodule Indian Journal of Dermatologyvol p skin metastases occurred during the immunotherapy Histology shows most commonly adenocarcinoma and thensquamoussmallcell followed by largecell carcinoma []Immunohistochemical markers are useful for the identification of the primary cancer or when a shorter diï¬erential isdesired AntiTTF is both sensitive and specific for primaryadenocarcinomas bronchioalveolar carcinomas and smallcell carcinomas when thyroid primary is excluded []CK7and CK20ˆ’ are sensitive but not specific for primaryadenocarcinomas and bronchioalveolar carcinomas eCK7CK20ˆ’tumors usually include the lung breast endometrium ovary thyroid salivary gland and mesothelioma [ ]Treatment of a single solitary skin lesion usually includessurgery alone or combined with chemotherapy andor radiation If lesions are more disseminated chemotherapy isthe primary option but may elicit an inadequate response[] Radiation can also be used alone andor in combinationwith chemotherapy andor surgery However despite thecombination of radiotherapy and chemotherapy patientswith lung cancer developing cutaneous metastases have apoor outcome Mean survival is short usually to monthsafter diagnosis of cutaneous metastasis []Conflicts of Intereste authors declare they have no conflicts of interestAcknowledgmentsis work was supported by a grant from the NationalNatural Science Foundation of China References[] T W Mollet C A Garcia and G Koester œSkin metastasesfrom lung cancer Dermatology Online Journal vol no [] S Sun J H Schiller and A F Gazdar œLung cancer in neversmokersa diï¬erent disease Nature Reviews Cancer vol no pp “ [] S Dreizen H M Dhingra D F Chiuten T Umsawasdi andM Valdivieso œCutaneous and subcutaneous metastases oflung cancer Postgraduate Medicine vol no pp “ [] M Khaja D Mundt R A Dudekula œLung cancerpresenting as skin metastasis of the back and hand a caseseries and literature review Case Reports in Oncology vol no pp “ [] W T McSweeney and K Tan œCutaneous metastases as apresenting sign of metastatic NSCLC Journal of Surgical CaseReports vol no [] M H Brownstein and E B Helwig œMetastatic tumors of theskin Cancer vol no pp “ [] R B McGrath S P Flood and R Casey œCutaneous metastases in nonsmall cell lung cancer BMJ Case Reportsvol [] V Jerome Marson J Mazieres O Groussard œExpression of TTF1 and cytokeratins in primary and secondaryepithelial lung tumours correlation with histological type andgrade Histopathology vol no pp “ 0c'

Thyroid_Cancer

"metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes Numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors Aspartate hydroxylase ASPH is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion ASPH also promotes tumor growth by stimulation ofangiogenesis and immunosuppression These effects are mainly achieved via the activation of Notch and SRCsignaling pathways ASPH expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact Therefore small molecule inhibitors of ASPH enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models ASPH can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation CD and CD4 T cells in mice The PAN3011 vaccine against ASPH has already been tested in aphase clinical trial in patients with prostate cancer In summary ASPH is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapyKeywords ASPH Small molecule inhibitor Metastasis ImmunotherapyBackgroundCancer is a multifactorial disease with an approximate million fatalities in Worldwide it is the secondleading cause of death [] The complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression Despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis [] delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer Since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential In thisapproachesthat Correspondence smahelmnaturcunicz1Department of Genetics and Microbiology Faculty of Science CharlesUniversity BIOCEV Vestec Czech RepublicFull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsAspartate hydroxylase ASPH has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] ASPH belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in “ of human solid tumors [“] The overexpressed ASPH is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the Cterminal regionto the extracellular environment where it is accessible toantibody binding Recently molecular targeted therapyhas been developed against this target using small molecule inhibitors SMI that can inhibit the catalytic site 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 0cKanwal Journal of Experimental Clinical Cancer Research Page of in the Cterminal region Moreover as antigenic epitopes that reside on the ASPH protein can efficientlystimulate cluster of differentiation CD and CD8 Tcell responses unique to tumor cells harboring ASPHthis enzyme can be used as a tumor associated antigenTAA in immunotherapy [ ]ThedioxygenasesStructure of the ASPH gene and isoformsASPH is a type II transmembrane protein of approxito the family of αmately kDa that belongsketoglutaratedependenthydroxylated products of ASPH hydroxylation were firstdetected in blood coagulation proteins [“] ASPHwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbEGFlike domains of various proteins[] Fig Thereafter the human ASPH gene wascloned and characterized [] This gene spanning base pairs long region of genomic DNA and containing exons is located at the position q123 of thehuman chromosome The ASPH sequence is highlyconserved in mammalian evolution The sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species[] The whole ASPH protein consists of five domainsan Nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a Cterminal catalytic domain [] Tissue specific transcription is directed from two putative promoters P1 and P2which differ in their regulation sequences [ ] Whilethe transcription from the P1 promoter was observed inmost human tissues the P2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor MEF2 particularly in muscle tissues []The ASPH gene undergoes extensive alternative splicingresulting in four protein isoforms ie ASPH humbugjunctate and junctin [ ] These proteins vary in theCterminal region which affects their function [ ]The two longest ASPH transcript variantsthat aretranscribed from the P1 and P2 promoters and differ inthe length of the ²untranslated region encode the fulllength ASPH protein This protein contains the catalyticCterminal domain that catalyzes the posttranslationalhydroxylation in the cbEGFlike domains of numerousproteins Supplementary Fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] Thetruncated isoforms humbug junctate and junctin sharethe Nterminal part with the ASPH protein but lackcatalytic function They are involved in calcium homeostasis [] Humbug has a potential role in cell adhesionand calcium flux and similar to ASPH its overexpressionhas been correlated with aggressive tumorcell behavior[]reticulummembranebound protein that is known for its functionin the regulation of the intracellular Ca2 concentrationJunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicJunctateisaLocalization in cells tissue distribution andexpression regulationASPH is predominantly a cellsurface protein [] that isalso localized in the endoplasmic and sarcoplasmicreticulum [] Furthermore a recent study identifiedmitochondriallocalization of ASPH in hepatocellularcarcinoma HCC In that study ASPH overexpressioncorrelated with an instability of mitochondrial DNA andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in HCC []ASPH is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands [] and has a potential role in placentalimplantation and fetal growth [] On the contrary theASPH expression in normal adult tissues is relativelylow or negligible However ASPH expression is inappropriately activated during oncogenesis when ASPH is required for generation of malignant and metastaticphenotypes The elevated expression of ASPH at bothFig ASPH catalytic reaction Aspartyl and asparaginyl residues in cbEGFlike domains are hydroxylated 0cKanwal Journal of Experimental Clinical Cancer Research Page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer Table The first study that demonstrated thesignificantly higher expression of both ASPH mRNAand protein in HCC and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by Lavaissiere [] Subsequently they verified the role of upregulated ASPH protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the NIH3 T3 cell line and animal models []The level of ASPH also correlated with cell motility andinvasiveness in in vitro experiments [ ] In thestudy by Maeda [] the overexpression of theASPH protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients Similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung [] and colon carcinomas[] and glioblastoma multiforme [] Recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples ASPH has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes [] In glioblastoma the prognostic significance ofASPH was suggested by profiling of alternative mRNAsplicing []ASPH gene expression is upregulated via Wntcatenin [] and insulininsulinlike growth factor IGF1insulin receptor substrate IRS1 signaling [ ]through extracellular signalregulated kinaseERKmitogenactivated protein kinase MAPK andphosphatidylinositol3kinaseprotein kinase B PI3KAkt pathways Fig for review see ref [] InsulinIGF1IRS1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorsTable Summary of the studies which have identified the elevated ASPH expression in human tumor tissuesStudyPositive cases of studied samples nnTumor tissuesDetectionmethodIHCAntibody recognized region ofASPH proteinFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab Nterminus or 15C7 Abcatalytic domainFB50 Ab NterminusmAb G3 hybridomaPolyclonalFB50 mAb NterminusIHCIHCIHCIHCRTqPCRIHCRTqPCRIHCIHCIHCRTqPCRIHCFB50 Ab NterminusLavaissiere []HepatocellularCholangiocarcinomaBreastColonPalumbo []Pancreatic adenocarcinomaSepe et al[]Primitive neuroectodermalmedulloblastoma neuroblastomaMaeda et al[]Cantarini []CholangiocarcinomaHepatocellularMonte et al[]HepatocellularYang et al[]Wang et al[]Dong et al[]Tang et al[]Lin et al[] types of tumor tissuesaHepatocellularPancreatic cancerHepatocellularBreast 75fold higher level of mRNAcompared to normal tissue 7fold higher level of mRNAcompared to normal tissuePancreatic ductal adenocarcinomaOgawa []aLiver kidney breast cervical ovarian Fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomaFB50 Ab NterminusIHC 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Regulation of ASPH expression and ASPH involvement in signaling pathways The expression of the ASPH protein can be regulated atseveral levels The ASPH gene can be amplified in tumor cells and its transcription activated by INIGF1 and Wnt catenin pathways or inducedby hypoxia At the posttranscriptional level miR200a and miR135a can downregulate ASPH expression Stability of the ASPH protein can bereduced by phosphorylation with GSK3 Conversely ASPH can enhance GSK3 activity by inhibition of its phosphorylation with AKT and p38kinases ASPH also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the Notch receptor and ligands ex JAG and interaction with pRb vimentin and ADAMs Finallyinactivation of NK cells by ASPH has been demonstrated Green arrow activation signal red bar inhibitory signal[] The catenindependent Wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies Upon aberrant activation of Wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus [] where an interaction between catenin and Tcellfactorlymphoidenhancerbinding factor TCFLEF proteins forms atranscriptional regulatory complex which enhances theexpression of Wnt target genes including IRS1 []ASPH was proposed as a common link between Wntcatenin and insulinIGF1IRS1 pathways and downstream signaling []The regulation of ASPH gene expression in tumorsmight also be affected by a copy number variation Inthe study by Kadota [] the ASPH gene locus hasbeen identified as one of the DNA regions with focalamplification in primary breast cancer In colorectal cancer ASPH gain or amplification was found in ofsamples [] Next a suppressant role of the microRNAmiR200a in posttranscription regulation of the ASPHexpression in hepatoma cells has been found [] MiR200a belongs to miR200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref[] Similarly miR135a has beenshown to suppress ASPH in endometrial cancer []Moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 GSK3casein kinase CK2 protein kinase A PKA and protein kinase C PKC on ASPH [] several studies demonstrated that phosphorylation can regulate the ASPHprotein expression [ “] Inhibition of the GSK3activity did not modify mRNA expression but increased 0cKanwal Journal of Experimental Clinical Cancer Research Page of the ASPH protein level [] Direct phosphorylation ofASPH by GSK3 probably decreases ASPH stability andthus reduces cell mobility [] ASPH protein expressionwas also increased by inhibitors of PKA PKC and CK2[] Mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofASPH phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] ThereforeASPH phosphorylation probably regulates the functionof this protein by various mechanismsASPH expression can also be regulated by hypoxia andoxidative stress In human neuronal cells this effect wasmediated by hypoxia inducible factor alpha HIF1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain PHD proteins and factorinhibiting HIF FIH are inactivated Consequently theHIF1 heterodimer made up of subunits HIF1α andHIF1 functions as a transcription factor likely enhancing ASPH expression by binding to hypoxiaresponsiveelements [] In hypoxic regions of glioblastoma bothHIF1α and ASPH were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of ASPH in mesenchymal transition [] Brewitz showed reducedASPH hydroxylation activity at low oxygen concentrations and suggested an ASPH role in oxygen hypoxiasensing ASPH upregulation induced by hypoxia couldcompensate for reduced enzymatic activity [] Moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon ASPHoverexpression which was reversed by silencing ASPHexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion []ASPH protein interactions and signaling pathwaysThe ASPH hydroxylation consensus sequence is confined within cbEGFlike domains that are found in proteins of diverse function including Notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3Axl family ofreceptor tyrosine kinases []The Notch signaling cascade is a remarkably conservedpathway Notch proteins Notch1 Notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment [] Notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells Dysregulation of the Notch pathway is directly linkedto cancer vascular disorders and congenital defects [] In mammals Notch signaling activated by binding ofone of two families of canonical Notch ligandsjaggedJAG1 and JAG2 and delta like DLL1 DLL3 and DLL4leads to the generation of the cleaved Notch intracellulardomain NICD fragment and its nuclear translocation Inthe nucleus the NICD fragment interacts with the DNAbinding complex CSL CBF1RBPjκ SuH Lag1 Thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bHLH transcription factor HES1 HESwith YRPW motif HEY1 CD44 epithelial cell adhesionmolecule EPCAM cmyc protooncogene matrix metallopeptidase MMP29 cyclin D1 cyclooxygenase vascular endothelial growth factor VEGF and proliferatingcell nuclear antigen PCNA [ ]Upregulation of ASPH results in enzymatic modification of the cbEGFlike repeats in the Notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof Notch signaling [ ] Furthermore the interactionof ASPH with a disintegrin and metallopeptidase domainADAM stabilizes this complex and enhances theS2 cleavage ofthe Notch receptors and subsequentNICD fragment release [] The activation of the targetgenes in malignant cells increases cell proliferation migration and invasion []through the epithelialtomesenchymal transition EMT that is probably upregulated by the interaction of ASPH with vimentin []Consequentlythis activation supports tumor growthand metastasis The ASPHNotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]The SRC kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis []ASPH has been identified as an SRC pathway activatorOverexpressed ASPH directly interacts with ADAM12 and strengthens the SRC activation by these proteinswhich promotes MMPmediated extracellular matrixdegradation and tumor invasiveness []ASPH can also contribute to malignant phenotype ofcells by interaction with other proteins Iwagami et alrevealed the interaction of ASPH with GSK3 that prevents GSK3 inactivation by phosphorylation with upstream kinases [] This mechanism was confirmed ina castrationresistant prostate cancer model [] GSK3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination Some types of tumors aresensitive to GSK3 inhibitors [] Recently Huang elucidated a direct binding of ASPH with retinoblastoma protein pRb leading to pRb phosphorylation[] They also showed that this effect was mediated byincreased binding of cyclindependent kinase CDK CDK4 and cyclins D1 and E with pRb and wasdependentAsASPH enzymaticonactivity 0cKanwal Journal of Experimental Clinical Cancer Research Page of phosphorylation of pRb inactivates its tumorsuppressorfunction ASPH can contribute to the progression of cellcycle via interaction with pRbEffect of ASPH on an immune systemTumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system [] In theseprocesses various mechanisms of both innate and adaptive immunity are included [] Immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties []As potential targets of ASPH hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when ASPH is overexpressed on cancercells Indeed such effect was demonstrated for humannatural killer NK cells by using recombinant ASPHwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively []Antibodies against ASPH inhibited these effectsInteraction of ASPH with other immune cells has notbeen studied However we suppose possible influence ofASPH on different tumorinfiltrating cells This assumption comes from the involvement of Notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells Forinstance Notch activation contributed to stimulation ofproinflammatoryantitumorigenic M1 polarization inboth bone marrowderived primary macrophages [] and tumorassociated macrophages[] WhenNotch signaling was abrogated protumorigenic M2polarization was induced even by stimulators of M1polarization [] miR125a has been identified as adownstream mediator of Notch signaling in macrophages [] Similarly the Notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of CD4 and CD8 T cells[] Different Notch receptors and their interactionwith different ligands contribute to these processes []Moreover noncanonical Notch signaling is implicatedin regulation of immune cells [] While activation ofNotch signaling in some cells eg T helper cells cytotoxic CD8 T cells and M1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory T cellsit leads to immunosuppression [] Thus immunostimulatory effect of Notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity [] Therapeutics affecting Notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedASPH as a therapeutic targetOncogenic abilities of ASPH have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with ASPHoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma [] pancreatic cancer [ ] glioma [] breast carcinoma [] castrationresistant prostatecancer [] and colorectal cancer [] In studies analyzingASPH function various approaches were utilized to revealsignaling pathways affected by ASPH Particularly ASPHexpression was diminished by using small interfering RNAs[ ] short hairpin RNAs [ ] or theCRISPRCas9 system [ ] The importance of ASPHenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by SMIs [ ] In vitro assays showed ASPH involvement in cell proliferation migration and invasion Cellularalterations included EMTinhibition of apoptosis andstemness acquisition Tumor growth and invasivenesscould further be supported by ASPHinduced extracellularmatrix degradation angiogenesis and transendothelial migration Notch and SRC signaling are probably major pathways influenced by ASPH Fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models Thus these studies also demonstrated thatASPH is a suitable target for cancer treatment especially bySMIs or immunotherapySmall molecule inhibitorsSMIs of ASPH Fig have been developed and used totest the role of ASPH in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] A small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches Not only can these inhibitors inhibit the catalytic activity of ASPH unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit ASPHcatalytic activity in the endoplasmic reticulum Differentcancers have different ASPH expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed []The first ASPH SMIs published were the tetronimidesMOI500 and MOI1100 Tetronimides were originallysynthesized in by Dahn [] and are redoxactivemimics of ascorbic acid and 2oxoglutarate MOI500 isa mixed inhibitor that inhibits both ASPH and the FatMass and Obesity protein FTO [] and is not onlyorally bioavailable but also can penetrate the blood 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Small molecule inhibitors of ASPHthereand kinasesbrain barrier MOI1100 is a more potent inhibitor ofASPH and is also more selective [] Despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for MOI1100 Enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup [] as in MOI1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inMOI1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters MOI1182 isreported to have the ability to suppress invasive activityat a concentration of nM [] SMIs of ASPH have acharacteristic in vitro concentration dependent profilewhere the activity of the SMI plateaus at value around viability [] emphasizing the noncytotoxic properties of this class of inhibitorsNatural products and inhibitors of other enzymes thathave been repurposed as ASPH inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthineCN2019101414327 and guaianolides related to nortrilobolideCN2019104575886CN2019101414219CN2019101414187 0cKanwal Journal of Experimental Clinical Cancer Research Page of sesquiterpene with complex anticancerBosutinib is a wellknown inhibitor of BCRABL and SRCtyrosine kinases approved for the treatment of chronic myelogenous leukemia [] Cepharanthine is a natural productactivityincluding AMPactivated protein kinase AMPK activationand nuclear factor kappa B NFκB inhibition [] Nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium ATPase SERCA inhibition [] Recently a family of potent pyridine dicarboxylates have alsobeen published [] utilizing a mass spectrometrybasedinhibition assay [] These compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate The synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include PHD2 FIH and lysinespecific demethylase 4EKDM4E in addition to ASPH with varying degrees of selectivity However while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface ASPH inhibitors that may nothave cell penetrating activity []immunity As a target of humoralImmunotherapyASPH can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression Since ASPH is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityASPH on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity ADCC complement dependent cytotoxicity CDCor antibodydependent cellular phagocytosis ADCP []When the ASPH antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen HLA class Ior class II molecules and recognized by CD8 or CD4 Tlymphocytes respectively [] that can be stimulated byimmunization breaking tolerance to selfantigens []Induction of ASPHspecific CD4 and CD8 T cells wasexamined in blood samples of HCC patients Using synthetic peptides derived from ASPH after prediction of HLAclass I and HLA class IIrestricted epitopes it has beenfound that ASPH is a highly immunogenic protein that activates both types of analyzed T cells [] Thus efficientantitumor reactions could be stimulated by immunizationThe first vaccine against ASPH was based on matureddendritic cells DC loaded with the ASPH protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma [] This study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis BothCD8 and CD4 cells contributed to an antitumor effectinduced in a mouse model of HCC by immunizationwith ASPHloaded DCs []The next antiASPH vaccine was based on a bacteriophage lambda display system The viral capsid proteingpD was fused with the N or Cterminus of ASPH andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models []The vaccine PAN3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer[] This study demonstrated safety and immunogenicity of PAN3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen PSAor PSA doubling time ASPHspecific immune responseswere mediated by both antibodies and T lymphocytesAs ASPH is a type II transmembrane protein its Cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes Development ofASPHspecific antibodies has been described in several s [“] The human IgG1 PAN622 recognizesthe catalytic domain of ASPH This antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells [] In the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results [] MouseIgG1 monoclonal antibody binding to the CterminalASPH domain mediated ADCC by human NK cells []Recently a secondgeneration antibody approach hasbeen disclosed The prepared antibody binds to the extreme Cterminus of ASPH US that is involved in specific substrate recognition [] Thereforethis antibody has direct ASPH inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activityPAN622 wasbioimagingusedforConclusionsASPH is an important enzyme in malignant transformationof cells It stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment Two main pathways Notch and SRCthrough which ASPH promotes the tumor growth havebeen identified It has also been shown that ASPH expression is induced by some growth factors and hypoxia and isregulated at various levels The overexpression of ASPHand its downstream targets has been detected in numeroushuman malignancies Since ASPH is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsFig Small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig ASPH as a therapeutic target ASPH expression is upregulated by growth factors and hypoxia Its enzymatic activity can be inhibited bySMIs or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionConsequently tumor growth and metastasis are also reduce

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

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

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

Thyroid_Cancer

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

genes of papillary thyroidcarcinoma by integrated bioinformatics analysisGang Xue11Department of Otorhinolaryngology Head and Neck Surgery The First Affiliated Hospital of Hebei North University Zhangjiakou China 2Department of Histology andEmbryology Hebei North University Zhangjiakou ChinaJingFang Wu2 Da Pei1 DongMei Wang2 Jing Zhang2 and WenJing Zhang2Xu Lin2Correspondence JingFang Wu wjfxg163comBackground Papillary thyroid carcinoma PTC is one of the fastestgrowing malignant tumor types of thyroid cancer Therefore identifying the interaction of genes in PTC is crucialfor elucidating its pathogenesis and finding more specific molecular biomarkersMethods Four pairs of PTC tissues and adjacent tissues were sequenced using RNASeqand differentially expressed genes were screened P005 logFC1 The enrichment analysis indicated that the vast majority of differentially expressed genes DEGs mayplay a positive role in the development of cancer Then the significant modules were analyzed using Cytoscape software in the protein“protein interaction network Survival analysisTNM analysis and immune infiltration analysis of key genes were analyzed And the expression of ADORA1 APOE and LPAR5 genes were verified by qPCR in PTC compared withmatching adjacent tissuesResults Twentyfive genes were identified as hub genes with nodes greater than Theexpression of genes were verified by the GEPIA database and the overall survival anddiseasefree survival analyses were conducted with Kaplan“Meier plotter We found onlythree genes were confirmed with our validation and were statistically significant in PTCnamely ADORA1 APOE and LPAR5 Further analysis found that the mRNA levels andmethylation degree of these three genes were significantly correlated with the TNM staging of PTC And these three genes were related to PTC immune infiltration Verification ofthe expression of these three genes by RTqPCR and Western blot further confirmed thereliability of our resultsConclusion Our study identified three genes that may play key regulatory roles in the development metastasis and immune infiltration of papillary thyroid carcinomaThese authors contributedequally to this work and shouldbe considered as cofirstauthorsReceived May Revised August Accepted August Accepted Manuscript online August Version of Record published August IntroductionThyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the Western world [] Thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma PTC follicular thyroid carcinoma FTC and anaplastic thyroid carcinoma ATC according to the histological subtype [] Clinicalresults vary across these subtypesThe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to PTC which accounts for “ of all thyroid carcinomas [] Inaddition patients with PTC suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately “ of cases may progress to a potentially fatal recurrentailment [] Due to these reasons uncovering the causes of PTC and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important 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 BSR20201555101042BSR20201555tasksWith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics [] Currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of PTC An examination of differentially expressedgenes DEGs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma As a form of molecular marker mRNA containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages [] Some studiesutilized public databases such as The Cancer Genome Atlas TCGA and the Gene Expression Omnibus GEO toidentify significant biomarkers of papillary thyroid carcinoma However these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the DEGsIn the present study we analyzed the DEGs in PTC tissues versus the matched adjacent tissues by RNASeq andbioinformatics methods to obtain the DEGs Then we screened out the key modules and extracted the key genes inthose modules by constructing DEGs interaction network Then the possible role of differentially expressed geneswas analyzed using GO annotation and KEGG pathway enrichment analysis The expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases Finally wefound that the three genes ADORA1 APOE and LPAR5 were highly expressed in PTC and were associated withPTC methylation TNM staging and immune infiltrationMethodsTissue samplesThirty pairs of PTC and adjacent tissues were collected from January to July at the First Affiliated Hospitalof Hebei North University This experiment was approved by the Ethical Committee of the First Affiliated Hospitaland all patients provided informed consent All tissues were frozen in liquid nitrogen after surgical resectionRNA library construction and sequencingTotal RNA was isolated from four adjacent normal and cancerous thyroid samples utilizing TRIzol reagent QiagenValencia CA USA as indicated by the manufacturer™s guidelines RNAs of PTC tissues and paracancerous tissuessample numbers 1C 1P 2C 2P 3C 3P 4C 4P the number represents different samples the œC indicates a cancersample and the œP represents a matched paracancerous tissue sample were used Six libraries were built utilizingan Illumina standard kit as indicated by the manufacturer™s protocol All sequencing was carried out on an IlluminaHiseq LC Bio ChinaDifferentially expressed genes screeningThe level of expression of mRNAs was evaluated using StringTie by calculating FPKM [] The DEGs between PTCand paracancerous tissue were screened with log2 fold change1 and P005 was regarded as statistically significant the analyses were conducted using the R package Ballgown []Functional enrichment analysis and pathway analysisTo reveal the functional roles of the DEGs the Annotation Visualization and Integrated Discovery function annotation tool DAVID httpdavidabccncifcrfgovhomejsp was used to perform Gene Ontology GO enrichmentanalysis and Kyoto Encyclopedia of Genes and Genomes KEGG pathway enrichment analysis P values less than were considered as cutoff criteriaPPI network construction and identification of hub genesPPI networks were constructed successively using STRING database tringdb [] The interactions ofDEGs with a combined score were set as significant and Cytoscape software version was utilized tovisualize and analyze the results of the STRING database To find key hub genes in this PPI network the significantmodule was analyzed by using the plugin MCODE of Cytoscape software The criteria for selection were set to thedefault The key genes were chosen with degrees ‰¥ Subsequently genes in that module were used to analyse theirfunctional roles with FunRich software 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 BSR20201555101042BSR20201555Table PCR primersGene symbolADORA1ActinAPOELPAR5bp base pair F forward primer R reverse primerPrimer sequenceF5cid3CCACAGACCTACTTCCACACC3cid3R5cid3TACCGGAGAGGGATCTTGACC3cid3F5cid3CACTCTTCCAGCCTTCCTTCC3cid3R5cid3AGGTCTTTGCGGATGTCCAC3cid3F5cid3 GTTGCTGGTCACATTCCTGG 3cid3R5cid3 GCAGGTAATCCCAAAAGCGACcid3F5cid3 CACTTGGTGGTCTACAGCTTG3cid3R5cid3 GCGTAGTAGGAGAGACGAACG3cid3Data validation and analysisTo verify the accuracy of our RNAseq results we used the Gene Expression Profiling Interactive Analysis databaseto verify the expression of key genes in PTC and adjacent tissues The overall survival and diseasefree survivalanalyses were performed by Kaplan“Meier plots for these PTCrelated hub genes Genetic alterations of hub genesin PTC and their correlations with other genes were analyzed utilizing the cBioPortal for Cancer Genomics Hub genesrelated to clinicopathological features were analyzed using the online database UALCAN httpualcanpathuabedu[] The correlation of ADORA1 APOE and LPAR5 expression with the immune infiltration level in PTC and theexpression of these three genes in different kinds of cancers was performed using the Tumor Immune EstimationResource database []For qRTPCR analysis total RNA was isolated from normal and cancerous papillary thyroid samples utilizingTRIzol reagent Qiagen Valencia CA USA cDNA was synthesized with RNA reverse transcription kit TIANGENBIOTECH Beijing China qRTPCR was performed with an ABI RealTime PCR System Applied Biosystems Life Technologies USA The expression of the genes of interest was normalized to actin The primers forADORA1 APOE LPAR5 and actin are shown in Table For Western blot RIPA buffer was used to extract protein from four pairs of tissue from PTC patients and theprotein concentrations were measured via BCA methods Briefly the SDSPAGE gel was used for electrophoresis andPDVF membrane was used for transmembrane transfer PDVF membrane was blocked and then incubated with primary antiADORA1 antibodies dilution Bioss bs6649R APOE dilution Bioss bs4892R LPAR5antibodies dilution Bioss bs15366R and actin dilution Bioss bs0061R at —¦C overnight followed by incubation with secondary antibodies Zhongshanjinqiao dilution at —¦C for h The signal wasdetected using ECL methodStatistical analysisAll the data were analyzed by R and SPSS SPSS Inc USA Kaplan“Meier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma The statistical difference was set at P ResultsDifferentially expressed genes screening based on RNASeqTo screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed RNASeq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes After RNASeq we acquire “ million reads for each sample The fold changesbetween PTC cancer tissues and matched paracancerous samples were calculated Setting the cutoff criterion as Pvalue and a fold change there were upregulated and downregulated genes These DEGswere considered to be candidate genes for subsequent study Figure 1A showed the expression of the top genes inPTC versus matched paracancerous tissuesFunctional enrichment analysis and pathway analysisConsidering that there were many falsepositive genes among these DEGs we verified our results one by onethrough the TCGA database We found that only genes in our data were consistent with the gene expression of the 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 BSR20201555101042BSR20201555Figure Identification of DEGs by RNAseqThe heat map A and PPI network of the DEGs B C The volcano plots of the DEGs D The most significant module was selectedby MCODE in Cytoscape Red represents the upregulated genes and blue represents the downregulated genesFigure GO and KEGG pathway enrichment analysis of DEGs through RNASeqA Bubble plot of Gene Ontology enrichment analysis of DEGs B Bubble plot of Kyoto Encyclopaedia of Genes and Genomespathway enrichment analysis of DEGsTCGA database To investigate the potential function of these DEGs in PTC genes functional enrichment was conducted by using GO and KEGG pathway analyses For the biological process category the DEGs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development Thecellular component category results showed PTCrelated DEGs were enriched in collagencontaining extracellularmatrix synaptic membrane cell“cell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane DEGs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity Figure 2AAs Figure 2B showed the KEGG pathway results showed DEGs were enriched in cytokine“cytokine receptorinteraction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing 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 BSR20201555101042BSR20201555Figure GO enrichment analysis and KEGG analysis for the key genesA Top elements involved in biological processes B Top elements involved in molecular function C Top elementsinvolved in cellular components D Top pathways related to the key genes through KEGG analysissyndrome parathyroid hormone synthesis secretion and action AGERAGE signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECM“receptor interaction arrhythmogenic right ventricular cardiomyopathyARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesPPI network construction and module analysisPPI networks were constructed successively by the database by loading the PTC related DGEs into the STRINGdatabase Figure 1BC Using Cytoscape we analyzed the most significant module in the PPI network Figure 1DThe PPI network consisted of nodes and edges Following the use of MCODE in Cytoscape the significantmodule was selected The top hub genes ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2CHGB CXCL12 EVA1A FAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF or MFI2 MFGE8 NMU OPRM1SERPINA1 SSTR3 TIMP1 and TNC were evaluated by degree in the PPI network Figure 1D The resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationGprotein coupled receptor activity cell adhesion molecule activity and GPCR ligand binding Figure Data analysis and validationAfter the key genes were selected the expression of key genes in PTC and its adjacent tissues were verified by theGEPIA database Figure ADORA1 APOE EVA1A LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1were positively related to the overall survival analysis of PTC patients while C5AR1 and GNAI1 were negativelyrelated Figure ADCY8 ADORA1 CHGB FN1 LPAR5 NMU and TNC showed positive associations withdiseasefree survival analysis of PTC patients but not APOE Figure Next we analyzed the alterations of the key genes by using the cBioPortal database Figure The key geneswere changed in of queried samples Figure 7B Figure 7A showed the frequency of alterations of eachPTC related key gene SSTR3 FN1 and ADORA1 were altered the most and respectively Figure 7Dshowed the network of the genes and their altered neighbouring genes in PTC patients out of a total of Among these genes only ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of PTC patients The qPCR experiments and Western blotdata verified that these three survivalrelated genes were all overexpressed in PTC Figure Then based on UAL 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 BSR20201555101042BSR20201555Figure Validation of the key DEGs in the GEPIA databaseADORA1 APOE CCL13 CDH2 CXCL12 EVA1A FAM20A FN1 GNAI1 LPAR5 MFGE8 NMU SERPINA1 TIMP1 and TNC areoverexpressed in PTC tissues compared with paracancerous tissue while GNAI and GPC3 are downregulated 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 BSR20201555101042BSR20201555Figure Overall survival analysis of key genes in PTC using Kaplan“Meier plotsExpression levels of ADORA1 APOE C5AR1 EVA1A FAM20A GNAI1 LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1are related to the overall survival of patients with PTCCAN the clinical features and degree of methylation of these three genes were analyzed The transcription levels ofADORA1 APOE and LPAR5 were significantly higher in PTC patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status Figure In addition ADOR1 and LPAR5 exhibiteda hypomethylation state in the cancer group but APOE showed a hypermethylation state in PTC samples Figure10ATo further clarify the role of these genes we conducted an analysis of immune infiltration The ADOR1 expression level was positively corelated with infiltrating levels of B cells r0111 P151e2 CD8 T cells r0246P396eˆ’ neutrophils r0162 P331eˆ’ and DCs r0232 P232eˆ’ The expression of APOE was 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 BSR20201555101042BSR20201555Figure Diseasefree survival analysis of key genes in PTC using Kaplan“Meier plotsExpression levels of ADCY8 ADORA1 APOE CHGB FN1 LPAR5 NMU and TNC are significantly related to the diseasefreesurvival of patients with PTCpositively associated with B cells r0228 P439eˆ’ CD8 T cells partialcor P930eˆ’ neutrophils r0197 P114eˆ’ and DCs partialcor P358eˆ’ LPAR5 expression level was positively related to B cells r0259 P815eˆ’ CD4 T cells r0238 P103eˆ’ macrophages r0175P105eˆ’ neutrophils r027 P142eˆ’ and DCs r0256 P104eˆ’ and negatively related to Purity r ˆ’ P294eˆ’ and CD8 T cells r ˆ’ P618eˆ’ Figure 10B These findings stronglysuggested that LPAR5 ADOR1 and APOE may play specific roles in immune infiltration in PTC especially those ofDCs Finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues Figure 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 BSR20201555101042BSR20201555Figure The key genes expression and mutation analysis in PTC by the cBioPortal for Cancer GenomicsA The genetic alterations of key genes of PTC samples Queried genes are altered in of queried patientssamplesB The expression heatmap of key genes C The alteration frequency of key genes in PTC D Network of key genesmutations and their frequently altered neighboring genes in PTC 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 BSR20201555101042BSR20201555Figure The mRNA and protein expressions of ADORA1 APOE and LPAR5 in PTC tissuesA“C Validation of expression levels of ADORA1 APOE and LPAR5 by RTqPCR in cases of PTC and matched adjacent tissuesD ADORA1 APOE and LPAR5 protein levels are increased in four cases of PTC and matched adjacent tissues as measured byWestern Blot P0001DiscussionPTC is a common cancer with great heterogeneity in morphological features and prognosis [] Although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features [] Activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of PTC To better clarify the molecular mechanism of PTC occurrence development and metastasis we identified key genes related to PTC progression through comprehensive bioinformaticsmethods and we screened three of the PTC prognosisrelated genes for a comprehensive analysisIn the present study we identified differentially expressed genes by RNASeq with GO enrichment analysis showing that the DEGs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cell“substrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cell“cell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity And KEGG pathway results showed DEGs were enrichedin cytokine“cytokine receptor interaction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing syndrome parathyroid hormone synthesis secretion and action AGERAGE signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECM“receptor interaction arrhythmogenic right ventricularcardiomyopathy ARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesTo further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a PPI regulatory network A total of DEGs with nodes greater than were screened out in the networkThe key genes were ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2 CHGB CXCL12 EVA1AFAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF MFGE8 NMU OPRM1 SERPINA1 SSTR3 TIMP1 andTNC Biological process and molecular function analyses of these key DEGs indicated that they were significantly 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 BSR20201555101042BSR20201555Figure Relative expression of ADORA1 APOE and LPAR5 in normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCANP0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentTo verify the credibility of the experiments and data the DEGs screened were verified by the GEPIA databaseAmong the selected genes genes showed expression differences consistent with our RNASeq data Among the 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 BSR20201555101042BSR20201555Figure Methylation level and immune infiltration level of ADORA1 APOE and LPAR5A Relative methylation level of ADORA1 APOE and LPAR5 based on normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCAN B The correlation between the three genes and TIICs TIMER TIICstumor infiltrating immune cells genes ADORA1 APOE CCL13 CDH2 EVA1A FAM20A FN1 LPAR5 MFGE8 NMU SERPINA1 TIMP1and TNC levels were overexpressed in PTC tissues while GPC3 and GNAI1 were downregulatedADORA1 belongs to the Gprotein coupled receptor family and protects human tissues and cells under physiological conditions [] Lin et al suggested that ADORA1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells [] Similarly Jayakar indicated that knockdown of APOE expression can reduce the level of MMPs by regulating the AP1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma [] Bioinformatics predictions were that APOE mRNAshows a significant increase in PTC [] CCL13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that CCL13 has a role in promoting the proliferation of tumorforming volumein nude mice [] CDH2 is overexpressed in various cancers Some research results indicate that overexpression ofCDH2 can increase the invasive ability and induce EMT in lung cancer cells [] Qiu et al confirmed CDH2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings [] EVA1A acts as a regulatorof programmed cell death and Shen et al indicates that EVA1A can inhibit the proliferation of GBM cells by inducing autophagy and apoptosis via inactivating the mTORRPS6KB1 signaling pathway [] FAM20A may play a keyrole in haematopoiesis There are few reports on the relationship between FAM20A and cancer and our experimentfound that FAM20A is more highly expressed in papillary thyroid carcinoma than in other cancers FN1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology [] Some researchersindicated that FN1 participates in regulating many types of cancer progression such as gastric cancer [] skin squamous cell carcinoma [] and papillary thyroid carcinoma [] It has been shown that LPAR5 is related tothe pathogenesis of pancreatic cancer [] Consistent with our study Zhang et al believes that LPAR5 may be involved in the development of papillary thyroid carcinoma [] According to previous reports MFGE8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer 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 BSR20201555101042BSR20201555Figure The expression of ADORA1 APOE and LPAR5 in thyroid cancer tissues and normal thyroid tissuesThe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the TIMER database P005P001 P001 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 BSR20201555101042BSR20201555[“] MFGE8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect[] Consistent with our data Zhang et al indicate that NMU is one of the DEGs of papillary thyroid carcinoma[] Recently a researcher has shown that abnormal expression of NMU is associated with a variety of cancers []For SERPINA1 there are currently six s pointing out that SERPINA1 may be a key gene for PTC consistentwith our results [“] Clinical studies have shown that high expression of TIMP1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers [] TNC is a component of theextracellular matrix ECM and is closely related to the malignant biological behavior of cancer In particular TNCoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer [] GPC3 belongs to the glypicans family It has been reported that overexpression of GPC3can promote the metastasis of hepatocellular carcinoma [] but we found that it is expressed at low levels in PTCSimilar to GPC3 some scholars believe that GNAI1 is a tumorpromoting gene and reported upregulated GNAI1mRNA in human glioma which is inconsistent with our data []Only the ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of PTC patients Considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes We found thatthe mRNA and methylation levels of these three genes were significantly correlated with TNM staging In additionADORA1 APOE and LPAR5 were all related to immune infiltration especially to dendritic cells Finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissuesHowever our research has certain limitations First only four pairs of cancer and adjacent tissues were analyzedusing RNAseq in this experiment so further research requires a larger sample size Second further experiments areneeded to validate the specific mechanisms of these key genesData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis study was supported by grants from the Hebei Provincial Department of Finance Specialist Capacity Building and SpecialistLeadership Program [grant number ] the Hebei Provincial Natural Science Foundation Project [grant number H201840505]and the Hebei North University Basic Research Business Expenses Project [grant number JYT2019015]Author ContributionXu Lin conducted the bioinformatics analysis Xu Lin and Gang Xue contributed as first authors Xu Lin wrote the manuscriptJingFang Wu and Gang Xue critically revised the Gang Xue and Da Pei obtained clinical specimens and the others contributed to verification of the RNAseq resultsAbbreviationsATC anaplastic thyroid carcinoma ECM extracellular matrix FTC follicular thyroid carcinoma PTC papillary thyroid carcinomaReferences Kitahara CM and Sosa JA The changing incidence of thyroid cancer Nat Rev Endocrinol “101038nrendo2016110 AschebrookKilfoy B Ward MH Sabra MM and Devesa SS Thyroid cancer incidence patterns in the United States by histologic type Thyroid “ 101089thy20100021 Pourseirafi S Shishehgar M Ashraf MJ and Faramarzi M Papillary Carcinoma of Thyroid with Nasal Cavity Metastases A Case Report IranJ Med Sci “ Ullmann TM Gray KD Moore MD Zarnegar R and Fahey III TJ Current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers Gland Surg “ 1021037gs20170908 Jin X Deng B Ye K et al Comprehensive expression profiles and bioinformatics analysis reveal special circular RNA expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy Mol Med Rep “103892mmr201910671 Rapisuwon S Vietsch EE and Wellstein A Circulating biomarkers to monitor cancer progression and treatment Comput Struct BiotechnolJ “ 101016jcsbj201605004 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 BSR20201555101042BSR20201555 Pertea M Pertea GM Antonescu CM et al StringTie enables improved reconstruction of a transcriptome from RNAseq reads NatBiotechnol “ 101038nbt3122 Frazee AC Pertea G Jaffe AE et al Ballgown bridges the gap between transcriptome assembly and expression analysis Nat Biotechnol “ 101038nbt3172 Von Mering C Huynen M Jaeggi D et al STRING a database of predicted functional associations between prot

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

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

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 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Thyroid_Cancer

ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl

Thyroid_Cancer

Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations Patients withCostello syndrome present craniofacial abnormalities cardiac defects and cancer predisposition as well as skinabnormalities including papillomas keratosis pilaris and eczematous dermatitis However the mechanisms underlyingthe dermatological abnormalities remain unclear Here we demonstrated that knockin mice expressing an Hras G12Smutation HrasG12S mice are susceptible to develop atopic dermatitis ADlike skin lesions including eczemapruritus elevated serum IgE levels acanthosis and the ltration of mast cells basophils and type2 innate lymphoidcells in the dermis after stimulation with house dust mite allergens Dermatophagoides farinae Dfb Reduced skinbarrier function increased proliferation of phosphorylated ERK pERKpositive epidermal cells and increased Th2typecytokines as well as epithelial cellderived cytokines including IL33 were observed in the skin tissue of HrasG12Smice compared with Hras mice Cultured HrasG12S keratinocytes exhibited increased IL33 expression after Dfbstimulation PD0325901 an MEK inhibitor ameliorated ADlike symptoms in HrasG12S mice showing decreasedproliferation of pERKpositive epidermal cells and decreased expression of IL33 Our findings indicate that theepidermis of HrasG12S mice stimulated by Dfb strongly induced IL33 expression and type2 innate lymphoid cellsresulting in ADlike skin lesions These results suggest that the epidermis of HrasG12S mice are prone to developmentof eczematous dermatitis stimulated with house dust mite allergensIntroductionThe skin is a stratified epithelium consisting of severallayers of cells in various stages of differentiation In orderto maintain normal skin homeostasis the proliferationdifferentiation and response of epidermal cells to externalstimuli must be tightly regulated1 The RASMAPK signaling pathway plays a crucial role in cell proliferationdifferentiation and apoptosis23 A strong activation of theRASMAPK pathway in skin is known to resultinCorrespondence Yoko Aoki aokiymedtohokuacjp1Department of Medical Genetics Tohoku University Graduate School ofMedicine Sendai Japan2Department of Pediatrics Tohoku University Graduate School of MedicineSendai JapanFull list of author information is available at the end of the Edited by E Candiepithelial cancers and melanoma45 Pigmented lesionshyperkeratosis pruritus curly hair and hyperplasia havealso been observed in vemurafenib a BRAF inhibitortreated patients6 The balance of the RASMAPK signaling pathway could be particularly important for epidermalhomeostasisCFCNoonan syndrome Costello syndrome and cardiofaciocutaneoussyndrome are phenotypicallyoverlapping genetic disorders characterized by craniofacial dysmorphia congenital heart defects and psychomotor retardation7 These syndromes are commonlycaused by germline mutations in components of the RASMAPK pathwaytermed RASopathies which constitutively activate the RASMAPK pathway89 Of thesesyndromes Costello syndrome is characterized by short 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 0cKatata Cell Death and Disease Page of stature craniofacial abnormalities congenital heart diseases hypertrophic cardiomyopathy and intellectual disability10 Approximately patients with Costellosyndrome develop malignant tumors including rhabdomyosarcoma and bladder carcinoma In we identified germline HRAS mutations in patients with Costellosyndrome11 A nucleotide change that cause the substitution of glycine at codon to serine pG12S in oneallele of HRAS has been observed in of Costellosyndrome patients The G12S mutation which has beenidentified in somatic cancer is an oncogenic mutationthat activates the downstream pathway Patients withCostello syndrome develop a variety of skin abnormalitiesincluding palmoplantar keratoderma acanthosis nigricans eczema loose skin cutis laxa darker skin colorand papillomata around nose and anus However thepathogenesis of dermatological abnormalities remainsunclearWe have recently generated a strain of knockin miceexpressing an Hras G12S mutation the most frequentmutation in Costello syndrome12 which exhibited facialdysmorphia cardiomyocyte hypertrophy and kidneyanomalies Impaired mitochondrial fatty acid oxidationwas observed in HrasG12S mice fed a highfat diet13Skin abnormalities including papillomas have not beenobserved in young HrasG12S mice weeks old underspecific pathogenfree conditions In contrast HrasG12Smice over weeks of age or highfat diet fedHrasG12Smice had cutaneous lesions due to scratching Supplementary Fig 1a under the same pathogenicfree condition Although we have not analyzed the histology of skinof HrasG12S mice over weeks of age gross appearances of the skin lesions and scratching behavior suggestthat they are atopic dermatitislike In the current studywe tested to generate experimentally induced dermatitisin HrasG12S mice and found that HrasG12S micedeveloped more severe atopic dermatitis ADlike lesionsthan Hras mice after treatment with house dust miteallergens Dermatophagoides farinae Dfb Furthermorethese ADlike skin lesions in HrasG12S mice werereversedbyan MEK inhibitorPD0325901treatment withResultsDfb ointment induces ADlike skin lesions in HrasG12SmiceWe first tested the effect of picryl chloride whichinduce contact dermatitis and imiquimod which inducepsoriasis on the skin of Hras and HrasG12S miceSupplementary Fig 1b c but no difference in skinlesions was observed between them Supplementary Fig1d In contrast the treatment with Dfb ointment developed severe dermatitisincluding severe erythemaOfficial journal of the Cell Death Differentiation Associationhemorrhage scarring and eczema in the dorsal skin ofHrasG12S mice but not in Hras mice Fig 1a andSupplementary Fig 2a The ears of HrasG12S micebecame thick with edema erosion and excoriationFig 1b The dermatitis score was significantly higher inDfbtreated HrasG12S mice than in any other group ofmice SDStreated control Hras mice DfbtreatedHras mice and SDStreated control HrasG12Smice on day Fig 1c and Supplementary Table Other dermatitis parameters including the ear swellingFig 1d and the scratching behavior Fig 1e increasedsignificantly in Dfbtreated HrasG12S mice comparedwith Dfbtreated Hras mice Serum IgE levels weresignificantly higher in HrasG12S mice compared toHras in nontreated baseline ± ngmL vs ± ngmL P Supplementary Fig Although the difference was not statistically significant in SDS treatment groups IgE levels were higher inHrasG12S mice compared to Hras ± ngmLvs ± ngmL P Fig 1f as well as in theDfb treatment groups ± ngmL vs ± ngmL P Fig 1f These symptoms werealso seen in Hras mice but skin lesions are moresevere in HrasG12 mice In both groups of mice the IgEelevations were triggered by Dfb ointmentWe next examined if Dfbinduced dermatitisinHrasG12S mice is caused by the same pathology as inHras miceHistological analysis revealed hyperkeratosis and epidermal hyperplasia in the dorsal skin of DfbtreatedHrasG12S mice Fig 2a The epidermis of HrasG12Smice became thicker than that of Hras mice althoughDfb treatment increased the epidermal thickness in bothHras and HrasG12S mice Supplementary Fig 2b Inthe ADlike skin lesions Dfbtreated HrasG12S micedisplayed increased number of mast cells toluidine blueand tryptase 1 a marked increase in the numbersof T cells CD4 and dendritic cells MHC class IIFig 2a b and Supplementary Fig 2c e Western blottinganalysis revealed that the levels of CD4 protein weresignificantly increased in Dfbtreated mice compared withcontrol mice Supplementary Fig 2d In line with theacanthosis of Dfbtreated HrasG12S mice an increasednumber of phosphohistone H3positivecells wereobserved in the suprabasal epidermis layers of HrasG12Smice Fig 2b Although phosphorylated ERK pERKpositive cells were also increased in the epidermis of Dfbtreated Hras and HrasG12S mice the immunostainedarea in HrasG12S mice was significantly larger than thatin Hras mice Fig 2b and Supplementary Fig 2f g Inaddition we examined the expression of filaggrin andclaudin1 as epidermal barrier markers in AD14“ Adecreased expression of claudin1 was observed in Dfb 0cKatata Cell Death and Disease Page of Fig Dfb treatment induces atopic dermatitislike skin lesions in HrasG12S mice a b Skin and ear features of mice on day after treatmentof Dfb ointment HrasG12S mice showed dermatitis by repeated application of Dfb a The severity of ear swelling responses to Dfb was strongerin HrasG12S than Hras mice b c Dermatitis scores of only SDStreated control Hras and HrasG12S and Dfbtreated Hras andHrasG12S mice n per group d Time course of ear thickness from Hras and HrasG12S mice after treatment with SDS or Dfb n pergroup e The number of scratching bouts per min assessed by the video n per group f Serum IgE levels in Hras and HrasG12S mice aftertreatment with SDS or Dfb n per group Data are presented as mean ± SD Significance in c d and f was analyzed by oneway ANOVAand the Tukeyˆ’Kramer method P P P and P HrasG12S Dfb vs Hras Dfb P P P and P HrasG12S Dfb vs HrasG12S SDS  P   P    P and     P Hras Dfb vs Hras SDSSignificance in e was analyzed by twotailed Student™s t test P treated HrasG12S mice compared with control HrasG12Smice Fig 2c d Together these results indicate that Dfbapplied HrasG12S mice exhibited more severe ADlikeskin lesions than Hras mice including acanthosis withhyperproliferation of pERKpositive cells in the epidermisas well as increased ‚ammatory cells and reducedclaudin1 expressionThe skin of Dfbapplied HrasG12S mice shows an increaseof itchassociated factors and ‚ammatory cytokinesTo further characterize the ADlike skin lesions weevaluated the levels of the itchassociated factors and‚ammatory cytokinesin the skin of DfbtreatedHrasG12S mice Itchrelated neuronal markers including skin Tac1 Klk7 and Klk14 mRNA levels or PAR2 andOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig Histological analysis reveals acanthosis with hyperproliferation of pERKpositive epidermal cells increased ‚ammatory cells andreduced claudin1 expression in the dorsal skin of Dfbtreated HrasG12S mice a Skin tissue stained with HE and TB b c Immunohistochemistryof CD4 tryptase 1 pERK pHH3 and claudin1 in the skin a“c Scale bars μm d Lysates from the skin were immunoblotted with antiClaudin1antibody Band intensities were quantified and compared among the four groups The expression levels were normalized to GAPDH n in eachgroup Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and the Tukeyˆ’Kramer method P P twotailedStudent™s t testIl1 pro‚ammatory cytokineEndothelin proteins17“ were significantly higher thanthose in control HrasG12S and Dfbtreated Hras miceFig 3a“c Regarding ‚ammatory cytokines the skinIl4mRNA levels ofTh2related cytokine and epidermalderived cytokinesIl33 and thymic stromal lymphopoietin Tslp were significantly elevated in Dfbtreated HrasG12S mice compared with control HrasG12S and Dfbtreated Hrasmice Fig 3d IL33 leads to the activation of type2innate lymphoid cells ILC2s through ST2 IL33 receptor21 In Dfbtreated HrasG12S mice the skin mRNAlevels of St2 were also significantly increased Fig 3dImmunohistochemistry analysis revealed that DfbtreatedHrasG12S mice showed enhanced expression levels ofIL33 and TSLP in the basal epidermal layers and thesurface of epidermis respectively Fig 3e Likewise theIL33 protein levels were significantly higher in the skin ofDfbtreated HrasG12S mice than in Dfbtreated Hrasmice Fig 3fIncreased numbers of ILC2 and increased IL33 expressionwere observed in the epidermis of HrasG12S miceTo investigate when and how ‚ammatory cytokinesare induced in HrasG12S mice we performed flowcytometry on skin and ear samples days after Dfbapplication that is once the ADlike skin lesions hadbegun to appear Fig 1c The accumulation of basophilsand ILC2s was observed on skin samples of DfbtreatedHrasG12S mice days after Dfb application Fig 4a Asignificant increase in mast cells eosinophils basophilsand ILC2s was also observed in the ears of HrasG12Smice Fig 4b On the other hand these immune cellswere hardly detected in Hras and HrasG12S mice days after SDS application Supplementary Fig The mRNA levels of ‚ammatory cytokines in the skinfrom HrasG12S mice days after Dfb applicationshowed that Il1 and Il33 were significantly elevated inthe skin of Dfbtreated HrasG12S mice compared withDfbtreated Hras mice Fig 5aAtopic dermatitis is characterized by increased serumIgE acanthosis loss of skin barrier function and ltration of immune cells including Th2 cells dendriticcells eosinophils basophils and mast cells2223 On thataccount we next examined the response of immune cellsin these mice HRAS is known to be highly expressed inOfficial journal of the Cell Death Differentiation Associationthe epidermal cells but not in immune cells2425 Indeedno significant difference was found in the population ofimmune cells from the spleen and lymph node LN Theproliferation of naive CD4 T cells and Th2 immuneresponse was comparable between Hras and HrasG12Smice at weeks of age suggesting that ADlike dermatitismay not be caused by different response of immune cellsFig 6a b Then we examined whether an increasedproduction of IL33 and TSLP was observed in primaryepidermal keratinocytes in response to Dfb Six hoursafter Dfb stimulation the mRNA level of Il1 not Tslpwas significantly elevated in cultured epidermal keratinocytes of Hras and HrasG12S mice Fig 5b NotaIl33 in the Dfbstimulatedblykeratinocytes of HrasG12S mice were significantlyincreased compared with those of nonstimulatedHrasG12S and Dfbstimulated Hras keratinocytessuggesting that epidermal keratinocytes with Hras G12Smutation have increased IL33 expression after stimulation with Dfb Fig 5bthe mRNA levels ofPD0325901 reduces skin lesions in DfbstimulatedHrasG12S miceamelioratesTreatment with MEK inhibitorstheabnormalities observed in RASopathyrelated modelmice26“ We investigated the effects of an MEK inhibitor PD0325901 on skin lesionsin DfbtreatedHrasG12S mice Supplementary Fig 5a Ten days oftreatment with PD0325901 or saline resulted in a significant improvement of the dermatitis score and earswelling in HrasG12S mice Fig 7a b PD0325901treatment resulted in a marked reduction of epidermalthickness mast cell numbers and pERKpositive epidermal cells as well as recovered expression levels ofclaudin1 Fig 7c d and Supplementary Fig 5b c ThemRNA levels of Il1 Il4 Il33 St2 and Klk14 were significantly lowerin the skin of PD0325901treatedHrasG12S mice than that of vehicle salinetreatedHrasG12S mice Fig 7e and Supplementary Fig 5dThese results suggest that ERK inhibition partially ameliorates Dfbinduced skin lesions in HrasG12S miceDiscussionHere we demonstrated that mice expressing a germlineHras G12S mutation but not Hras mice developed 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig Expression of itchassociated factors and ‚ammatory cytokines is enhanced in Dfbinduced skin lesions in HrasG12S mice a c dRelative mRNA expression related to neuronal factors a Tac1 and Ngf skin proteases c Klk5 Klk7 and Klk14 and ‚ammation d Il1 Il4 Tslp Il33and St2 in the dorsal skin mRNA levels were normalized to those of 18s rRNA n per group b f Protein extracts from dorsal skin wereimmunoblotted with antiPGP95 antiPAR2 antiEndothelin and antiIL33 antibody n in each group GAPDH same data as in Fig 2d f Bandintensities were quantified and compared among the four groups Expression levels were normalized to GAPDH e Immunohistochemistry of IL33and TSLP Scale bars μm Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and the Tukeyˆ’Kramer method P P P and P P twotailed Student™s t testADlike skin lesions under conditions of Dfb exposureThe levels of IL1 and epithelial cellderived cytokinesIL33 and TSLP were also increased in DfbtreatedHrasG12S mice In addition an increased production ofIL1 IL4 and IL33 as well as ‚ammatory cellsbasophils and ILC2s was observed in the dorsal skin ofHrasG12S mice before the development of ADlike skinlesions Analysis of the underlying mechanism revealedthat Dfbstimulated keratinocytes in HrasG12S miceinduced IL33 production while in naive CD4 T cellsfrom the spleen the Th2 immune response was comparable between Hras and HrasG12S mice Finallythe inhibition of ERK activation by PD0325901 treatmentameliorated the ADlike skin lesions and IL33 production Together these data indicate that germline HrasG12S activating mutation causes ADlike skin lesions viathe ERKIL33 axis Fig cellsepidermalIn the present study after repeated stimulation withDfb HrasG12S mice showed hyperproliferation of pERKpositiveand increased IL33expression in the dorsal skin Increased IL33 expressionwas also observed in primary epidermal keratinocytesfrom HrasG12S mice after Dfb stimulation which issimilar to the reports that the activation of RASMAPKsignaling was associated with increased IL33 expressionin cancer cells2930 Recently IL33 was found to inducethe Th2 ‚ammatory response in allergic diseasesespecially AD31 Excess IL33 is also associated with skinbarrier dysfunction and ILC2 functions which is partiallyregulated by the RASMAPK signaling pathway3233Epithelialspecific IL33 transgenic mice have been foundto develop ADlike dermatitisincluding acanthosispruritus increased IgE serum levels reduced claudin1expression and increased production of eosinophils mastcells and ILCs3334 Of note IL33 and its receptor ST2are highly expressed in the skinderived ILC2s of ADpatients and lung ILC2s of patients with allergic airwaydiseases respectively3536 Importantly consistent with thephenotype of IL33 transgenic mice and AD patients Dfbtreated HrasG12S mice showed ADlike skin lesionsreduced claudin1 expression increased IL33 expressionhyperproliferation of pERKpositive epidermal cells andincreased ILC2 production Collectively excess IL33could lead to ERK activation resulting in an increase ofILC2s and impaired skin barrier Fig Official journal of the Cell Death Differentiation AssociationThe pathogenesis of skin lesions observed in Costellosyndrome includes dermal connective tissue abnormalities cutis laxa and deep palmer and plantar creaseshyperproliferative skin disease palmoplantar keratoderma cutaneous papilloma and acanthosis nigricansand ‚ammatory skin abnormalities sensitive skineczema and pruritus A previous study showed that inthe skin fibroblasts of Costello syndrome elastic fiberswere not assembled due to a functional deficiency of theelastinbinding protein as a result of an unusual accumulation ofchondroitin sulfatebearing proteoglycans3738 Regarding the hyperproliferative skin lesions ithas been reported that the root cause of papillomashyperkeratosis and epidermal hyperplasia such as psoriasis is the activation of the RASMAPK pathway39“However the pathophysiological mechanism of ‚ammatory skin abnormalities in Costello syndrome remainsunclear In the present study Dfbtreated HrasG12S micedisplayed pruritus and eczema Recently mice withepidermisspecific BRAFRAF1 deficient also showed ADlike dermatitis which is characterized by increased serumIgE levels and a Th2 response43 The elevated IgE levelshave not been systematically examined or reported inCostello syndrome patients However it is possible thatthe allergic reaction stimulated by house dust mites couldbe involved in the development of ‚ammatory skinabnormalities in Costello syndrome patientsincludingsensitive skin pruritus and eczemaimprove HRASdrivenAt present acitretin has been reported to treat palmoplantar keratosis in patients with Costello and CFC syndrome4445 Several reports have demonstrated that MEKtumorigenesis46inhibitorsimpaired enamel formation in the teeth27 and longtermdepression in the hippocampus in Hras G12V knockinmice28 as well as hyperkeratosis and hyperplasia in theforestomach of BrafQ241R mice26 In the present studyPD0325901 treatment of the ADlike skin lesions inHrasG12S mice was found to reverse these lesions byreducing hyperproliferation of pERKpositive epidermalcells and the production of ‚ammatory cells and cytokines including IL1 IL4 and IL33 Treatment withU0126 an MEK inhibitor in human keratinocytes hasalso been found to restore the reduced expression levels ofclaudin1 and filaggrin and increase ERK activationthrough excess IL333347Indeed reduced claudin1 0cKatata Cell Death and Disease Page of Fig An increase in ILC2s and basophils observed in the skin and ear of Dfbtreated HrasG12S mice a b Flow cytometric analysis of skin aand ear b cells from Hras and HrasG12S mice collected days after Dfb application Eosinophils CD45 SSChi siglecF Basophils CD45siglecFˆ’ FcεRIα DX5 Mast cells CD45 siglecFˆ’ FcεRIα DX5ˆ’ ILC2 CD45 Linˆ’ CD3ε CD4 CD8a CD11c FcεRIα NK11 CD19 Ter119 CD5 F4 Gr1 Sca1 GATA3 n in each group Data are presented as mean ± SD Significance was analyzed by twotailed Student™s t test P P expression was improved in Dfbtreated HrasG12S miceafter PD0325901 treatment Recently hypertrophic cardiomyopathy in Noonan syndrome patients were treatedby MEK inhibitor48 So MEK inhibitors could be effectivein patients with RASopathies The most common sideeffect of trametinib MEK inhibitor in human patients isOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig Expression of ‚ammatory cytokines is enhanced in the skin of Dfbtreated HrasG12S mice in the early stage of dermatitisa Relative mRNA expression related to ‚ammation including Il1 Il4 Il13 Tslp Il33 and St2 in the dorsal skin of Hras and HrasG12S mice on day after treatment of Dfb mRNA levels were normalized to those of 18s rRNA n per group b Relative mRNA expression of Il1 Tslp and Il33 inDfbstimulated ngμl h or vehicletreated PBS h keratinocyte from Hras and HrasG12S mice mRNA levels were normalized to those ofGapdh Results represent five independent experiments Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and theTukeyˆ’Kramer method P P and P skin rush and common toxicity associated with vemurafenib BRAF inhibitor is cutaneous abnormalities suchas keratoacanthoma and squamous cell carcinoma by themechanism of paradoxical MAPK pathway activation49Therefore the balance of RASMAPK signaling plays animportant role in the emersion of skin abnormalitiesAdjusting dosage of MEK inhibitors may be effective onskin lesions of patients with Costello syndromeHere Dfbtreated HrasG12S mice exhibited increasedIL33 expression through hyperproliferation of pERKpositive epidermal cells Additionally we show thatPD0325901 treatment ameliorated the ADlike skinlesions in HrasG12S mice under conditions of exposureto Dfb Thus it will be interesting to investigate whethertreatment with IL33 antibody reduces the ADlike skinlesions in HrasG12S mice Our findings provide additional perspective that HrasG12S mice will serve as avaluable model to study pathophysiology and potentialtherapeutic approaches in ADMaterials and methodsMiceHrasG12S mice on a C57BL6J background have beendescribed previously13 Male mice were analyzed inthis studyGenotypingThe genomic DNA was extracted from the tail tissueusing a Maxwell Mouse Tail DNA Purification KitPromega Madison WI USA or the alkaline lysismethod For the alkaline lysis method a small piece ofeach tail mm was incubated in mM NaOH for min at °C After the addition of M TrisHClpH the extracts were used for PCR Genotyping ofOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig No significant difference was found in Th2 cytokine responses and the proliferation of naive CD4 T cells between Hras andHrasG12S mice a Naive CD4 T cells were isolated from the spleen of Hras and HrasG12S mice at weeks of age After that the cells werestained with CSFE and cultured with mouse antiCD3 monoclonal antiCD28 monoclonal recombinant mouse IL2 recombinant mouse IIL4 andantiIFNγ antibodies for days After incubation the cells were fixed permeabilized and stained with IL5 and IL13 antibodies Flow cytometricanalysis was performed n in each group b After days culture of purified naive T cells from spleen of Hras and HrasG12S mice theproliferation index was measured by flow cytometry n in each group Data are presented as mean ± SD Significance was analyzed by twotailedStudent™s t testHras and HrasG12S mice was performed by PCRusing KOD FX Neo TOYOBO Osaka Japan The primers used for PCR have been described previously1350Induction of dermatitisAtopic dermatitislike skin lesions were induced in malemice at weeks of age according to the manufacturer™sinstructions The mice were anesthetized with isofluraneand their dorsal hair was removed using an electric clipper2000AD Natsume Seisakusho Tokyo Japan For sensitization mg of Biostir AD Biostir Inc Kobe Japanan ointment of Dfb extract was applied to the shaveddorsal skin and ears Three or four days later hair growthwas removed with an electric clipper and μl of SDS SigmaAldrich St Louis MO USA was applied tothe shaved dorsal skin and ears to disrupt the skin barrierAfter h mg of Biostir AD was applied to theirshaved dorsal skin and ears to induce ADlike skin lesionsThese procedures were repeated twice a week for daysThe mice were sacrificed on day to collect skin and earsamples Supplementary Fig 2a To assess the effect ofDfb ointment to Hras and HrasG12S mice they wererandomly divided into four groups SDStreatedHras SDStreated HrasG12S Dfbtreated Hrasand Dfbtreated HrasG12S using single blinding testEvaluation of dermatitis and ear thicknessThe dermatitis scores were evaluated twice a weekaccording to the development of four symptoms erythemahemorrhage of dorsal skin scarringdryness ofdorsal skin edema of ear and excoriationerosion ofear51 The total dermatitis score maximum score wasdefined as the sum of individual scores none mild moderate severe for each symptom SupplementaryTable Ear thickness was measured with a digimaticmicrometer CLM115QM Mitutoyo Kanagawa JapanMeasurement of scratching behaviorOn day scratching behavior was monitored by videoGZHM890 JVC Kanagawa Japan for min TheOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig MEK inhibitor reduces the clinical severity of dermatitis in Dfbstimulated HrasG12S mice a b Gross appearances dermatitis score andear thickness in vehicle saline or PD0325901treated Hras and HrasG12S mice after days of daily injections n per group All of the micein these figures were treated with Dfb The circle and square show vehicle group without MEKi with same amount of saline c Skin tissuestained with HE and TB in Hras and HrasG12S mice with PD0325901 or vehicle treatment n per group d Immunohistochemistry of pERKand claudin1 in skin c d Scale bars μm e Relative mRNA expression of Il1 Il4 and Il33 in dorsal skin mRNA levels were normalized to those of18s rRNA vehicle group n PD0325901 group n Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and theTukeyˆ’Kramer method P P and P HrasG12S PD0325901 vs HrasG12S vehicle P HrasG12S vehicle vs Hrasvehicle  P onetailed Student™s t testFig Germline HrasG12S mutation causes ADlike skin lesions via ERKIL33 axis Exposure to Dfb allergen induces ADlike skin lesionsincluding eczema acanthosis and pruritus in HrasG12S mice Dfbtreated HrasG12S keratinocytes show increased IL33 expression throughhyperproliferation of pERKpositive epidermal cells Excess IL33 activates basophil and ILC2containing ST2 receptors These activated immune cellsinduce the production of type2 ‚ammatory cytokines such as IL4 Furthermore excess IL33 can activate ERK signaling resulting in reducedclaudin1 expression and skin barrier dysfunction DC dendritic cellsnumber of scratching bouts was assessed by replaying thevideo Each incidence of scratching behavior was definedas rubbing of ears and dorsal skin with forepaws hindpaws and mouthHistology and immunohistochemistryThe dorsal skins were fixed in neutral bufferedformalin for paraffinembedded specimen and paraformaldehyde for frozen specimen Embedded tissueswere sectioned at μm paraffinembedded specimensor μm frozen specimens Paraffinembedded specimens were stained with hematoxylin and eosin HEand toluidine blue TB Epidermalthickness wasmeasured in five randomly selected areas × μmof each HEstained sample Mast cells were counted inten randomly selected areas × μm of each TBstained sample For immunohistochemistry the paraffinembedded sections were deparaffinized using xylene andrehydrated with ethanol Frozen specimens were driedsufficiently with a dryer Antigens were activated using aHistofine simple stain kit Nichirei Bio Sciences TokyoJapan The antibodies used are described in Supplementary Table Signals were visualized with a DABSubstrate KitNichirei Bio Sciences Sections werecounterstained with hematoxylin pERK immunostainedareaepidermis was measured in five randomlyOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of selected areas × μm of each pERKstainedsampleSpleen and LN immune cell preparation and flowcytometryQuantitative reverse transcriptionPCRTotal RNA extraction and purification of keratinocyteswas performed according to the standard proceduresusing an RNeasy Mini Kit Qiagen Hilden Germany andQIAshredder Qiagen The extraction and purification ofthe total RNA from the dorsal skin and cDNA synthesiswere performed as previously described26 Quantitativerealtime PCR was performed using THUNDERBIRDSYBR qPCR MIX TOYOBO in a StepOnePlus ThermoFisher Scientific Waltham MA USA The amplificationprimers are described in Supplementary Table Eachsample was run in duplicateWestern blottingSkin tissue was homogenized in lysis buffer mM TrisHCl pH and SDS containing phosphatase and protease inhibitor P5726 and P8340 SigmaAldrich Supernatants were collected after centrifugation and the proteinconcentration was determined using a BioRad ProteinAssay BioRad Laboratories Hercules CA Western blotanalyses were performed as previously described26 Brieflythe proteins were transferred onto nitrocellulose membranesand blocked with nonfat milk in Trisbuffered salinewith Tween20 mmolL TrisHCl pH mmolLNaCl and Tween20 for h at room temperature Themembranes were incubated overnight at °C with theantibodies described in Supplementary Table All membranes were visualized using the Western Lightning ECLPlus Kit PerkinElmer Waltham MA USA The bandintensities were quantified using NIH ImageJ softwareELISASerum IgE was determined using LBIS Mouse IgEELISA Kit FUJIFILM Wako Shiba

Thyroid_Cancer

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

Thyroid_Cancer

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

"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

Moringa oleifera L from the Moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions M oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine With no reports of side effects in doses achievable by ingestion different parts of M oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders This plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women The seed and leaves powder has water purification properties through flocculation It also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries So M oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil According to the holistic or traditional medicine M oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients It is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life The high and specific immunological potential of M oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against Covid19 diseaseKeywords Moringa oleifera a0· Drumstick tree a0· Miracle tree a0· Medicinal plant a0· Cosmetics a0· Food supplementDiana Meireles and Jo£o Gomes authors contributed equally to this workdianameireleslivecompt Diana Meireles ICBAS Institute of a0Biomedical Sciences Abel Salazar University of a0Porto Rua de Je Viterbo Ferreira no a0Porto Portugal YIDAO Acupuncture and a0TCM Center Porto Portugal CIIMAR Interdisciplinary Centre of a0Marine and a0Environmental Research University of a0Porto Porto Portugal CBSin Center of a0BioSciences in a0Integrative Health Porto PortugalIntroductionMoringa oleifera L Moringa pterygosperma G wellknown as the œdrumstick or œhorseradish tree is native of Northwest India its main producer but can also be found in South Africa Northeast Africa Madagascar Tropical Asia Southwest Asia and Latin America The Moringa genus comprises species M arborea M longituba M borziana M pygmaea M hildebrandtii M drouhardii M longituba M peregrina M stenopetala M rivae M ruspoliana M Ovalifolia M Concanensis and M ole­fera Rani a0 From the Moringaceae family M oleifera is the most known studied and used species Anwar Olson with human and animal applications The various resources obtained from this plant”leaves flowers seeds pods bark and roots”can be used for cooking or in traditional medicine to treat several pathologies M oleifera has the capability to survive in humid or dry hot climates Vol01234567891 0c D a0Meireles et aland poor soils Anwar et a0al Mainenti M oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries Zongo ValdezSolana et a0al Gopalakrishnan Debajyoti et a0al M oleifera gained the title of œMiracle Tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry Tables a0 and In fact when compared to other plants from a0g of dry leafs of M oleifera we can obtain times more vitamin C than from oranges times more vitamin A than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach Oduro et a0al a0 a0Rockwood Saini et a0al Table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the United States Department of Agriculture USDA database although it is Table Nutritional values per a0g of the edible portion of M oleifera pods and leavesComponentsPer a0gRaw podsa“Raw leavesa““““Energy kcalWater gProtein gTotal lipid gCarbohydrate by difference gFibre total dietary gFatty acids total saturated gFatty acids total monounsaturated gFatty acids total polyunsaturated gFatty acids total trans gCholesterol mgVitamin A RAE µgVitamin D D2 D3 µgVitamin D IUThiamin mgRiboflavin mgNiacin mgPantothenic acid mgVitamin B6 mgVitamin B12 µgVitamin E mgVitamin C total ascorbic acid mgFolate total µgFolic acid µgSodium mgPotassium mgCalcium mgPhosphorus mgMagnesium mgIron mgZinc mgCopper mgManganese mgSelenium µga Information obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June b Average values and standard deviation published by Witt Only two values were found Witt Dried leavesb ± ± ± ± ± ““““““ ± ““““““ ± “ ± ± ± ± ± ± ± ± ““ 0cTable Amino acids and flavonols per a0g of the edible raw portion of M oleifera leavesPer a0gRaw leavesComponentsAmino acids a0Tryptophan g a0Threonine g a0Isoleucine g a0Leucine g a0Lysine g a0Methionine g a0Cystine g a0Phenylalanine g a0Tyrosine g a0Valine g a0Arginine g a0Histidine g a0Alanine g a0Aspartic acid g a0Glutamic acid g a0Glycine g a0Proline g a0Serine gFlavonols a0Isorhamnetin mg a0Kaempferol mg a0Myricetin mg a0Quercetin mgInformation obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June known that the nutrient content varies according to the plantation site Aslam and seasons Witt The nutritional value of dried leaves not existent in USDA database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by Witt From leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds Anwar et a0al Leone et a0al 2015a b Saini et a0al Fahey Debajyoti et a0al Divya et a0al M oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant Fahey Abdull Razis et a0al Divya et a0al Anwar et a0al published a table with various traditional medicinal uses of the different parts of M oleifera Anwar et a0al The attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans According the revision by Stohs and Hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of M oleifera leaves and leaf extracts achievable by oral ingestion Although there was not any report of major adverse side effects there are some important information that should be registered In fact there are some studies suggesting that M Oleifera cannot be used in combination with other modern medicines in humans A research by Gholap et a0al concluded that M oleifera has been noted to be a good regulator of insulin Thus according Sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using M oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications Another study suggests that when treating thyroids M oleifera compounds of the leaf may improve thyroid function Tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactionA research work concerning the œAcceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of M oleifera on children girls in Zambia Barichella et a0al With regards to safety concerns supplementation of a0g per day of M oleifera powder was deemed safe for children and adolescents both in the short and long term This research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of M oleifera daily showing to be still an inadequate and symptomatic dose in childrenOther studies suggest that M oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function Das et a0al Kelly Sileshi et a0al Despite the numerous positive health benefits associated with M oleifera phytochemicals there are suspicions that it contains harmful substances Fahey et a0al Annongu et a0al Maizuwo It contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects Maizuwo et a0al Some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin Fahey et a0al There are unconfirmed reports that M oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women Dutta It is also suspected that it can prevent implantation in women hence it must be avoided A review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c D a0Meireles et alTable Compilation of food supplements containing M oleifera tree parts or extracts in June Tree partsProduct informationBrandProductNaturingaMoringa capsulesLeavesMoringa teaMoringa kids multivitamin complexMoringa powderBioheraDried seeds extract Moringa capsulesLeavesMoringa syrupLeavesLeavesMiracle treeMoringa anic teaanic Moringa superfood supplements”capsulesMoringa Superfood PowderMoringa Superfood SticksIswariMoringa powder anicDrasanviLeaves dry extract Nutrabasics”MoringaRegulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditionDelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindStrengthens the immune system rich in vitamins and minerals stimulates natural defensesAdds nutritional value source of fiber protein vitamins and minerals improves physical conditionStrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoIt is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids Daily use of Moringa can help to restore your imbalances in your dietThe Moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundThe richness of its active ingredients helps maintain blood glucose levels Provides Flavonoids and Polyphenols by those attempting to conceive as it functioning as an abortifacient Nath et a0al Dutta Finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rat™s cells was verified at a0mgkg Asare et a0al However all mentioned side effects were verified with doses that far exceed the amounts used in food intake Asare et a0al So research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of M oleifera extracts and products on both human and animal models Adedapo et a0al Stohs et a0al Many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of M oleifera Fahey In fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as Ayurveda and Traditional Chinese Medicine TCM Karadi et a0al Kasote et a0al Debajyoti et a0al As an endemic source with highly digestible protein Ca Fe Vitamin C and carotenoids is considered as a suitable natural product to be used by undernourishment populations Dixit The resources obtained from a0M oleifera tree on a0a a0conventional approachLeaves and a0podsIn some countries leaves and fruits are commonly used in culinary as vegetables Leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption Moyo Olson et a0al In all ways of use and conservation M oleifera does not lose nutritional value Mahmood Leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants Table a0 Anwar Rebufa et a0al Phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids Table a0 M oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content Moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0cTable Compilation of cosmetics containing M oleifera tree parts or extracts as ingredients in June Cosmetic ingredientsProduct informationProduct brandnameSkinSecretAntiwrinkle face creamAntiaging moisturizer face creamHand creamBody milkLushAfrican paradise body conditionerQueen bee hair honeyMagical Moringa facial moisturizerCharity pot Hand and body lotionPassion fruit lip balmGo faster feet foot lotionTwinkle toes foot powderLush gardener cold pressed soapLaboratoires S©robiologiquesPURISOFT®Body shopMoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubMoringa eau de toilette Moringa body mistPurifying and protective action against environmental stress such as smoke and pollutionMoisturizing nourishingDeodorizingRemove dirt moisturizing nourishingSkin cleansingpurification protects skin against pollution heavy metals cigarette smokeSkin feels smooth and restoredDelicately scent your skin in a crisp floral aroma with MoringaLeavesM pterygosperma oilM pterygosperma leaf infusionoilM pterygosperma powderActive ingredient peptide from Moringa seedsM pterygosperma oilM oleifera leaf extractM pterygosperma seed extractM oleifera seeds and oilM oleifera leaf extractoilM pterygosperma seed extractM pterygosperma extractClarinsExtracomfort antipollution cleansing cream Eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionNeutralizes the effects of pollution and purifies the skin to restore its natural radiancePurifies and refines while preserving your skin™s natural moisture balance Neutralizes the harmful effects of pollutionOnestep facial cleanserExfoliating body scrubOnestep gentle exfoliating cleanserWater purifycomfort onestep cleanserDaily energizer cleansing gelNaturingaMoringa soap bioMoringa exfoliating face scrubMoringa O2Herbal moisturizing lotionfacial tonersoapherbal shampooconditionerShu UemuraAntiOxi pollutant and dullness clarifying cleansing oilUrban moisture hydronourishing shampooconditionerdouble serumdeep treatment masqueHigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissueMoringa seeds peel and exfoliate the skin while Moringa oil moisturizes and regenerates the skinRejuvenate nourish and protects skinRepairs strengthens reduces hair fallEnhanced power to remove micro impurities and stubborn makeup antipollution breakthroughHighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c Table continuedCosmetic ingredientsM oleifera seed extractProduct brandnameBioBeaut©Antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating gelDualphase waterproof eye makeup removerD a0Meireles et alProduct informationRemoves makeup pollution particles and excess sebum while leaving the skin well moisturized The Seed of Moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination This extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health Anwar et a0al Table a0 In dried M oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet In the leaves is found greater a variety and quantity of proteins when comparing to other tree parts Rebufa et a0al Wang et a0al Due to its nutritional rich values M oleifera can be a good enriching food additive to human diet and also an animal feed fortifier Moyo Adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition Bhargave Studies of acceptance by the consumer of enriched foodssnacks with M oleifera have been obtaining good results Ellis Jung M oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children Phytosterols from M oleifera increase estrogen production that enhance the activity of the mammary glands ducts Gopalakrishnan Doses of a0mgg of body weight given to mice result in increased milk production Also the pup weight augment with increasing doses of M oleifera leaf powder intake Titi et a0al Titi and Nurjanah Studies of toxicity in animals show that M oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes M oleifera may cause toxicity by accumulation of some elements a0Ali et a0al The amount of a0g of M oleifera dried leaf per day is the maximum recommended doseage AsieduGyekye et a0 al Table a0 compiles some food supplements based on M oleifera tree parts or extracts A hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice Table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis Bharali et a0al M oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer Gopalakrishnan et a0al carotene the major component reported from the drumsticks of the M oleifera plant as well as the presence of vitamin A and C suggest an action in the induction of antioxidant and antiinflammatory profiles Geervani and Devi Bharali et a0al Praengam et a0al It was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model Gupta et a0al Kraiphet et a0al Studies in rats showed that M oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function Kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases Sutalangka et a0al It was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a M oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days Kaur et a0al This effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitor”SSRI according to Sutalangka et a0al and Kaur et a0al 2015The influence of M oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function Vauzour et a0al Other studies with consumption of M oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects William reduction on post prandial blood glucose Ghiridhari increased insulin secretion in healthy subjects Anthanont et a0al decreased total plasma cholesterol and increased HDL Nambiar The presence of sitosterol in M oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces Jain Mbikay 0cnoitcudorpmubes niks ni noitcudeRledom laminAdna ortiv nI la a0te amreV a0 a0 pytivitca tnadixoitnAledom laminAAN yehaFnoitcefni la a0te ruaK la a0te akgnalatuSelfiorp dipil no tcapmi evitisoPledom laminA a0 a0 p raibmaN ledomnamuH a0 a0 pyattasiri yehaF la a0te lukaK la a0te ruozuaV la a0te roknoD la a0te la a0te eednoDnesodU hanajruNdna iti T la a0te itiT la a0te inor™aS la a0te nanhsirkalapoGledom laminA a0 a0 p ledomnamuH a0 a0 p la a0te tnonahtnA mailliW irahdirihGnilusni esaercnI doolb laidnarp tsop no noitcudeR ni slihportuen gnitalucric esaercnIseiduts ortiv nI a0 a0 pesoculgledom namuH a0 a0 p la a0te eurDledom laminAsserts etuca a0 a0 pledom laminA a0 a0 p lariv uehritna recnac ni romutitnAdna lairetcab itna msitamRSShti serpeditnA wnoitanibmoc ni tnas recnahne evitingoC tnatcetorporuen ralucsavorbereCairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaerTsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercnIselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo M tnereffid fo snoitca lacigolocamrahp fo elbat yrammuS elbaTarefielo M fo snoitca lacigolocamrahP ledomnamuH a0 a0 pmsidioryhtrepyh etalugeR ledomnamuH a0 a0 pamehtyre niks ni ecudeRseiduts ortiv nI a0 a0 pa ilA la a0te ilA la a0te dammahuMgnigaitna nikSstcartxe suoeuqa fognilaehdnuoW tsaerb tnadixoitna yrotammaflniitnAicrac noloc ni sisotpopa ecudnI la a0te ilarahB la a0te tehpiarK la a0te inailihaTledom laminA a0 a0 pnoitacfiixoted dnaledom laminA a0 a0 p ledomnamuHc ilA a0 a0 pamonytivitca laiborcimitnAledom laminA a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnAdna la a0te nanhsirkalapoG la a0te iramsAlAseiduts ortiv nIselknirwitnA a0 a0 pniap tnioj dna aehrraid taerT la a0te ilAledom namuH a0 a0 p la a0te nanhsirkalapoG a0 a0 pAN raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitnA la a0te lednoroTnoitardyh niks ni esaercnI ledomnamuH a0 a0 p ni laitnetop evitneverpomehCrecnac neelps dna citapeh nanhsirkalapoG la a0te ilarahBseiduts ortiv nI a0 a0 p ledomnamuHb ilA a0 a0 p dna tnadixoitna citebaiditnAseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonnikS tnadixoitna la a0te yebaR l Ednaledom laminA a0 a0 p ledomnamuHANammaflni gnul etuca fo esaerceD la a0te thginKcMledom laminA a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerceD ilarahB eDdna atpugsaD la a0teledom laminA a0 a0 p yebaR l Edna iklaMlAledom laminA a0 a0 pdik lla detaroilema dna desaercnIsretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihnIledom laminA a0 a0 pseiduts ortiv nI a0 a0 p azmaH la a0te areiV iklaMlAnajahaM narmI dna meedaNsevaeLsdoPsdeeSA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiuGledom laminA la a0te reugiuGledom laminA inamkcuRledom laminA hzimahtabnI la a0te nanhsirkalapoGseiduts ortiv nIANdipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorPstceffe evitaxalyticixototapeh decudni”lomatecarap no evitcetorpotapeH etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnArecnac eruc dna cimeloretselohcopyHsmelborp yraniruarefielo M fo snoitca lacigolocamrahPdeunitnoc elbaT eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecudeR la a0te idaraK dna ytivitca evitcetorpotycyrotercesitna recluitnA la a0te yrahduohCAN a0p a0evitcetorpotapeHipilitna dna cimeretselohcitnAledom laminA a0 a0 p amruKledom laminA a0 a0 pledom laminA a0 a0 p ahceneSledom laminA a0 a0 pcimed tcart yraniru fo tnemeganaMsmotpmys snoitcefni hgni Sdna ayruaM la a0te alkuhSledom laminA a0 a0 p ledomnamuH a0 a0 p serec¡Cledom laminAlairetcabitnA a0 a0 p la a0te reffaZseiduts ortiv nIANtceffe noitatnalpmiitnA a0p a0yrotammaflniitnA a0p a0yticixototapeHdecudnI ”lomatecaraPno evitcetorpotapeHaropsorueN tsniaga lagnufitnA inamkcuRledom laminA a0 a0 pseiduts ortiv nI ahJ a0 a0 pD a0Meireles et alSeveral M oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive Stohs and Hartman The antioxidant activity derives from the high amounts of polyphenols Leone et a0al 2015a b Verma et a0al Leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing Reactive Oxygen Species ROS production only in cancer cells which leads to cell apoptosis Gopalakrishnan The active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and G2M enrichment AlAsmari et a0al Some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model Krishnamurthy et a0al Table a0In traditional medicine a paste made of leaves is applied externally in wounds Siddhuraju and Becker Some scientific studies have shown that leave extracts have beneficial properties in skin Aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing Muhammad et a0al A M oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts Gothai et a0al A hydroalcoholic extract of M oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds Sunscreen and photo protective characteristics were studied very recently Baldisserotto et a0al When applying a cream with this extract it was also verified a reduction in sebum production Ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration Ali et a0al 2013a b c Wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with M oleifera leaf extract Ali et a0al The compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins A C and B Jadoon et a0al M oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema Ali et a0al 2013a b c Table a0 M oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed Torondel et a0al The efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarceA leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing ANyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutSsrewolFstooRkraB 0crate size and resistance on those plants and fruits Bhargave M oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that Moringa tea has adaptogenic capabilities in cases of stress Drue et a0al Table a0 Previous studies using dried Moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis McKnight et a0al An ethanolic extract of Moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of M oleifera leaves may have produced its anxiolytic effects via multiple mechanisms Bhat SeedsSeeds collected from pods can be eaten raw or cooked From M oleifera seeds a rich vegetable oil can be produced M oleifera seed oil or BehenBen oil is produced through the cold pressing of the M oleifera seeds M oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry Rashid et a0al The oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils Ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic Anwar It is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties It is also a good skin cleansing product Nadeem and Imran Table a0 details some cosmetic brands that use M oleifera leaves oil or active extracts as ingredients in the composition of their products This oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals Milled M oleifera seed shells can be used as a natural exfoliating agentMoringa oleifera seeds can also help diabetic patients Table a0 Some studies by AlMalki and El Rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of M oleifera seeds powderkg body weight during a0weeks At the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids Moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters In fact M Oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the CCl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity Hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities Hamza Treatment with M oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity Histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening From all above results and observations it can be concluded that the seeds possess promising antiarthritic property Mahajan et a0al These seeds have others appeals to the daily life and industry Seed powder showed capacity to purify water and remove heavy metals and anic compounds Sharma et a0al through low molecular weight cationic proteins mediated precipitation Kansal and Kumari There was a reduction of “ in the turbidity of the water and “ of bacterial reduction Bhargave Lea The remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value Lea Compounds such as pterygospermin moringine and benzyl isothiocya

Thyroid_Cancer

Moringa oleifera L from the Moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions M oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine With no reports of side effects in doses achievable by ingestion different parts of M oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders This plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women The seed and leaves powder has water purification properties through flocculation It also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries So M oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil According to the holistic or traditional medicine M oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients It is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life The high and specific immunological potential of M oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against Covid19 diseaseKeywords Moringa oleifera a0· Drumstick tree a0· Miracle tree a0· Medicinal plant a0· Cosmetics a0· Food supplementDiana Meireles and Jo£o Gomes authors contributed equally to this workdianameireleslivecompt Diana Meireles ICBAS Institute of a0Biomedical Sciences Abel Salazar University of a0Porto Rua de Je Viterbo Ferreira no a0Porto Portugal YIDAO Acupuncture and a0TCM Center Porto Portugal CIIMAR Interdisciplinary Centre of a0Marine and a0Environmental Research University of a0Porto Porto Portugal CBSin Center of a0BioSciences in a0Integrative Health Porto PortugalIntroductionMoringa oleifera L Moringa pterygosperma G wellknown as the œdrumstick or œhorseradish tree is native of Northwest India its main producer but can also be found in South Africa Northeast Africa Madagascar Tropical Asia Southwest Asia and Latin America The Moringa genus comprises species M arborea M longituba M borziana M pygmaea M hildebrandtii M drouhardii M longituba M peregrina M stenopetala M rivae M ruspoliana M Ovalifolia M Concanensis and M ole­fera Rani a0 From the Moringaceae family M oleifera is the most known studied and used species Anwar Olson with human and animal applications The various resources obtained from this plant”leaves flowers seeds pods bark and roots”can be used for cooking or in traditional medicine to treat several pathologies M oleifera has the capability to survive in humid or dry hot climates Vol01234567891 0c D a0Meireles et aland poor soils Anwar et a0al Mainenti M oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries Zongo ValdezSolana et a0al Gopalakrishnan Debajyoti et a0al M oleifera gained the title of œMiracle Tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry Tables a0 and In fact when compared to other plants from a0g of dry leafs of M oleifera we can obtain times more vitamin C than from oranges times more vitamin A than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach Oduro et a0al a0 a0Rockwood Saini et a0al Table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the United States Department of Agriculture USDA database although it is Table Nutritional values per a0g of the edible portion of M oleifera pods and leavesComponentsPer a0gRaw podsa“Raw leavesa““““Energy kcalWater gProtein gTotal lipid gCarbohydrate by difference gFibre total dietary gFatty acids total saturated gFatty acids total monounsaturated gFatty acids total polyunsaturated gFatty acids total trans gCholesterol mgVitamin A RAE µgVitamin D D2 D3 µgVitamin D IUThiamin mgRiboflavin mgNiacin mgPantothenic acid mgVitamin B6 mgVitamin B12 µgVitamin E mgVitamin C total ascorbic acid mgFolate total µgFolic acid µgSodium mgPotassium mgCalcium mgPhosphorus mgMagnesium mgIron mgZinc mgCopper mgManganese mgSelenium µga Information obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June b Average values and standard deviation published by Witt Only two values were found Witt Dried leavesb ± ± ± ± ± ““““““ ± ““““““ ± “ ± ± ± ± ± ± ± ± ““ 0cTable Amino acids and flavonols per a0g of the edible raw portion of M oleifera leavesPer a0gRaw leavesComponentsAmino acids a0Tryptophan g a0Threonine g a0Isoleucine g a0Leucine g a0Lysine g a0Methionine g a0Cystine g a0Phenylalanine g a0Tyrosine g a0Valine g a0Arginine g a0Histidine g a0Alanine g a0Aspartic acid g a0Glutamic acid g a0Glycine g a0Proline g a0Serine gFlavonols a0Isorhamnetin mg a0Kaempferol mg a0Myricetin mg a0Quercetin mgInformation obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June known that the nutrient content varies according to the plantation site Aslam and seasons Witt The nutritional value of dried leaves not existent in USDA database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by Witt From leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds Anwar et a0al Leone et a0al 2015a b Saini et a0al Fahey Debajyoti et a0al Divya et a0al M oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant Fahey Abdull Razis et a0al Divya et a0al Anwar et a0al published a table with various traditional medicinal uses of the different parts of M oleifera Anwar et a0al The attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans According the revision by Stohs and Hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of M oleifera leaves and leaf extracts achievable by oral ingestion Although there was not any report of major adverse side effects there are some important information that should be registered In fact there are some studies suggesting that M Oleifera cannot be used in combination with other modern medicines in humans A research by Gholap et a0al concluded that M oleifera has been noted to be a good regulator of insulin Thus according Sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using M oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications Another study suggests that when treating thyroids M oleifera compounds of the leaf may improve thyroid function Tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactionA research work concerning the œAcceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of M oleifera on children girls in Zambia Barichella et a0al With regards to safety concerns supplementation of a0g per day of M oleifera powder was deemed safe for children and adolescents both in the short and long term This research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of M oleifera daily showing to be still an inadequate and symptomatic dose in childrenOther studies suggest that M oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function Das et a0al Kelly Sileshi et a0al Despite the numerous positive health benefits associated with M oleifera phytochemicals there are suspicions that it contains harmful substances Fahey et a0al Annongu et a0al Maizuwo It contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects Maizuwo et a0al Some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin Fahey et a0al There are unconfirmed reports that M oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women Dutta It is also suspected that it can prevent implantation in women hence it must be avoided A review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c D a0Meireles et alTable Compilation of food supplements containing M oleifera tree parts or extracts in June Tree partsProduct informationBrandProductNaturingaMoringa capsulesLeavesMoringa teaMoringa kids multivitamin complexMoringa powderBioheraDried seeds extract Moringa capsulesLeavesMoringa syrupLeavesLeavesMiracle treeMoringa anic teaanic Moringa superfood supplements”capsulesMoringa Superfood PowderMoringa Superfood SticksIswariMoringa powder anicDrasanviLeaves dry extract Nutrabasics”MoringaRegulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditionDelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindStrengthens the immune system rich in vitamins and minerals stimulates natural defensesAdds nutritional value source of fiber protein vitamins and minerals improves physical conditionStrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoIt is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids Daily use of Moringa can help to restore your imbalances in your dietThe Moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundThe richness of its active ingredients helps maintain blood glucose levels Provides Flavonoids and Polyphenols by those attempting to conceive as it functioning as an abortifacient Nath et a0al Dutta Finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rat™s cells was verified at a0mgkg Asare et a0al However all mentioned side effects were verified with doses that far exceed the amounts used in food intake Asare et a0al So research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of M oleifera extracts and products on both human and animal models Adedapo et a0al Stohs et a0al Many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of M oleifera Fahey In fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as Ayurveda and Traditional Chinese Medicine TCM Karadi et a0al Kasote et a0al Debajyoti et a0al As an endemic source with highly digestible protein Ca Fe Vitamin C and carotenoids is considered as a suitable natural product to be used by undernourishment populations Dixit The resources obtained from a0M oleifera tree on a0a a0conventional approachLeaves and a0podsIn some countries leaves and fruits are commonly used in culinary as vegetables Leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption Moyo Olson et a0al In all ways of use and conservation M oleifera does not lose nutritional value Mahmood Leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants Table a0 Anwar Rebufa et a0al Phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids Table a0 M oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content Moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0cTable Compilation of cosmetics containing M oleifera tree parts or extracts as ingredients in June Cosmetic ingredientsProduct informationProduct brandnameSkinSecretAntiwrinkle face creamAntiaging moisturizer face creamHand creamBody milkLushAfrican paradise body conditionerQueen bee hair honeyMagical Moringa facial moisturizerCharity pot Hand and body lotionPassion fruit lip balmGo faster feet foot lotionTwinkle toes foot powderLush gardener cold pressed soapLaboratoires S©robiologiquesPURISOFT®Body shopMoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubMoringa eau de toilette Moringa body mistPurifying and protective action against environmental stress such as smoke and pollutionMoisturizing nourishingDeodorizingRemove dirt moisturizing nourishingSkin cleansingpurification protects skin against pollution heavy metals cigarette smokeSkin feels smooth and restoredDelicately scent your skin in a crisp floral aroma with MoringaLeavesM pterygosperma oilM pterygosperma leaf infusionoilM pterygosperma powderActive ingredient peptide from Moringa seedsM pterygosperma oilM oleifera leaf extractM pterygosperma seed extractM oleifera seeds and oilM oleifera leaf extractoilM pterygosperma seed extractM pterygosperma extractClarinsExtracomfort antipollution cleansing cream Eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionNeutralizes the effects of pollution and purifies the skin to restore its natural radiancePurifies and refines while preserving your skin™s natural moisture balance Neutralizes the harmful effects of pollutionOnestep facial cleanserExfoliating body scrubOnestep gentle exfoliating cleanserWater purifycomfort onestep cleanserDaily energizer cleansing gelNaturingaMoringa soap bioMoringa exfoliating face scrubMoringa O2Herbal moisturizing lotionfacial tonersoapherbal shampooconditionerShu UemuraAntiOxi pollutant and dullness clarifying cleansing oilUrban moisture hydronourishing shampooconditionerdouble serumdeep treatment masqueHigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissueMoringa seeds peel and exfoliate the skin while Moringa oil moisturizes and regenerates the skinRejuvenate nourish and protects skinRepairs strengthens reduces hair fallEnhanced power to remove micro impurities and stubborn makeup antipollution breakthroughHighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c Table continuedCosmetic ingredientsM oleifera seed extractProduct brandnameBioBeaut©Antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating gelDualphase waterproof eye makeup removerD a0Meireles et alProduct informationRemoves makeup pollution particles and excess sebum while leaving the skin well moisturized The Seed of Moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination This extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health Anwar et a0al Table a0 In dried M oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet In the leaves is found greater a variety and quantity of proteins when comparing to other tree parts Rebufa et a0al Wang et a0al Due to its nutritional rich values M oleifera can be a good enriching food additive to human diet and also an animal feed fortifier Moyo Adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition Bhargave Studies of acceptance by the consumer of enriched foodssnacks with M oleifera have been obtaining good results Ellis Jung M oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children Phytosterols from M oleifera increase estrogen production that enhance the activity of the mammary glands ducts Gopalakrishnan Doses of a0mgg of body weight given to mice result in increased milk production Also the pup weight augment with increasing doses of M oleifera leaf powder intake Titi et a0al Titi and Nurjanah Studies of toxicity in animals show that M oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes M oleifera may cause toxicity by accumulation of some elements a0Ali et a0al The amount of a0g of M oleifera dried leaf per day is the maximum recommended doseage AsieduGyekye et a0 al Table a0 compiles some food supplements based on M oleifera tree parts or extracts A hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice Table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis Bharali et a0al M oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer Gopalakrishnan et a0al carotene the major component reported from the drumsticks of the M oleifera plant as well as the presence of vitamin A and C suggest an action in the induction of antioxidant and antiinflammatory profiles Geervani and Devi Bharali et a0al Praengam et a0al It was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model Gupta et a0al Kraiphet et a0al Studies in rats showed that M oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function Kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases Sutalangka et a0al It was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a M oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days Kaur et a0al This effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitor”SSRI according to Sutalangka et a0al and Kaur et a0al 2015The influence of M oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function Vauzour et a0al Other studies with consumption of M oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects William reduction on post prandial blood glucose Ghiridhari increased insulin secretion in healthy subjects Anthanont et a0al decreased total plasma cholesterol and increased HDL Nambiar The presence of sitosterol in M oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces Jain Mbikay 0cnoitcudorpmubes niks ni noitcudeRledom laminAdna ortiv nI la a0te amreV a0 a0 pytivitca tnadixoitnAledom laminAAN yehaFnoitcefni la a0te ruaK la a0te akgnalatuSelfiorp dipil no tcapmi evitisoPledom laminA a0 a0 p raibmaN ledomnamuH a0 a0 pyattasiri yehaF la a0te lukaK la a0te ruozuaV la a0te roknoD la a0te la a0te eednoDnesodU hanajruNdna iti T la a0te itiT la a0te inor™aS la a0te nanhsirkalapoGledom laminA a0 a0 p ledomnamuH a0 a0 p la a0te tnonahtnA mailliW irahdirihGnilusni esaercnI doolb laidnarp tsop no noitcudeR ni slihportuen gnitalucric esaercnIseiduts ortiv nI a0 a0 pesoculgledom namuH a0 a0 p la a0te eurDledom laminAsserts etuca a0 a0 pledom laminA a0 a0 p lariv uehritna recnac ni romutitnAdna lairetcab itna msitamRSShti serpeditnA wnoitanibmoc ni tnas recnahne evitingoC tnatcetorporuen ralucsavorbereCairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaerTsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercnIselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo M tnereffid fo snoitca lacigolocamrahp fo elbat yrammuS elbaTarefielo M fo snoitca lacigolocamrahP ledomnamuH a0 a0 pmsidioryhtrepyh etalugeR ledomnamuH a0 a0 pamehtyre niks ni ecudeRseiduts ortiv nI a0 a0 pa ilA la a0te ilA la a0te dammahuMgnigaitna nikSstcartxe suoeuqa fognilaehdnuoW tsaerb tnadixoitna yrotammaflniitnAicrac noloc ni sisotpopa ecudnI la a0te ilarahB la a0te tehpiarK la a0te inailihaTledom laminA a0 a0 pnoitacfiixoted dnaledom laminA a0 a0 p ledomnamuHc ilA a0 a0 pamonytivitca laiborcimitnAledom laminA a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnAdna la a0te nanhsirkalapoG la a0te iramsAlAseiduts ortiv nIselknirwitnA a0 a0 pniap tnioj dna aehrraid taerT la a0te ilAledom namuH a0 a0 p la a0te nanhsirkalapoG a0 a0 pAN raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitnA la a0te lednoroTnoitardyh niks ni esaercnI ledomnamuH a0 a0 p ni laitnetop evitneverpomehCrecnac neelps dna citapeh nanhsirkalapoG la a0te ilarahBseiduts ortiv nI a0 a0 p ledomnamuHb ilA a0 a0 p dna tnadixoitna citebaiditnAseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonnikS tnadixoitna la a0te yebaR l Ednaledom laminA a0 a0 p ledomnamuHANammaflni gnul etuca fo esaerceD la a0te thginKcMledom laminA a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerceD ilarahB eDdna atpugsaD la a0teledom laminA a0 a0 p yebaR l Edna iklaMlAledom laminA a0 a0 pdik lla detaroilema dna desaercnIsretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihnIledom laminA a0 a0 pseiduts ortiv nI a0 a0 p azmaH la a0te areiV iklaMlAnajahaM narmI dna meedaNsevaeLsdoPsdeeSA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiuGledom laminA la a0te reugiuGledom laminA inamkcuRledom laminA hzimahtabnI la a0te nanhsirkalapoGseiduts ortiv nIANdipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorPstceffe evitaxalyticixototapeh decudni”lomatecarap no evitcetorpotapeH etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnArecnac eruc dna cimeloretselohcopyHsmelborp yraniruarefielo M fo snoitca lacigolocamrahPdeunitnoc elbaT eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecudeR la a0te idaraK dna ytivitca evitcetorpotycyrotercesitna recluitnA la a0te yrahduohCAN a0p a0evitcetorpotapeHipilitna dna cimeretselohcitnAledom laminA a0 a0 p amruKledom laminA a0 a0 pledom laminA a0 a0 p ahceneSledom laminA a0 a0 pcimed tcart yraniru fo tnemeganaMsmotpmys snoitcefni hgni Sdna ayruaM la a0te alkuhSledom laminA a0 a0 p ledomnamuH a0 a0 p serec¡Cledom laminAlairetcabitnA a0 a0 p la a0te reffaZseiduts ortiv nIANtceffe noitatnalpmiitnA a0p a0yrotammaflniitnA a0p a0yticixototapeHdecudnI ”lomatecaraPno evitcetorpotapeHaropsorueN tsniaga lagnufitnA inamkcuRledom laminA a0 a0 pseiduts ortiv nI ahJ a0 a0 pD a0Meireles et alSeveral M oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive Stohs and Hartman The antioxidant activity derives from the high amounts of polyphenols Leone et a0al 2015a b Verma et a0al Leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing Reactive Oxygen Species ROS production only in cancer cells which leads to cell apoptosis Gopalakrishnan The active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and G2M enrichment AlAsmari et a0al Some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model Krishnamurthy et a0al Table a0In traditional medicine a paste made of leaves is applied externally in wounds Siddhuraju and Becker Some scientific studies have shown that leave extracts have beneficial properties in skin Aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing Muhammad et a0al A M oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts Gothai et a0al A hydroalcoholic extract of M oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds Sunscreen and photo protective characteristics were studied very recently Baldisserotto et a0al When applying a cream with this extract it was also verified a reduction in sebum production Ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration Ali et a0al 2013a b c Wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with M oleifera leaf extract Ali et a0al The compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins A C and B Jadoon et a0al M oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema Ali et a0al 2013a b c Table a0 M oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed Torondel et a0al The efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarceA leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing ANyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutSsrewolFstooRkraB 0crate size and resistance on those plants and fruits Bhargave M oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that Moringa tea has adaptogenic capabilities in cases of stress Drue et a0al Table a0 Previous studies using dried Moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis McKnight et a0al An ethanolic extract of Moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of M oleifera leaves may have produced its anxiolytic effects via multiple mechanisms Bhat SeedsSeeds collected from pods can be eaten raw or cooked From M oleifera seeds a rich vegetable oil can be produced M oleifera seed oil or BehenBen oil is produced through the cold pressing of the M oleifera seeds M oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry Rashid et a0al The oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils Ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic Anwar It is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties It is also a good skin cleansing product Nadeem and Imran Table a0 details some cosmetic brands that use M oleifera leaves oil or active extracts as ingredients in the composition of their products This oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals Milled M oleifera seed shells can be used as a natural exfoliating agentMoringa oleifera seeds can also help diabetic patients Table a0 Some studies by AlMalki and El Rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of M oleifera seeds powderkg body weight during a0weeks At the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids Moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters In fact M Oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the CCl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity Hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities Hamza Treatment with M oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity Histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening From all above results and observations it can be concluded that the seeds possess promising antiarthritic property Mahajan et a0al These seeds have others appeals to the daily life and industry Seed powder showed capacity to purify water and remove heavy metals and anic compounds Sharma et a0al through low molecular weight cationic proteins mediated precipitation Kansal and Kumari There was a reduction of “ in the turbidity of the water and “ of bacterial reduction Bhargave Lea The remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value Lea Compounds such as pterygospermin moringine and benzyl isothiocya

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

Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly citedCutaneous metastases are unusual presenting symptoms of lung cancer erefore they are prone to be misdiagnosed and missede report describes a case of a fortynineyearold female with painful zosteriform rashes showing multiple vesiclelike papuleslocalized on the left breast for days e patient had been diagnosed as lung adenocarcinoma at the department of oncology oneyear ago Skin biopsy revealed blue nodular lesions in the dermis composed of clustered heterogeneous tumor cells with glandularformation Immunohistochemical stains confirmed the diagnosis of metastatic lung adenocarcinoma IntroductionLung cancer can metastasize to almost all ans butmore often invades the hilar nodes liver adrenal glandsbones and brain [] e incidence of lung cancer withmetastases to the skin varies between “ [] A lungcancer metastasis is usually classified only as adenocarcinoma squamous cell carcinoma SCC or undiï¬erentiated carcinoma Until the 1980s SCC was reported asthe most common type of lung cancer However adenocarcinoma has replaced SCC as the most common lungcancer subtype especially in women and in neversmokers Sun reported that the type of adenocarcinoma was times more frequent than that of SCC []Skin metastases can appear on any cutaneous surface andthe most common sites are the chest abdomen head andneck [ ] Cutaneous metastases have various manifestations such as single papulesnodules or multiplelesions on anywhere of the skin while other rare formsmay show plaquelike lesions erysipelaslike papuleszosteriform lesions and scars [ ] Case PresentationA fortynineyearold nonsmoker female was admitted toour department with multiple painful papules localizedon the left breast ey appeared eruptively for about days and initially diagnosed as herpes zoster in anotherhospital e patient had been diagnosed as lung adenocarcinoma at the department of oncology one year agoShe was given oxitinib mesylate a targeted drug for thetreatment of nonsmallcell lung cancer In addition thepatient exhibited symptom of pain signs of weight lossanorexia and fatiguePhysical examination showed zosteriform vesiclelikepapules measuring “ cm on the left breast elesions were pink or red firm and tender Figure Excisional biopsy was performed revealing blue nodularlesions ltrating in the dermis composed of clusteredheterogeneous tumor cells with glandular formationSome tumor cells were detected within vessels or lymphatic vessels Some cells were transparent Mitosis wassignificant Figures 2a“2c In immunohistochemistumor cells were positive for cytokeratin CKtrycytokeratin7 CK7thyroid transcription factor1TTF1 and EMA and negative for cytokeratin20CK20 carcinoembryonic antigen CEA and grosscysticGCDFP15Figures 3a“3c Proliferative index as measured byKi67 was approximately “ oftumor cellsAccording to the clinical and pathological features cutaneous metastatic lung adenocarcinoma was madeprotein15diseasefluid 0cCase Reports in Dermatological MedicineFigure Zosteriform vesiclelike papules measuring “ cm on the left breast Pink or red firm and tenderabcFigure Skin biopsy revealed a blue nodular lesions ltrating in the dermis composed of clustered heterogeneous tumor cells withglandular formation HE magnification — b some tumor cells were detected within vessels or lymphatic vessels HE magnification — c some cells were blue and transparent and mitosis was significant HE magnification —abcFigure Immunohistochemical stain highlighting the tumor cells showing a CK7 b EMA and c TTF1 positive DiscussionSkin metastases suggest the progression of primary cancerand portend a poor clinical prognosis Skin metastases fromlung cancer are rare e percentage of patients with lungcancer that develops cutaneous metastases ranges from to percent [] It is seen more often in men than in women[] It does not show any specific presentation It is oftenpainless and less likely to be noticed making it more difficultto be diagnosed correctly which may delay treatment Although described cases show that metastatic nodules arepainless our patient showed severe pain e presence ofzosteriform painful vesiclelike lesions really mimics herpeszoster clinically in our casee mechanisms determining the metastasis of lungcancer in skin remain unknown Pathogenesis is suggested tobe by lymphovascular invasion with poor diï¬erentiationand upper lobe tumors increasing the risk [] Usually skinmetastasis develops after initial diagnosis of the primarymalignancy and late in the course of the disease Occasionally skin lesions that arise from lung cancer may develop before the primary tumor is recognized In our case 0cCase Reports in Dermatological Medicine[] R Koca Y Ustundag E Kargi G Numanoglu andH C Altinyazar œA case with widespread cutaneous metastases of unknown primary origin grave prognostic findingin cancer Dermatology Online Journal vol no p [] N A Babacan S Kilickap S Sene œA case of multifocalskin metastases from lung cancer presenting with vasculitictype cutaneous nodule Indian Journal of Dermatologyvol p skin metastases occurred during the immunotherapy Histology shows most commonly adenocarcinoma and thensquamoussmallcell followed by largecell carcinoma []Immunohistochemical markers are useful for the identification of the primary cancer or when a shorter diï¬erential isdesired AntiTTF is both sensitive and specific for primaryadenocarcinomas bronchioalveolar carcinomas and smallcell carcinomas when thyroid primary is excluded []CK7and CK20ˆ’ are sensitive but not specific for primaryadenocarcinomas and bronchioalveolar carcinomas eCK7CK20ˆ’tumors usually include the lung breast endometrium ovary thyroid salivary gland and mesothelioma [ ]Treatment of a single solitary skin lesion usually includessurgery alone or combined with chemotherapy andor radiation If lesions are more disseminated chemotherapy isthe primary option but may elicit an inadequate response[] Radiation can also be used alone andor in combinationwith chemotherapy andor surgery However despite thecombination of radiotherapy and chemotherapy patientswith lung cancer developing cutaneous metastases have apoor outcome Mean survival is short usually to monthsafter diagnosis of cutaneous metastasis []Conflicts of Intereste authors declare they have no conflicts of interestAcknowledgmentsis work was supported by a grant from the NationalNatural Science Foundation of China References[] T W Mollet C A Garcia and G Koester œSkin metastasesfrom lung cancer Dermatology Online Journal vol no [] S Sun J H Schiller and A F Gazdar œLung cancer in neversmokersa diï¬erent disease Nature Reviews Cancer vol no pp “ [] S Dreizen H M Dhingra D F Chiuten T Umsawasdi andM Valdivieso œCutaneous and subcutaneous metastases oflung cancer Postgraduate Medicine vol no pp “ [] M Khaja D Mundt R A Dudekula œLung cancerpresenting as skin metastasis of the back and hand a caseseries and literature review Case Reports in Oncology vol no pp “ [] W T McSweeney and K Tan œCutaneous metastases as apresenting sign of metastatic NSCLC Journal of Surgical CaseReports vol no [] M H Brownstein and E B Helwig œMetastatic tumors of theskin Cancer vol no pp “ [] R B McGrath S P Flood and R Casey œCutaneous metastases in nonsmall cell lung cancer BMJ Case Reportsvol [] V Jerome Marson J Mazieres O Groussard œExpression of TTF1 and cytokeratins in primary and secondaryepithelial lung tumours correlation with histological type andgrade Histopathology vol no pp “ 0c'

Thyroid_Cancer

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

Thyroid_Cancer

"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

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

Thyroid_Cancer

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

Thyroid_Cancer

Moringa oleifera L from the Moringaceae family is a perennial tree widely cultivated in many tropic regions and easily grown even in adverse conditions M oleifera is also known as the miracle tree which for centuries has been indicated for traditional medicine With no reports of side effects in doses achievable by ingestion different parts of M oleifera is used to treat several conditions such as malnutrition diabetes blindness anemia hypertension stress depression skin arthritis joints and a0kidney stones disorders This plant also showed capacity of helping in maintenance of the cardiovascular system health bloodglucose levels and providing antioxidant antiinflammatory and anticancer activity as well as the regulation of urinary tract and lactation in nursing women The seed and leaves powder has water purification properties through flocculation It also supplements the food in the human diet and in the fortification of livestock feed especially in developing countries So M oleifera properties have also been applied to cosmetic and byproducts industries due to the high nutritive and protective properties of its seed oil According to the holistic or traditional medicine M oleifera has very relevant therapeutic properties and applications depending on the constitution somatic and psychological needs of patients It is usually referred as a natural product that can treat different physical and psychological health aspects offering an energetic action and structural rebuilder of the body and promoting emotions of highly positive attitudes towards life The high and specific immunological potential of M oleifera leads us to suggest an indepth study to assess the hypothesis of conferring a supportive effect against Covid19 diseaseKeywords Moringa oleifera a0· Drumstick tree a0· Miracle tree a0· Medicinal plant a0· Cosmetics a0· Food supplementDiana Meireles and Jo£o Gomes authors contributed equally to this workdianameireleslivecompt Diana Meireles ICBAS Institute of a0Biomedical Sciences Abel Salazar University of a0Porto Rua de Je Viterbo Ferreira no a0Porto Portugal YIDAO Acupuncture and a0TCM Center Porto Portugal CIIMAR Interdisciplinary Centre of a0Marine and a0Environmental Research University of a0Porto Porto Portugal CBSin Center of a0BioSciences in a0Integrative Health Porto PortugalIntroductionMoringa oleifera L Moringa pterygosperma G wellknown as the œdrumstick or œhorseradish tree is native of Northwest India its main producer but can also be found in South Africa Northeast Africa Madagascar Tropical Asia Southwest Asia and Latin America The Moringa genus comprises species M arborea M longituba M borziana M pygmaea M hildebrandtii M drouhardii M longituba M peregrina M stenopetala M rivae M ruspoliana M Ovalifolia M Concanensis and M ole­fera Rani a0 From the Moringaceae family M oleifera is the most known studied and used species Anwar Olson with human and animal applications The various resources obtained from this plant”leaves flowers seeds pods bark and roots”can be used for cooking or in traditional medicine to treat several pathologies M oleifera has the capability to survive in humid or dry hot climates Vol01234567891 0c D a0Meireles et aland poor soils Anwar et a0al Mainenti M oleifera is a highly nutritious plant being ideal to treat malnutrition in developing countries Zongo ValdezSolana et a0al Gopalakrishnan Debajyoti et a0al M oleifera gained the title of œMiracle Tree and commercial attention supported on several properties such as nutritional values amino acids and flavonols content which can be used in food supplements and cosmetic industry Tables a0 and In fact when compared to other plants from a0g of dry leafs of M oleifera we can obtain times more vitamin C than from oranges times more vitamin A than from carrots times more calcium than in milk a0times more protein than in yoghurt times more potassium than from bananas and times more iron than the obtained from spinach Oduro et a0al a0 a0Rockwood Saini et a0al Table a0 shows the nutritional values for the edible parts of raw pods and leaves obtained from the United States Department of Agriculture USDA database although it is Table Nutritional values per a0g of the edible portion of M oleifera pods and leavesComponentsPer a0gRaw podsa“Raw leavesa““““Energy kcalWater gProtein gTotal lipid gCarbohydrate by difference gFibre total dietary gFatty acids total saturated gFatty acids total monounsaturated gFatty acids total polyunsaturated gFatty acids total trans gCholesterol mgVitamin A RAE µgVitamin D D2 D3 µgVitamin D IUThiamin mgRiboflavin mgNiacin mgPantothenic acid mgVitamin B6 mgVitamin B12 µgVitamin E mgVitamin C total ascorbic acid mgFolate total µgFolic acid µgSodium mgPotassium mgCalcium mgPhosphorus mgMagnesium mgIron mgZinc mgCopper mgManganese mgSelenium µga Information obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June b Average values and standard deviation published by Witt Only two values were found Witt Dried leavesb ± ± ± ± ± ““““““ ± ““““““ ± “ ± ± ± ± ± ± ± ± ““ 0cTable Amino acids and flavonols per a0g of the edible raw portion of M oleifera leavesPer a0gRaw leavesComponentsAmino acids a0Tryptophan g a0Threonine g a0Isoleucine g a0Leucine g a0Lysine g a0Methionine g a0Cystine g a0Phenylalanine g a0Tyrosine g a0Valine g a0Arginine g a0Histidine g a0Alanine g a0Aspartic acid g a0Glutamic acid g a0Glycine g a0Proline g a0Serine gFlavonols a0Isorhamnetin mg a0Kaempferol mg a0Myricetin mg a0Quercetin mgInformation obtained from United States department of agriculture nutrient database ndbnalusdagovndbfoods in June known that the nutrient content varies according to the plantation site Aslam and seasons Witt The nutritional value of dried leaves not existent in USDA database is presented as an average of values with standard deviation calculated from diverse papers that was compiled and published by Witt From leaves to roots it is possible to obtain good quantities of important minerals proteins vitamins carotene amino acids and phenolic compounds Anwar et a0al Leone et a0al 2015a b Saini et a0al Fahey Debajyoti et a0al Divya et a0al M oleifera extracts have been studied with different medicinal purposes antiinflammatory antihypertensive diuretic antimicrobial antioxidant antidiabetic antihyperlipidemic antineoplastic antipyretic antiulcer and hepatoprotectant Fahey Abdull Razis et a0al Divya et a0al Anwar et a0al published a table with various traditional medicinal uses of the different parts of M oleifera Anwar et a0al The attractive properties of this plant led to studies of side effects and medical interactions in animal models and humans According the revision by Stohs and Hartman until this date none of the human in a0vitro studies or extrapolations of animal studies to humans reported adverse effects with doses of M oleifera leaves and leaf extracts achievable by oral ingestion Although there was not any report of major adverse side effects there are some important information that should be registered In fact there are some studies suggesting that M Oleifera cannot be used in combination with other modern medicines in humans A research by Gholap et a0al concluded that M oleifera has been noted to be a good regulator of insulin Thus according Sileshi et a0al patients suffering from lack of insulin will probably have adverse reductions of sugar levels when using M oleifera for medicinal purposes suggesting that it could decrease the blood sugar to even lower levels when used in combination with other modern medications Another study suggests that when treating thyroids M oleifera compounds of the leaf may improve thyroid function Tahiliani et a0al this well proving evidence further suggests that it can possibly conflict with other thyroid medication triggering drug interactionA research work concerning the œAcceptability and safety of shortterm daily supplementation in a group of malnourished girls assessed the use acceptability and safety of M oleifera on children girls in Zambia Barichella et a0al With regards to safety concerns supplementation of a0g per day of M oleifera powder was deemed safe for children and adolescents both in the short and long term This research also noted that mild nausea was reported in of the children at various age groups when meals were supplemented with a0g of M oleifera daily showing to be still an inadequate and symptomatic dose in childrenOther studies suggest that M oleifera could adverse and slowly breaking down the pharmaceutical drugs in the liver and thus a0may develop cirrhosis and liver failure resulting in malnutrition and weight loss as well as decreased cognitive function Das et a0al Kelly Sileshi et a0al Despite the numerous positive health benefits associated with M oleifera phytochemicals there are suspicions that it contains harmful substances Fahey et a0al Annongu et a0al Maizuwo It contains specific chemical compounds such as alkaloids and other phytotoxins which when consumed in high doses presents potentially nerveparalysing properties and other adverse effects Maizuwo et a0al Some of these phytochemicals include moringine moringinine estrogen pectinesterase and phenols including tannin Fahey et a0al There are unconfirmed reports that M oleifera stems roots and flowers potentially contain harmful phytochemical constituents especially during pregnancy which may promote uterus contraction leading to miscarriages in pregnant women Dutta It is also suspected that it can prevent implantation in women hence it must be avoided A review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c D a0Meireles et alTable Compilation of food supplements containing M oleifera tree parts or extracts in June Tree partsProduct informationBrandProductNaturingaMoringa capsulesLeavesMoringa teaMoringa kids multivitamin complexMoringa powderBioheraDried seeds extract Moringa capsulesLeavesMoringa syrupLeavesLeavesMiracle treeMoringa anic teaanic Moringa superfood supplements”capsulesMoringa Superfood PowderMoringa Superfood SticksIswariMoringa powder anicDrasanviLeaves dry extract Nutrabasics”MoringaRegulates the gastrointestinal transit natural antiinflammatory lowers cholesterol levels improves diabetic conditionDelays the ageing process ensures proper digestion high antioxidant power helps healing process tonifies body and mindStrengthens the immune system rich in vitamins and minerals stimulates natural defensesAdds nutritional value source of fiber protein vitamins and minerals improves physical conditionStrengthens the immune system helps to reverse the aging process beautifies the skin reduces the appearance of wrinkles and fine lines maintains the normal glucose level stimulates brain function and concentration increases libidoIt is a nutritionally complex whole food naturally rich in vitamins minerals and amino acids Daily use of Moringa can help to restore your imbalances in your dietThe Moringa leaf boasts a vast array of beneficial nutrients making this tree one of the highest plant sources of vitamins and minerals aroundThe richness of its active ingredients helps maintain blood glucose levels Provides Flavonoids and Polyphenols by those attempting to conceive as it functioning as an abortifacient Nath et a0al Dutta Finally cytotoxicity was verified in experiments with human peripheral blood mononuclear cells only at a0mgkg of an aqueous leaf extract genotoxicity on blood rat™s cells was verified at a0mgkg Asare et a0al However all mentioned side effects were verified with doses that far exceed the amounts used in food intake Asare et a0al So research on the adverse side effects with doses achievable by oral ingestion should still go on since currently there are no scientifically confirmed clear toxic and harmful effects of M oleifera extracts and products on both human and animal models Adedapo et a0al Stohs et a0al Many studies on nutrition phytotherapy disease treatment and prevention goals have been published thus supporting scientific basis about the efficiency of traditional uses of M oleifera Fahey In fact records about symptoms signs and treatment strategies in different diseases are found in several ancient texts of traditional medicines such as Ayurveda and Traditional Chinese Medicine TCM Karadi et a0al Kasote et a0al Debajyoti et a0al As an endemic source with highly digestible protein Ca Fe Vitamin C and carotenoids is considered as a suitable natural product to be used by undernourishment populations Dixit The resources obtained from a0M oleifera tree on a0a a0conventional approachLeaves and a0podsIn some countries leaves and fruits are commonly used in culinary as vegetables Leaves can also be dried and used in infusions or grounded into powder allowing easier conservation and consumption Moyo Olson et a0al In all ways of use and conservation M oleifera does not lose nutritional value Mahmood Leaves and pods are low in calories and rich in minerals vitamins and natural antioxidants Table a0 Anwar Rebufa et a0al Phytochemicals like flavonoids are also present in leaves as well as a significant percentage of essential amino acids Table a0 M oleifera leaves contain a high quantity of polyunsaturated fatty acids and low saturated fatty acids content Moyo which combined with diuretic lipid and blood pressure lowering properties from leaves and pods contribute to the maintenance 0cTable Compilation of cosmetics containing M oleifera tree parts or extracts as ingredients in June Cosmetic ingredientsProduct informationProduct brandnameSkinSecretAntiwrinkle face creamAntiaging moisturizer face creamHand creamBody milkLushAfrican paradise body conditionerQueen bee hair honeyMagical Moringa facial moisturizerCharity pot Hand and body lotionPassion fruit lip balmGo faster feet foot lotionTwinkle toes foot powderLush gardener cold pressed soapLaboratoires S©robiologiquesPURISOFT®Body shopMoringa range shower gel oil body butter body milk body sorbet hand cream soap body scrubMoringa eau de toilette Moringa body mistPurifying and protective action against environmental stress such as smoke and pollutionMoisturizing nourishingDeodorizingRemove dirt moisturizing nourishingSkin cleansingpurification protects skin against pollution heavy metals cigarette smokeSkin feels smooth and restoredDelicately scent your skin in a crisp floral aroma with MoringaLeavesM pterygosperma oilM pterygosperma leaf infusionoilM pterygosperma powderActive ingredient peptide from Moringa seedsM pterygosperma oilM oleifera leaf extractM pterygosperma seed extractM oleifera seeds and oilM oleifera leaf extractoilM pterygosperma seed extractM pterygosperma extractClarinsExtracomfort antipollution cleansing cream Eliminates traces of pollution detoxifies the epidermis and protects the skin from the harmful effects of pollutionNeutralizes the effects of pollution and purifies the skin to restore its natural radiancePurifies and refines while preserving your skin™s natural moisture balance Neutralizes the harmful effects of pollutionOnestep facial cleanserExfoliating body scrubOnestep gentle exfoliating cleanserWater purifycomfort onestep cleanserDaily energizer cleansing gelNaturingaMoringa soap bioMoringa exfoliating face scrubMoringa O2Herbal moisturizing lotionfacial tonersoapherbal shampooconditionerShu UemuraAntiOxi pollutant and dullness clarifying cleansing oilUrban moisture hydronourishing shampooconditionerdouble serumdeep treatment masqueHigh antioxidant value slowing skin ageing exfoliate dead cells by regenerating the tissueMoringa seeds peel and exfoliate the skin while Moringa oil moisturizes and regenerates the skinRejuvenate nourish and protects skinRepairs strengthens reduces hair fallEnhanced power to remove micro impurities and stubborn makeup antipollution breakthroughHighly concentrated in nutrients vitamins and antioxidants intensely hydrates deep within strandsA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c Table continuedCosmetic ingredientsM oleifera seed extractProduct brandnameBioBeaut©Antipollution micellar cleansing watergentle cleansing foamcleansing oil gelgentle exfoliating gelDualphase waterproof eye makeup removerD a0Meireles et alProduct informationRemoves makeup pollution particles and excess sebum while leaving the skin well moisturized The Seed of Moringa extract selected contains purifying peptides which on the surface limit the adhesion of the pollution particles and in depth activate their elimination This extract acts as a protective shield capable of preserving the good bacteria from the cutaneous flora against the aggression of pollutionof cardiovascular health Anwar et a0al Table a0 In dried M oleifera leaves it was also found a high content in calcium and iron which is normally residual in other plants used in our diet In the leaves is found greater a variety and quantity of proteins when comparing to other tree parts Rebufa et a0al Wang et a0al Due to its nutritional rich values M oleifera can be a good enriching food additive to human diet and also an animal feed fortifier Moyo Adding fresh or dried leaves to the feed of milk cows increased milk production and respectively that fact would be of great importance in developing countries to fight deficiencies in nutrition Bhargave Studies of acceptance by the consumer of enriched foodssnacks with M oleifera have been obtaining good results Ellis Jung M oleifera can also help lactating mothers produce more milk and help to treat malnutrition in young children Phytosterols from M oleifera increase estrogen production that enhance the activity of the mammary glands ducts Gopalakrishnan Doses of a0mgg of body weight given to mice result in increased milk production Also the pup weight augment with increasing doses of M oleifera leaf powder intake Titi et a0al Titi and Nurjanah Studies of toxicity in animals show that M oleifera dried leaf extract might be safe for consumption although in high doses and prolonged intakes M oleifera may cause toxicity by accumulation of some elements a0Ali et a0al The amount of a0g of M oleifera dried leaf per day is the maximum recommended doseage AsieduGyekye et a0 al Table a0 compiles some food supplements based on M oleifera tree parts or extracts A hydroalcoholic extract of green pods increased liver enzymes involved in the detoxification of xenobiotic substances in mice Table a0 suggesting a chemo preventive potential of a drumstick extract against chemical carcinogenesis Bharali et a0al M oleifera pods are also valuable to treat digestive and obesity problems and thwart colon cancer Gopalakrishnan et a0al carotene the major component reported from the drumsticks of the M oleifera plant as well as the presence of vitamin A and C suggest an action in the induction of antioxidant and antiinflammatory profiles Geervani and Devi Bharali et a0al Praengam et a0al It was suggested that carotene and sterols present in the plant pods acts as potent inhibitors on the formation of reactive oxygen intermediates a prerequisite for tumorigenesis and so inducing apoptosis in the mouse colon carcinoma model Gupta et a0al Kraiphet et a0al Studies in rats showed that M oleifera leaves extract might act as potential neuroprotectant via decreased oxidative stress and the enhanced cholinergic function Kirisattayakul et a0al and function as a cognitive enhancer hence being used in dementia cases Sutalangka et a0al It was also found an antidepressant activity in mouse models of depression when giving orally a a0mgkgday of a M oleifera alcoholic extract plus a0mgkgday fluoxetine for a0days Kaur et a0al This effect can be increased when combined with fluoxetine as a selective serotonin reuptake inhibitor”SSRI according to Sutalangka et a0al and Kaur et a0al 2015The influence of M oleifera may be due to the action of antioxidants and flavonoids through radical scavenging since its action is verified in other studies on animal models with cerebrovascular diseases exerting a multiplicity of neuroprotective actions within the brain and suppressing neuroinflammation and thus suggesting a great potential to promote memory learning and cognitive function Vauzour et a0al Other studies with consumption of M oleifera leaf powder revealed properties in human an animal models such as decreased blood glucose levels on diabetic type two subjects William reduction on post prandial blood glucose Ghiridhari increased insulin secretion in healthy subjects Anthanont et a0al decreased total plasma cholesterol and increased HDL Nambiar The presence of sitosterol in M oleifera leaves may be one of the reasons for decreasing plasma cholesterol since phytosterols cause less intestinal absorption of dietary cholesterol and increase its excretion on feces Jain Mbikay 0cnoitcudorpmubes niks ni noitcudeRledom laminAdna ortiv nI la a0te amreV a0 a0 pytivitca tnadixoitnAledom laminAAN yehaFnoitcefni la a0te ruaK la a0te akgnalatuSelfiorp dipil no tcapmi evitisoPledom laminA a0 a0 p raibmaN ledomnamuH a0 a0 pyattasiri yehaF la a0te lukaK la a0te ruozuaV la a0te roknoD la a0te la a0te eednoDnesodU hanajruNdna iti T la a0te itiT la a0te inor™aS la a0te nanhsirkalapoGledom laminA a0 a0 p ledomnamuH a0 a0 p la a0te tnonahtnA mailliW irahdirihGnilusni esaercnI doolb laidnarp tsop no noitcudeR ni slihportuen gnitalucric esaercnIseiduts ortiv nI a0 a0 pesoculgledom namuH a0 a0 p la a0te eurDledom laminAsserts etuca a0 a0 pledom laminA a0 a0 p lariv uehritna recnac ni romutitnAdna lairetcab itna msitamRSShti serpeditnA wnoitanibmoc ni tnas recnahne evitingoC tnatcetorporuen ralucsavorbereCairetsyh sa sredrosid suovren airalamdna diohpit fo tnemtaerTsesaesid negortse noitcudorp rosrucerpdna noitatcal esaercnIselpicnirp lanoitnevnoc ot gnidrocca krab dna stoor srewofl sdees sdop sevael su hcus strap eert arefielo M tnereffid fo snoitca lacigolocamrahp fo elbat yrammuS elbaTarefielo M fo snoitca lacigolocamrahP ledomnamuH a0 a0 pmsidioryhtrepyh etalugeR ledomnamuH a0 a0 pamehtyre niks ni ecudeRseiduts ortiv nI a0 a0 pa ilA la a0te ilA la a0te dammahuMgnigaitna nikSstcartxe suoeuqa fognilaehdnuoW tsaerb tnadixoitna yrotammaflniitnAicrac noloc ni sisotpopa ecudnI la a0te ilarahB la a0te tehpiarK la a0te inailihaTledom laminA a0 a0 pnoitacfiixoted dnaledom laminA a0 a0 p ledomnamuHc ilA a0 a0 pamonytivitca laiborcimitnAledom laminA a0 a0 psamonicrac latceroloc tsniaga ytivitca recnacitnAdna la a0te nanhsirkalapoG la a0te iramsAlAseiduts ortiv nIselknirwitnA a0 a0 pniap tnioj dna aehrraid taerT la a0te ilAledom namuH a0 a0 p la a0te nanhsirkalapoG a0 a0 pAN raelc dna paos ot lauqe pu lortnoc eht fognihsaw tnereffiddnah ni seitreporp lairetcabitnA la a0te lednoroTnoitardyh niks ni esaercnI ledomnamuH a0 a0 p ni laitnetop evitneverpomehCrecnac neelps dna citapeh nanhsirkalapoG la a0te ilarahBseiduts ortiv nI a0 a0 p ledomnamuHb ilA a0 a0 p dna tnadixoitna citebaiditnAseitreporp citirhtraitnagnisnaelc evitcetorp gnizirutsiom gnihsiruonnikS tnadixoitna la a0te yebaR l Ednaledom laminA a0 a0 p ledomnamuHANammaflni gnul etuca fo esaerceD la a0te thginKcMledom laminA a0 a0 pnoit dna setaidemretni noitadixorep dipil fonegyxo evitcaer esaerceD ilarahB eDdna atpugsaD la a0teledom laminA a0 a0 p yebaR l Edna iklaMlAledom laminA a0 a0 pdik lla detaroilema dna desaercnIsretemarap snoitcnuf yen ecuder sisorbfi revil detaroilemaesadixorepoleymytivitca citapeh yticixototapeh decudni tibihnIledom laminA a0 a0 pseiduts ortiv nI a0 a0 p azmaH la a0te areiV iklaMlAnajahaM narmI dna meedaNsevaeLsdoPsdeeSA review of a0properties nutritional and a0pharmaceutical applications of a0Moringa oleifera¦ 0c detneverp dna elfiorp la a0te reugiuGledom laminA la a0te reugiuGledom laminA inamkcuRledom laminA hzimahtabnI la a0te nanhsirkalapoGseiduts ortiv nIANdipil niagdevorpm thgiewi dna ytilitom lanitsetni etomorPstceffe evitaxalyticixototapeh decudni”lomatecarap no evitcetorpotapeH etatsorp fo sledom tnednepedni negordna ni ytivitca recnacitnArecnac eruc dna cimeloretselohcopyHsmelborp yraniruarefielo M fo snoitca lacigolocamrahPdeunitnoc elbaT eht dna etalaxo syendik eht ni noitisoped enotsyraniru eht ecudeR la a0te idaraK dna ytivitca evitcetorpotycyrotercesitna recluitnA la a0te yrahduohCAN a0p a0evitcetorpotapeHipilitna dna cimeretselohcitnAledom laminA a0 a0 p amruKledom laminA a0 a0 pledom laminA a0 a0 p ahceneSledom laminA a0 a0 pcimed tcart yraniru fo tnemeganaMsmotpmys snoitcefni hgni Sdna ayruaM la a0te alkuhSledom laminA a0 a0 p ledomnamuH a0 a0 p serec¡Cledom laminAlairetcabitnA a0 a0 p la a0te reffaZseiduts ortiv nIANtceffe noitatnalpmiitnA a0p a0yrotammaflniitnA a0p a0yticixototapeHdecudnI ”lomatecaraPno evitcetorpotapeHaropsorueN tsniaga lagnufitnA inamkcuRledom laminA a0 a0 pseiduts ortiv nI ahJ a0 a0 pD a0Meireles et alSeveral M oleifera studies with leaf powder or extracts on animals revealed other properties beyond the previously referred antioxidant chemoprotectant and antihypertensive Stohs and Hartman The antioxidant activity derives from the high amounts of polyphenols Leone et a0al 2015a b Verma et a0al Leaves extracts have revealed anticancer properties with antineoproliferative activity by inducing Reactive Oxygen Species ROS production only in cancer cells which leads to cell apoptosis Gopalakrishnan The active compounds present in extracts from leaves and bark revealed anticancer activity against breast and colorectal cancer cell lines through diverse mechanisms as decreased cell mobility decreased colony formation low cell survival high apoptosis and G2M enrichment AlAsmari et a0al Some extract fractions with anticancer activity have already been isolated characterized and tested in a0vitro and in a0vivo rat model Krishnamurthy et a0al Table a0In traditional medicine a paste made of leaves is applied externally in wounds Siddhuraju and Becker Some scientific studies have shown that leave extracts have beneficial properties in skin Aqueous leaves extract increased human dermal fibroblasts proliferation leading to faster wound healing Muhammad et a0al A M oleifera leave extract fraction with ethyl acetate in low concentration a0µgml showed in a0vitro effect in skin healing by increasing proliferation of human dermal fibroblasts Gothai et a0al A hydroalcoholic extract of M oleifera leaves used in a cream showed antiaging characteristics due to phenolic compounds Sunscreen and photo protective characteristics were studied very recently Baldisserotto et a0al When applying a cream with this extract it was also verified a reduction in sebum production Ali et a0al 2013a b c and in transepidermal water loss allowing to increase hydration Ali et a0al 2013a b c Wrinkles and other signs of lack of skin vitality where improved during a0months of using the same topic formulation with M oleifera leaf extract Ali et a0al The compounds responsible for this improvement in skin surface appear to be phenolics eg kaempferol and quercetin and antioxidants such as vitamins A C and B Jadoon et a0al M oleifera leaf extract cream was also tested for potential skin irritation by a a0h semioccluded patch test and proved to be nonirritant and well accepted by the volunteers also reducing skin erythema Ali et a0al 2013a b c Table a0 M oleifera leaf powder can be used to clean hands when four grams of wet more efficient or dried powder are applied on hands and rubbed Torondel et a0al The efficacy results were the same as for nonmedicated soap revealing potential to help in hand hygiene and prevent pathogen transmission in developing countries where hygiene products are scarceA leave extract sprayed in plant crops revealed another utility for this plant having beneficial effects on the growing ANyb detneserper era seulav dnuof tonelbacilppanon a pyb detneserper era eulav tnacfiingis yllacitsitats htiw seidutSsrewolFstooRkraB 0crate size and resistance on those plants and fruits Bhargave M oleifera leaf tea studies demonstrated alterations in blood circulating neutrophils and conclude that Moringa tea has adaptogenic capabilities in cases of stress Drue et a0al Table a0 Previous studies using dried Moringa leaves tea in mouse model with acute lung inflammation showed that mice that had decreased lung inflammation marked by alterations in cytokine production leukocyte migration and neutrophil apoptosis McKnight et a0al An ethanolic extract of Moringa leaves has antianxiety effect in swiss albino mice the ethanolic extract of M oleifera leaves may have produced its anxiolytic effects via multiple mechanisms Bhat SeedsSeeds collected from pods can be eaten raw or cooked From M oleifera seeds a rich vegetable oil can be produced M oleifera seed oil or BehenBen oil is produced through the cold pressing of the M oleifera seeds M oleifera oil can be used to cook as a source to prepare biodiesel as a lubricant and in the cosmetic industry Rashid et a0al The oil name comes from its high content on behenic acid which confers more resistance to oxidative degradation comparing to other vegetable oils Ben oil is rich in oleic acid up to palmitic but also stearic behenic and arachidic Anwar It is used in various cosmetic formulations as emollient and confers nourishing moisturizing antioxidant and protective properties It is also a good skin cleansing product Nadeem and Imran Table a0 details some cosmetic brands that use M oleifera leaves oil or active extracts as ingredients in the composition of their products This oil is also used in the enfleurage process allowing the extraction of fragrances and active compounds from difficult sources as flower petals Milled M oleifera seed shells can be used as a natural exfoliating agentMoringa oleifera seeds can also help diabetic patients Table a0 Some studies by AlMalki and El Rabey showed its antidiabetic activity by reducing the blood glucose level when rats where treated with or a0mg of M oleifera seeds powderkg body weight during a0weeks At the same time ingestion lead to an increase in antioxidant enzymes and consequently the compound content such as glucomoringin phenols and flavonoids Moreover the same authors treating these diabetic rats significantly increased and ameliorated all kidney functions parameters In fact M Oleifera seeds ameliorated liver fibrosis in rats reducing liver damage and symptoms of liver fibrosis decrease the CCl4induced elevation of serum aminotransferase activities and globulin level as well as reduce the elevated hepatic hydroxyproline content and myeloperoxidase activity Hamza improving the indices of hepatoxicity in rats such as malonialdehyde level and total antioxidant capacity glutathione content catalase and superoxide dismutase activities Hamza Treatment with M oleifera seeds also altered oxidative stress in relation to its antiinflammatory activity Histopathological observations showed mild or less infiltration of lymphocytes angiogenesis and synovial lining thickening From all above results and observations it can be concluded that the seeds possess promising antiarthritic property Mahajan et a0al These seeds have others appeals to the daily life and industry Seed powder showed capacity to purify water and remove heavy metals and anic compounds Sharma et a0al through low molecular weight cationic proteins mediated precipitation Kansal and Kumari There was a reduction of “ in the turbidity of the water and “ of bacterial reduction Bhargave Lea The remaining paste after the oil extraction still has the same flocculation properties serving both purposes and adding value Lea Compounds such as pterygospermin moringine and benzyl isothiocya

Thyroid_Cancer

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

Thyroid_Cancer

"Follicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that mostly occurs in systemiclymph nodes FDCS in the uterine cervix has not yet been reportedCase presentation A 49yearold woman was referred to our department with a cervical tumor which washistologically suspected to be undifferentiated carcinoma She underwent hysterectomy salpingooophorectomyand pelvic lymphadenectomy after neoadjuvant chemotherapy with paclitaxel and carboplatin The resectedspecimen contained high numbers of spindle cells and was immunohistochemically confirmed to be FDCS Thetumor was completely resected and recurrence was not detected at a 16month followupConclusion FDCS is an extremely rare malignant tumor in the uterine cervix and an accurate diagnosis andcomplete resection are essential for a good prognosisKeywords Follicular dendritic cell sarcoma Cervical cancerBackgroundFollicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that was initially reported in []Three types of tumors are derived from dendritic cellsFDCS derived from follicular dendritic cells that presentantigens to B lymphocytes in lymph follicles [] interdigitating dendritic cell sarcoma derived from the Tcellzones of lymphoid ans such as the paracortex anddeep cortex of the lymph nodes and fibroblastic reticular cell sarcoma derived from a reticular network oflymphoid ans [] An accurate diagnosis is challenging without an appropriate series ofimmunohistochemistry therefore some tumors may be diagnosed asundifferentiated carcinoma The accuracy of a diagnosisinfluences the prognosis of patients because of the lowresponse rate to established chemotherapy and radiotherapy [“] Correspondence myoshihara1209mednagoyauacjp2Department of Obstetrics and Gynecology Nagoya University GraduateSchool of Medicine Tsurumacho Showaku Nagoya Aichi JapanFull list of author information is available at the end of the Although previous studies described FDCS in the cervical lymph nodes liver stomach and tonsils FDCS inthe uterine cervix has not yet been reported We hereinpresent a 49yearold woman diagnosed with FDCS inthe uterine cervix which was successfully treated bycomplete surgery without postoperative adjuvant therapybased on a precise pathological diagnosis This is thefirst case of FDCS arising from the uterine cervix andwe described the time course of this patient from herinitial admission to diagnosis and treatment We alsoemphasized the importance of considering FDCS as adifferential diagnosis for cervical tumors because an accurate diagnosis and complete resection are essential fora good prognosisCase presentationA 49yearold woman gravida para with abnormalgenital bleeding was referred to our department with acervical mass She had a previous history of uterine myomectomy and cervical polypectomy which were both benign diseases Pelvic magneticimagingrevealed a — mm solid tumor developing from theresonance The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cNakamura BMC Women's Health Page of findingsrevealed aIntraoperativeand carboplatin AUC5 mgbody Massive genitalbleeding occurred months after the admission therefore the patient underwent extended total hysterectomybilateral salpingooophorectomy and pelvic lymphadenectomysmallamount of bloody ascites and the resected specimen hada necrotic mass at the posterior side of the cervix Fig Tumor cells exhibited the same histopathological characteristics as those in the previous biopsy Fig 3a Additionalimmunohistochemistry revealed cells that werepositive for both CD68 and FDC Fig 3b which wasconsistent with the diagnosis of FDCS The tumor wascompletely resected and lymphovascular invasion wasnot detected There was also no evidence of lymph nodemetastasis She did not receive adjuvant therapy and herCA19“ level decreased to within almost normal limitsTable Tumor recurrence was not detected at the month followup after admission The consent of the patient for publication was recorded according to the Ethics Committee of Nagoya University and the principlesof the Declaration of HelsinkiDiscussionThis is the first case of FDCS in the uterine cervix Afterreported surgery and chemotherapy we reached a finaldiagnosis Our experience demonstrates the difficultiesassociated with accurately diagnosing FDCS due to alack of familiarity with the pathologies of rare tumorsincluding sarcoma Although FDCS in the gynecologicalsystem is markedly rarer than other carcinomas it needsto be considered as a differential diagnosis because of itspotentiallyappropriatetreatmentprognosiswithoutfatalFDCS has been reported in approximately casesworldwide with an age range of to years and amalefemale ratio of The primary lesion of FDCS isFig Surgical specimen of the tumor The tumor with a necroticlesion arose from the posterior side of the cervix arrowheadsFig Magnetic resonance imaging of the patient at the initialpresentation A solid tumor developing from the posterior of theuterine cervix was confirmed arrowheadsposterior of the uterine cervix Fig and — and — mm spaceoccupying lesions were also detectedaround the rectus which were suspected to be lymphnode metastases Chestabdominal computed tomography CT showed no enlarged lymph nodes or distantmetastasis except for the tumors already detected Positron emission tomographyCT revealed the accumulation offluorodeoxyglucose maximum standardizeduptake value at the cervical tumor only Serumtumor marker levels were as follows carcinoembryonicantigen ngml cancer antigen125 UmLcancer antigen199 Uml squamous cell carcinomaantigen ngml soluble interleukin2 receptor Uml The other results of the blood examination including renal function liver enzymes and electrolyteswere within normal limitsBiopsy of the cervical tumor was performed and ahistopathological examination indicated that the tumorhad atypically spindle ovoid and polygonal cells with aneosinophilic hyalinerich cytoplasm with whorled andcordlike forms Nuclei were oval to round and had mildatypia Inflammatory cells were scattered to various degrees around tumor cells The results of immunohistochemical staining were as follows cytokeratin AE1AE3positive epithelial membrane antigen EMA negativeCAM52 negative cytokeratinMNF116 positive S100negative vimentin positive actin negative desminnegative cluster of differentiation CD negative CD8negative CD10 negative CD5 negative CD20 negativeCD79a negative PgR PgR inhibin negative thyroid transcription factor1 negative EpsteinBarr virusencoded RNA in situ hybridization negativeSince the tumor was suspected to be undifferentiatedcarcinoma of the uterine cervix the patient receivedchemotherapy with four cycles of paclitaxel mgm2 0cNakamura BMC Women's Health Page of Fig The histopathology of the cervical tumor The tumor had atypically spindle ovoid and polygonal cells with an eosinophilic hyalinerichcytoplasm with whorled and cordlike forms Nuclei were oval to round and had mild atypia a Tumor cells were positive for FDC bmostly in the lymph nodes in the neck axilla and mediastinum The remainder of FDCS originate from extralymphatic lesions such as the liver mesenterium stomach smallintestine pharynx tonsils retroperitoneumand ovary [“] however FDCS in the uterine cervixhas not yet been reported in the English literatureDue to the wide range of primary lesions there is noknown specific initial symptom of FDCS some patientsmay exhibit symptoms associated with an increase intumor volume such as lymph node swelling [] Sincethere are no specific findings in blood examinations orimaging studies difficulties are associated with makingan accurate diagnosis prior to the initiation of treatmentThe diagnosis of FDCS is pathologically confirmed usingbiopsy or resected specimens however due to the rarityof this tumor it is important to conduct appropriate immunohistochemistry in consideration of FDCS from thefindings of hematoxylin and eosin staining The tumorcells of FDCS are spindle to epithelioid in shape with thecoexistence of multinucleated cells Single nuclear inflammatory cells infiltrate and form bundles flowers andswirls structures Some giant cells and ReedSternbergcells are also detected in these tumors To reach a definitive diagnosis of FDCS FDC markers such as CD21CD35 KiM4p and CNA42 are used to distinguish itfrom other tumors that may have a mesenchymal structure such as undifferentiated carcinoma meningiomaand paraganglioma [ ] Alternatively FDCS may befound within lesions of Castleman™s disease particularlyits hyalinevascular type [] It has been reported thatFDCS occurred after the excision of a lesion of hyalinevascular type Castleman™s disease and occupied most ofthe lesions It is also important to distinguish FDCSfrom inflammatory pseudotumorlike FDCS associatedwith EpsteinBarr virus This tumor is often found in theabdominal ans particularly in the liver and spleenand has the characteristics of positive FDCS markersand the detection of EBV by in situ hybridization It progresses more slowly than FDCS and longterm survivalhas been reported even after recurrence []The treatment for cervical cancer principally involvessurgeryradiation and chemotherapy New surgicaltechniques such as laparoscopic radical hysterectomyand sentinel lymph node biopsy have also become available [ ] On the other hand a basic therapeuticstrategy for FDCS is prioritized to guarantee completesurgical tumor resection because of the low responserate to established chemotherapy and radiotherapy [“] Saygin examined patients with FDCS andreported 2year survival rates of and forearlylocally advanced and distant metastasis diseaserespectively Patients who underwent complete tumorresection had a better prognosis than those with unresectable localized tumors Furthermore no prognosticdifference was observed between the surgery group andpostoperative radiotherapy groups A large tumor sizelarger than cm and lymphoplasmacytic cell invasionhave been identified as poor prognostic factors [] InTable Level of tumor markers in each followup periodTumor marker Normal rangeCA125 UmL AdmissionCA199 UmL CEA UmL SCC ngmL At surgery m after admissionAt final followup m after admissionsIL2R UmL Abbreviation CA cancer antigen CEA carcinoembryonic antigen SCC squamous cell carcinoma antigen sIL2R soluble interleukin2 receptor 0cNakamura BMC Women's Health Page of the present case as the tumor remained localized andwas completely resected the patient could follow favorable clinical course without any tumor recurrenceConclusionFDC sarcoma is a rare tumor that may develop in theuterine cervix FDCS needs to be considered when confirming a mesenchymal cervical tumor and appropriateimmunohistochemistry needs to be performed for bothan accurate diagnosis and selection of a therapeuticstrategyAbbreviationsFDC Follicular dendritic cell sarcoma FDCS Follicular dendritic cellCT Computed tomography CD Cluster of differentiationAcknowledgmentsThe authors thank Dr Sakata and Dr Hirata for supporting the managementof the case with patience and knowledgeAuthors™ contributionsTN and MY developed the study concept interpreted the data and wrotethe manuscript ST and HK participated in designing the study and plannedthe investigations FK drafted the study design and supervised all of thework All authors have approved the final manuscriptShia J Chen W Tang LH Carlson DL Qin J Guillem JG Extranodalfollicular dendritic cell sarcoma clinical pathologic and histogeneticcharacteristics of an underrecognized disease entity Virchows Arch “Ruco LP Gearing AJ Pigott R Pomponi D Burgio VL Cafolla A et alExpression of ICAM1 VCAM1 and ELAM1 in angiofollicular lymph nodehyperplasia Castleman's disease evidence for dysplasia of folliculardendritic reticulum cells Histopathology “ Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemicalrecognition of human follicular dendritic cells FDCs in routinely processedparaffin sections J Histochem Cytochem “Arber D Kamel O van de Rijn M Davis RE Medeiros LJ Jaffe ES et alFrequent presence of the EpsteinBarr virus in inflammatory PseudotumorHum Pathol “ Casarin J Bogani G Papadia A Ditto A Pinelli C Garzon S et alPreoperative Conization and risk of recurrence in patients undergoinglaparoscopic radical hysterectomy for early stage cervical cancer amulticenter study J Minim Invasive Gynecol “ Rossetti D Vitale SG Tropea A Biondi A Lagan  AS New procedures for theidentification of sentinel lymph node shaping the horizon of futureManagement in Early Stage Uterine Cervical Cancer Updat Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNo funding was obtained for this studyAvailability of data and materialsNot applicableEthics approval and consent to participateNot requiredConsent for publicationWritten consent to publish this information was obtained from studyparticipantsCompeting interestsAll authors declare that there are no competing interestsAuthor details1Department of Obstetrics and Gynecology Anjo Kosei Hospital Anjo AichiJapan 2Department of Obstetrics and Gynecology Nagoya UniversityGraduate School of Medicine Tsurumacho Showaku Nagoya AichiJapanReceived April Accepted August References Monda L Warnke R Rosai J A primary lymph node malignancy withfeatures suggestive of dendritic reticulum cell differentiation A report of cases Am J Pathol “Tew JG Kosco MH Burton GF Szakal AK Follicular dendritic cells asaccessory cells Immunol Rev “Saygin C Uzunaslan D Ozguroglu M Senocak M Tuzuner N Dendritic cellsarcoma a pooled analysis including cases with presentation of ourcase series Crit Rev Oncol Hematol “Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Tumoursof histiocytes and accessory dendritic cells an immunohistochemicalapproach to classification from the international lymphoma study groupbased on cases Histopathology “Chan JK Fletcher CD Nayler SJ Cooper K Follicular dendritic cell sarcomaClinicopathologic analysis of cases suggesting a malignant potentialhigher than currently recognized Cancer “ 0c"

Thyroid_Cancer

evaluation of biochemical and hematological parameters in adults with Down syndromeDavid de Gonzalo‘calvo123 Isabel Barroeta45 Madalina Nicoleta Nan6 Jos Rives6 Diana Garzn45 Mar­a Carmona‘Iragui457 Bessy Benejam457 Laura Videla457 Susana fern¡ndez7 Miren Altuna45 S­lvia Valldeneu45 Rafael Blesa45 Alberto Lle45 Francisco Blanco‘Vaca689 Juan Fortea457 Mireia Tondo6Down syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting live‘born infants In addition to intellectual disability individuals with DS have other comorbidities and complex medical conditions The increase in the life expectancy of patients with DS requires expanding the knowledge about their clinical characteristics and related laboratory parameters Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism Therefore our main goal was to describe the biochemical and hematological findings in a DS cohort and to compare the values to those of a control population A total of DS individuals and control subjects were enrolled DS individuals had a higher frequency of several clinical conditions compared to control individuals and presented with significant differences with respect to the controls in both biochemical and hematological parameters We found age‘ and sex‘related differences in several of the parameters A good understanding of the differences in our cohort might be of aid in the clinical follow‘up of adults with DS especially considering that the lifespan of DS individuals may reach years of age in developed countriesAbbreviationsAD AF ALT AST B12 CKDEPI DS ESR FT4 eGFR GGT HbA1c Alzheimer™s disease Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Vitamin B12 Chronic kidney disease epidemiology collaboration Down syndrome Erythrocyte sedimentation rate Free thyroxine Estimated glomerular filtration rate Gammaglutamyl transferase Glycated hemoglobin1Biomedical Research Institute Sant Pau IIB Sant Pau Barcelona Spain 2Institute of Biomedical Research of Barcelona IIBB Spanish National Research Council CSIC Barcelona Spain 3Translational Research in Respiratory Medicine University Hospital Arnau de Vilanova and Santa Maria IRBLleida Lleida Spain 4Sant Pau Memory Unit Department of Neurology Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau Universitat Aut²noma de Barcelona Barcelona Spain 5Center of Biomedical Investigation Network for Neurodegenerative Diseases CIBERNED Madrid Spain 6Department of Biochemistry Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau CSant Quint­ Barcelona Spain 7Barcelona Down Medical Center Fundaci Catalana de S­ndrome de Down Barcelona Spain 8Center of Biomedical Investigation Network for Diabetes and Metabolic Diseases CIBERDEM Madrid Spain 9Department of Biochemistry and Molecular Biology Universitat Aut²noma de Barcelona Barcelona Spain email mtondosantpaucatScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cHDLc LDLc MCH MCHC MCV MDRD4 MPV K RDW Na TG TSH Highdensity lipoprotein cholesterol Lowdensity lipoprotein cholesterol Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Modification of diet in renal disease Mean platelet volume Potassium Red blood cell distribution width Sodium Triglycerides Thyroid stimulating hormoneDown syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants with an estimated birth prevalence of per live births1“ Despite the shorter life expectancy when compared to healthy subjects and adults with other causes of intellectual disability4 there has been a progressive increase in the life expectancy of patients with DS in recent decades currently reaching nearly years5 This fact has increased the need to expand the knowledge about the clinical characteristics of DS individuals and the health problems differentiating them from both pediatric and adult populations6 DS is associated with a distinct phenotype involving many body systems In addition to intellectual disability individuals with DS present with a high number of comorbidities and complex medical conditions whose frequencies are modified throughout the lifespan of the individuals7 The increase in life expectancy has led to a higher prevalence of agerelated pathologies including premature Alzheimer™s disease AD8Since optimal medical management is associated with improved quality of life and functioning among persons with DS910 medical professionals including pediatricians and other physicians should closely supervise this population throughout their lifespan and evaluate their laboratory results Previous investigations in DS cohorts have focused on select biochemical parameters such as uric acid and thyroid function biomarkers bone mineral density nutritional zinc status gonadal and endocrine function and glucose and lipid metabolism parameters11“ However no previous work has described a comprehensive panel of biochemical and hematological parameters in a large cohort of DS patientsOur hypothesis is that a thorough analysis of the biochemical and hematological parameters will provide a basis to establish whether commonly observed alterations in DS individuals are intrinsic of the disease or have clinical implications similarly as for the general population Therefore our goals were to describe the biochemical and hematological findings in our DS cohort and to compare the values to those of a control populationMaterial and methodsStudy participants This was a singlecenter descriptive study of adults with DS recruited at Barcelona Down Medical Center Fundaci Catalana S­ndrome de Down and Hospital de la Santa Creu i Sant Pau Barcelona in Catalonia Spain according to a populationbased health plan to screen for neurological comorbidities1718 The Down Medical Center provides medical care specifically for individuals with DS and possesses over medical records more than of the estimated Down syndrome population in Catalonia therefore it reflects the population with DS in our geographic area The period of patient recruitment for this study was February to June In adults with DS ‰¥ a0years a biochemical and hematological analysis was performed as part of their annual health plan visit A total of patients were enrolled in the study Six further patients were ultimately excluded for presenting with conditions unrelated to DS according to their medical records patients with hepatitis C patient with hepatitis B and patient with breast cancer resulting in a final total number of DS individuals included age range “ a0years A total of healthy control participants in the same age range “ a0years were enrolled in the study Volunteers were recruited from the SPIN Sant Pau Initiative on Neurodegeneration cohort santp aumem oryun itcomourresea rchspincohor t or social media SantPauMemory Further details on the clinical protocol of the SPIN cohort can be found elsewhere19Based on current guidelines1720 associated clinical conditions were obtained through a systematic review of the medical records including the following history of arterial hypertension dyslipidemia diabetes mellitus congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology psychiatric pathology epilepsy and Alzheimer™s disease Treatment data with a special focus on the treatment of hypothyroidism were also collectedBiochemical and hematological data Analyzed biochemical and hematological parameters were selected according to a defined laboratory blood profile as recommended in the guidelines for management of patients with DS1720Blood collection and processing were performed in accordance with the Standard Operating Procedures for Serum and Plasma Collection from the Early Detection Research Network EDRN Consensus Statement and Standard Operating Procedure Integration Working Group21 Blood samples were collected by venipuncture after an overnight fastWhole blood samples were collected in VACUTAINER tubes and fractionated by centrifugation at a0g for a0min at room temperature to obtain serum Serum was aliquoted into a0mL tubes and the following parameters were measured according to standard commercially available assays adapted to an Architect C4000 Abbott Diagnostics USA using automated procedures thyroid stimulating hormone TSH free thyroxine FT4 Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cAge yearsMalefemaleArterial hypertensionDyslipidemiaDiabetes mellitusCongenital heart diseaseGastrointestinal pathologyDermatological pathologyBone pathologyHypothyroidismTreatment for hypothyroidismHearing problemsOtolaryngology pathologyOphtalmological pathologyPsychiatric pathologyEpilepsyAlzheimer™s disease “ Controln Median P25“P75n “ Down syndromenMedian P25P754n “ pvalue Table Characteristics of the Study Population Data are presented as frequencies percentages for categorical variables Continuous variables are presented as median interquartile range Differences between groups were analyzed using Wilcoxon ranksum test or Fisher™s exact testsodium Na potassium K glucose urea creatinine total bilirubin triglycerides TG total cholesterol aspartate aminotransferase AST alanine aminotransferase ALT alkaline phosphatase AF gammaglutamyl transferase GGT total proteins vitamin B12 and folate The estimated glomerular filtration rate eGFR was calculated according to the MDRD4 Modification of Diet in Renal Disease and CKDEPI Chronic Kidney Disease Epidemiology Collaboration formulasWhole blood samples in EDTAK3 were also obtained for determining blood cell count and indices The tubes were immediately inverted times to mix the anticoagulant additive with blood The blood was processed within a0h of extraction Using the impedance channel of the automated hematology analyzer Sysmex XE2100 Roche Diagnostics Kobe Japan the following parameters were determined red blood cell count RBC white blood cell count WBC platelet count hemoglobin hematocrit mean volume MCV mean corpuscular hemoglobin concentration MCHC mean corpuscular hemoglobin MCH red blood cell distribution width RDW and mean platelet volume MPV The erythrocyte sedimentation rate ESR was calculated with a VES cube Sysmex Analyzer Roche Diagnostics Kobe JapanValues were compared to normal reference ranges used in our laboratory established in a healthy population from our geographical area according to standardized guides22Statistical analysis Descriptive statistics were used to summarize the characteristics of the study population Data are presented as medians [25th percentile P25“75th percentile P75] for continuous variables and as frequencies percentages for categorical variables Data normality was analyzed using the Kolmogorov“Smirnov test Continuous variables were compared between groups using the Wilcoxon ranksum test ANCOVA models adjusted for age and sex were used to compare continuous variables across the study groups Variables were logtransformed to achieve a normal distribution For clarity the original values are shown Categorical variables were compared between groups using Fisher™s exact test Spearman™s rho coefficient was used to assess the correlation between continuous variables The statistical software package R wwwrproje ct was used for statistical analyses A Pvalue was considered statistically significantEthical aspects The study was approved by the Sant Pau Ethics Committee following the standards for medical research in humans recommended by the Declaration of Helsinki and in accordance with Spanish legislation for research in people with intellectual disabilities All participants or their legally authorized representatives gave written informed consent before enrolment in accordance with the guidelines of the local ethics committeeResultsStudy cohort characteristics We enrolled a total of individuals with DS males and females with a median age of “ years and control subjects males and females with a median age of “ years The clinical features of the DS and control populations are listed in Table a0 The frequency of the following clinical conditions was significantly higher in the DS group than in the control group history of congenital heart disease gastrointestinal pathology dermatologiScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0ccal pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology epilepsy and AD No differences were observed in the frequency of diabetes mellitus or psychiatric pathology for either group DS individuals presented with a lower frequency of arterial hypertension and dyslipidemia compared to the control group See Table a0 for further details on the cohort characteristicsBiochemical and hematological parameters in patients with Down syndrome We performed a detailed biochemical and hematological analysis of the DS cohort and compared the profiles obtained with our control population The reference values of the studied parameters the number and percentage of patients out of range and the median P25“P75 of the whole study population are shown in Table a0 Seventythree percent of the studied hematological parameters and of the studied biochemical parameters were significantly different between the DS individuals and the control population The DS individuals presented with higher TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW values and lower TG total cholesterol folate eGFR MPV and WBC values These differences remained significant or close to signification after adjusting for confounding factors such as age and sex Statistical differences for RBC and MCHC were observed after adjustment An additional analysis to evaluate the impact of hypothyroidism treatment on TSH was performedNo differences were observed for TSH between both studied groups treated DS individuals “ vs untreated DS individuals “ Pvalue For categorical variables the percentage of DS individuals out of range for some parameters was also statistically significant compared to the control population Parameters with a higher percentage of values out of range in the DS group were TSH urea creatinine total proteins RBC MCV ESR MCH and WBC whereas those with a lower percentage of values out of range were K TG total cholesterol and ASTThe differences in the biochemical and hematological parameters and the number and percentage of patients out of range between DS individuals and the control population according to sex are displayed in Supplemental Tables a0 and For the female DS cohort parameters with significantly higher values were TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol GGT eGFR RBC MPV and WBC For categorical variables parameters with significantly higher percentages of values out of range were TSH creatinine total proteins MCV ESR MCH and WBC whereas those with a significantly lower percentage of values out of range were total cholesterol and B12 Supplemental Table a0 For the male DS cohort parameters with significantly higher values were TSH hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol eGFR MPV and WBC Regarding categorical variables parameters with significantly higher percentages of values out of range were TSH ESR and MCH whereas those with significantly lower percentages of values out of range were K TG total cholesterol and GGT Supplemental Table a0The differences in the biochemical and hematological parameters between males and females as well as the frequency and percentage of patients out of range in the control and DS groups are displayed in Supplemental Table a0 and Table a0 respectively For the control group parameters with significantly higher values in the male subgroup were K creatinine TG ALT hemoglobin hematocrit RBC and MCHC whereas those with significantly lower values were AF eGFR and ESR Among the categorical variables K had a significantly higher percentage of values out of range in the male subgroup and ESR had a significantly lower percentage of values out of range Supplemental Table a0 For the DS cohort parameters with significantly higher values in the male subgroup were creatinine total bilirubin TG ALT GGT hemoglobin hematocrit RBC MCHC and WBC whereas those with significantly lower values were folate MCV ESR RDW platelet count and MPV Regarding categorical variables parameters with significantly higher percentages of values out of range in the male subgroup were total bilirubin B12 RBC and MPV whereas those with significantly lower percentages of values out of range were MCV ESR and MCHC Table a0The correlation between the biochemical and hematological data with age was also explored in both study groups As shown in Table a0 for the control population urea creatinine total cholesterol and AST showed a significant positive correlation with age while eGFR showed a significant negative correlation For the DS population Na urea creatinine TG total cholesterol AST AF MCV ESR MCH and RDW showed a significant positive correlation with age while eGFR ALT B12 hemoglobin hematocrit RBC MCHC and platelet count showed a significant negative correlationDiscussionThe present study evaluated several biochemical and hematological parameters in a large sample of adults with DS Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism11“ DS is among the most complex genetic conditions compatible with life characterized by accelerated aging and affecting gene expression beyond chromosome The sheer number of affected genes and epigenetic changes suggests that numerous pathways of human metabolism are altered and subsequently might be reflected in laboratory test parameters Here we performed a comprehensive approach by analyzing parameters related to different physiological mechanisms We found significant differences with respect to nontrisomic controls in both biochemical and hematological parameters even after adjusting for potential confounding factors Furthermore we found age and sexrelated differences in several of the parameters The fact that women with DS experience menopause earlier than healthy women24 may explain some of these sexrelated differencesClinically and as previously described48925 our DS cohort presented with a higher incidence of congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism otolaryngology pathology ophthalmological pathology epilepsy and AD than the control population Arterial Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT UL““““ ‰¤ a0years “ a0years “Females Males Females Males Females Males Females “Males “Females Males “““Total proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109LFemales “Males “Females “Males “Females “Males “““““““““nn OOR “ “ “ “ “ “ “ “ “ “n OOR “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ControlDown syndromeReference valuesMedian P25“P75nMedian P25“P75pvalue categoricalpvalue continuouspvalue continuous adjusted Table Biochemical and hematological parameters in the control group and the cohort of patients with Down Syndrome Differences between groups were analyzed using Wilcoxon Ranksum test ANCOVA models adjusted for age and sex or the Fisher™s exact test OOR out of range NA not applicablehypertension and dyslipidemia were less prevalent whereas no difference was observed regarding the diabetes mellitus incidence as discussed belowWith respect to laboratory studies the hematological profile was largely altered in DS individuals when compared to the control population Of note significant differences were found for almost all the hematological parameters when comparing males and females suggesting the need to consider sex when evaluating the hematological profile in a DS individual It is well known that trisomy impacts hematopoietic cell biology through multiple and complex pathways In adults the metabolic and redox derangements observed in the RBCs from individuals with DS have been previously linked to alterations in cell survival and size in particular macrocytosis26 Different studies have also proposed that the additional copy of chromosome has a profound impact on fetal hematopoiesis which ultimately impacts the function and number of hematopoietic lineages27“ Additionally between and of newborn infants with DS develop transient myeloproliferative disorder32“ Although the disease usually resolves without treatment in the first few months of life it is estimated that “ of individuals with transient myeloproliferative disorder will go on to develop subsequent leukemia3536 Finally the fact that folate concentrations are significantly lower in DS individuals matches the observed hematological alterations Taken together these impaired hematological parameters suggest the existence of abnormalities Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cFemalenVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109L n OOR Median P25“P75n OOR Median P25“P75pvalue categoricalpvalue continuousMalen “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Table Differences between sex in the Down syndrome group Differences between groups were analyzed using Wilcoxon Ranksum test or the Fisher™s exact test OOR out of range NA not applicablein hematopoiesis and provide information on how an extra copy of chromosome may lead to phenotypic consequencesConcerning the biochemical profile our results support the findings from previous independent studies We showed that of our DS individuals presented with values out of range for TSH level Of those out of were treated for hypothyroidism Impaired TSH and FT4 levels have been largely described in DS populations37 Moreover subclinical hypothyroidism in children with DS is an abundantly common occurrence with a prevalence of approximately and has been attributed to the dysregulation of the hypothalamicpituitarythyroid axis37 Regarding urea metabolism of our DS individuals presented with a high urea concentration which may be due to impaired renal function among other causes Indeed and as previously reported39 almost of our DS individuals also presented with impaired creatinine values Serum creatinine is the most reliable parameter for detecting kidney damage due to its high diagnostic specificity From its concentration and based on formulas in which age sex and weight are taken into account it is possible to estimate the glomerular filtration rate eGFR Our DS cohort also presented with a lower eGFR which is in agreement with a previous study exploring renal disease in DS individuals40 Despite the significantly altered parameters related to renal function our DS individuals presented with a very low frequency of arterial hypertensionConcerning the lipid profile we found significantly lower total cholesterol and TG concentrations in DS individuals compared to the control population It would have been interesting to study the fractioned forms of cholesterol together with their apolipoprotein concentrations however because the current study was not designed to answer questions regarding lipid metabolism lowdensity lipoprotein cholesterol LDLc and highdensity lipoprotein cholesterol HDLc were not measured Several works measuring circulating total cholesterol Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cControlnSpearman™s rhoDown syndromenSpearman™s rhopvaluepvalueBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal Proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC — 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count — 109LMPV fLWBC — 109Lˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ Table Correlations between biochemical and hematological parameters and age NA not applicableLDLc HDLc and TG concentrations in the DS population exist However they report contradictory results and prevent firm conclusions from being drawn Some studies have reported an unfavorable41“ or favorable lipid profile46 However most of the studies reported no change in serum TC LDLc or HDLc in individuals with DS compared to a control group or to population norms414547“ In our study these lower total cholesterol and TG concentrations may have translated into a significantly lower prevalence of hyperlipidemia in DS individuals It has been described that DS individuals may be protected against atherosclerosis4752“ leading to a low incidence of cardiovascular events53 However a work carried out with individuals with DS found that they were at high risk of cerebrovascular events but a lower risk of coronary events in males55 Therefore risk of major cerebrovascular events in people with DS should not be ruled out Concerning diabetes mellitus a similar incidence of type diabetes mellitus50 and a higher incidence of type diabetes mellitus has been described for individuals with DS56 We found no difference in type diabetes mellitus frequency among our DS and control populations as previously described in a different study16 In regard to arterial hypertension prevalence our results are in line with numerous studies that have described a lower incidence of this condition in DS individuals50515758 Despite these observations cholesterol fractioned forms and glycated hemoglobin HbA1c concentrations were not measured making it difficult to draw conclusions regarding dyslipidemia and diabetes mellitus in our cohort Yet an increased degree of hypolipidemia should not be ruled out Overall future studies elucidating the mechanisms behind the low cholesterol and TG concentrations and lower prevalence of arterial hypertension observed in our DS cohort should be performedIt is important to emphasize that our main goal was to help determining if the observed biochemical and hematological alterations have direct clinical implications for DS individuals While the altered biochemical and hematological profiles may be developmental features ie a consequence of the specific genetic characteristics of individuals with DS or the result of accelerated aging it should be recalled that they may also be reflecting comorbidities or the use of medication From a clinical standpoint to elucidate if the observed differences are consequence of concomitant conditions or features of the syndrome itself could be of help in the management of DS individuals Unfortunately due to the design of our study these questions remain unanswered Future Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cstudies focusing on specific areas of metabolism of DS individuals with different comorbidities could shed some light on this matterOur study has several strengths We collected relevant clinical biochemical and hematological data in a large DS cohort and performed a systematic analysis The fact that our controls were chosen from a healthy background broadens the actual differences and strengthens the present results Ultimately according to the wide inclusion criteria and the broad range of represented ages we believe that the results from our study may help clinicians when interpreting laboratory analyses in DS individuals Some limitations should also be taken into account The control and DS populations were not strictly age and sex matched and the control group had a reduced number of males when compared to females Nonetheless both populations were within the same age range and additional analysis including adjustment for age and sex were performed Furthermore despite our large cohort of DS individuals the number was still not sufficient to perform statistical analysis stratification according to the observed clinical conditions Moreover and as stated previously some of the observed biochemical andor hematological alterations may have been a consequence of the use of drugs for the treatment of other comorbidities Finally our defined clinical biochemical and hematological profiles were somehow general and unable to cover all the possible comorbidities present in DS individualsIn conclusion adults with DS show a specific profile of biochemical and hematological parameters A good understanding of the differences in our cohort with those in the general population might aid in the clinical followup of adults with DS especially considering that the life span of DS individuals can now reach a0years of age in developed countriesReceived March Accepted July References Parker S E et al Updated national birth prevalence estimates for selected birth defects in the United States “ Birth Canfield M A et al National estimates and raceethnicspecific variation of selected birth defects in the United States “ Defects Res A Clin Mol Teratol “ Birth Defects Res A Clin Mol Teratol “ Besser L M Shin M Kucik J E Correa A Prevalence of down syndrome among children and adolescents in metropolitan Atlanta Birth Defects Res A Clin Mol Teratol “ Yang Q Rasmussen S A Friedman J M Mortality associated with Down™s syndrome in the USA from to A populationbased study Lancet “ Bittles A H Glasson E J Clinical social and ethical implications of changing life expectancy in Down syndrome Dev Med Child Neurol “ Morris J K Alberman E Trends in Down™s syndrome live births and antenatal diagnoses in England and Wales from to Analysis of data from the National Down Syndrome Cytogenetic Register BMJ b3794 Startin C M et al Health comorbidities and cognitive abilities across the lifespan in Down syndrome J Neurodev Disord “ Hithersay R et al Association of dementia with mortality among adults with Down syndrome older than years JAMA Neurol Bull M J Health supervision for children with Down syndrome Pediatrics “ Roizen N J Patterson D Down™s syndrome Lancet “ Hawli Y Nasrallah M ElHajj Fuleihan G Endocrine and musculoskeletal abnormalities in patients with Down syndrome Nat Rev Endocrinol “ Sakadamis A Angelopoulou N Matziari C Papameletiou V Souftas V Bone mass gonadal function and biochemical assessment in young men with trisomy Eur J Obstet Gynecol Reprod Biol “ Niegawa T et al Evaluation of uric acid levels thyroid function and anthropometric parameters in Japanese children with Down syndrome J Clin Biochem Nutr “ Costa R et al Bone mineral density distribution curves in Spanish adults with Down syndrome J Clin

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

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

"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935

Thyroid_Cancer

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

Thyroid_Cancer

EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

"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

High burden of depression among cancerpatients on chemotherapy in University ofGondar comprehensive hospital and FelegeHiwot referral hospital Northwest EthiopiaAdhanom Gebreegziabher BarakiID1 Getahun Mengistu Tessema2 EyayawAdisu Demeke3 Department of Epidemiology and Biostatistics College of Medicine and Health Sciences Institute of PublicHealth University of Gondar Gondar Ethiopia Department of Internal Medicine College of Medicine andHealth Sciences School of Medicine University of Gondar Gondar Ethiopia Department ofPhysiotherapy Bahirdar University Bahirdar Ethiopia adsh04gmailcomAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Baraki AG Tessema GM Demeke EA High burden of depression among cancerpatients on chemotherapy in University of Gondarcomprehensive hospital and Felege Hiwot referralhospital Northwest Ethiopia e0237837 101371journalpone0237837Editor Nu¨lu¨fer Erbil Ordu University TURKEYReceived September Accepted August Published August Copyright Baraki 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 containspotentially identifying characteristics as well assensitive patient information eg HIV statusTherefore please send all data requests to theDirector of School of Medicine Dr MezgebuSilamsew at msilamsawgmailcom orPostgraduate committee Mr Getasew Amare atgetasewa23gmailcomFunding The authors received no specificfunding for this workIntroductionCancer the most stressful event a person may experience often triggers depressionDepression among these groups of people in turn affects chemotherapy adherence lengthof hospitalization quality of life and cancer treatment outcome Even though the problem isenormous studies that address it are limited Therefore this study was conducted to determine the prevalence of depression and associated factors among cancer patients on chemotherapy in FelegeHiwot referral hospital and University of Gondar referral hospitalNorthwest EthiopiaMethodsAn institutionbased crosssectional study was conducted from April to May A total of cancer patients on chemotherapy were included Depression was assessed using thepatient health questionnaire PHQ9 Binary logistic regression was used to select variablesand determine Crude Odds Ratio COR Variables with P value were entered into multivariable logistic regression Adjusted Odds Ratio AOR with confidence intervals forvariables with Pvalue was estimated to show factors affecting depression amongcancer patients The fitness of the model was checked by using the HosmerLemeshowgoodnessoffit testResultsThe prevalence of depression among cancer patients on chemotherapy was CI Educational status of college and above AOR CI Jobless AOR CI UnderweightAOR CI PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaCompeting interests The authors have declaredthat no competing interests existAbbreviations AOR Adjusted Odds Ratio CICrude Odds Ratio COR Crude Odds Ratio PHQPatient Health Questionnaire SD StandardDeviation UoGCSH University of GondarComprehensive Specialized Hospitalchemotherapy duration � months or more AOR CI were notablyassociated with depressionConclusionThe burden of depression among cancer patients in this study was high We recommendconcerned bodies working to curve the problem to intervene based on the identified risk factors Improving educational status reducing work stress and maintaining normal weightwould reduce depressionIntroductionThe global burden of cancer has risen to million new cases and million deaths in Worldwide the total number of people who are alive within years of a cancer diagnosis isestimated to be million []Depression is a common mental disorder characterized by persistent sadness and a loss ofinterest in activities that one normally enjoys accompanied by an inability to carry out dailyactivities for at least two weeks More than million people are now living with depressionan increase of more than between and [] The national prevalence of depression among the general population in Ethiopia was []Cancer the most stressful event that a person may experience often triggers depression [] The prevalence of depression among cancer exceeds that observed in the general population [] and it ranges from to [ “] Depression among cancer patientsaffects treatment since they have to take medications for both cancer and depression [] affectacceptance of adjuvant cancer treatment [] adherence [] extend hospitalization reducesthe quality of life [ ] and increases the risk of suicide [] Depression also predicts cancerprogression and mortality [ ]Several factors affect depression among cancer patients these include age sex marital status educational status occupation pain type of cancer phase of treatment [ ] andsocial support [ ]Even though routine screening of distress is recommended internationally for good cancercare [] less emphasis is given in the study area and most of the care focuses on cancer Studies on the magnitude and the contributing factors are also limited Therefore this study wasconducted to fill this information gap by determining the prevalence of depression among cancer patients and factors affecting itMethodsStudy design and periodAn institutionbased crosssectional study was conducted among cancer patients from Aprilto May Study areaThis study was conducted on cancer patients who are getting treatment and have followed upat the oncology unit of the University of Gondar comprehensive specialized hospitalUoGCSH and FelegeHiwot referral hospital FHRH The two hospitals are found in theAmhara region northwest Ethiopia km and km away from the capital Addis AbabaPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiarespectively The oncology unit of UoGCSH currently has beds for the management of cancer patients whereas the oncology unit of FHRH has currently beds for inpatient treatmentof cancer patientsParticipantsThe source populations were all adult cancer patients visiting the oncology unit and treatedwith chemotherapy in these hospitals All adults with any type of cancer patients under chemotherapy treatment and follow up during the study period were included in the studySample size and sampling procedureA final sample size of was found by using single population proportion formula with population correction for total cancer patients of in the two hospitals using the followingassumptions the prevalence of depression Za2 for confidence interval andmargin of error of The final sample size was proportionally allocated to the two hospitals for UoGCSHand 176FHRH A systematic random sampling method was employed to select every 3rdpatients who were coming to the oncology unit during the data collection period and full fillthe inclusion criteria We had a plan to randomly select and replace the nonresponders butno study participant refused to participateVariablesThe dependent variable of depression was measured using the widely used Patient HealthQuestionnaire PHQ9 The Amharic Local language version of the scale has been validatedin Ethiopia sensitivity and specificity [] We have used a cutoff point of toclassify patients as having depression or notIndependent variables like Age sex marital status average monthly income educationallevel smoking habit alcoholic habit physical activity were collected by intervieweradministered questionnaire whereas variables like Body Mass Index Type of cancer clinicalstagetype of chemotherapy Duration of chemotherapy and comorbidities like Hypertension DMHIV and Anemia were collected from patient charts The smoking and Alcohol use habitswere assessed by asking the patients if they ever smoke cigarette or drink alcohol and socialsupport was evaluated using the Oslo item social support scale with scores ranging from to poor “ moderate “ and strong “ []Data collection procedure and quality assuranceThe data was collected by interviewing the participants using a structured pretested questionnaire and chart review The data was collected by three nurses working in each oncology unitData collectors were trained for one day about the objective of the study and ethical considerations Data collectors were supervised by the principal investigator Data was reviewed andchecked for completeness accuracy and consistency after each day of data collectionData processing and analysisData were entered into Epiinfo version and STATA version was used for analysis Frequencies and percentages were computed for all variables Data were presented in tables andgraphs Binary logistic regression was used to select variables and to determine Crude OddsRatio COR Variables with P value were entered into multivariable logistic regressionAdjusted Odds Ratio AOR with confidence intervals for variables with Pvalue PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiawas estimated to show factors affecting depression among cancer patients The fitness of themodel was checked by using the HosmerLemeshow goodnessoffit testEthics statementEthical approval to conduct the study was received from the University of Gondar ethicalreview board School of Medicine Reference number SOM12372019 A permission letterwas received from the two hospitals To keep the privacy of participants™ name and other personal identifiers were not collected Consent to participate in the study was also orally takenfrom patients Patients with depression were also linked to the psychiatry clinicResultsSociodemographic characteristic of study participantsA total of patients participated in the study From the total respondents the majority ofthem were females were married and were housewivesRegarding to age distribution the mean and standard deviation of participant™s age was SD Table Behavioral and comorbidity characteristicsFour participants were declared that they were smoking cigarettes daily and were alcohol consumers Most of the participants were physically active Most ofthe participants were in normal weight category based on their BMI whereasTable Baseline characteristics of cancer patients in UoGCSH and FHRH FrequencyPercentageVariablesSexMarital statusMaleFemaleSingleMarriedDivorcedWidowedEducational levelOccupationNo educationPrimary educationSecondary educationCollege and aboveGovernment employeeMonthly Income ETBFarmerMerchantUnemployed ““ 101371journalpone0237837t001PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Behavioral factors and comorbidities among study participant at UoGCSH and FHRH VariablesSmoking statusNoYesAlcohol drinkingNoYesPhysical activityNoYesBody mass index “ComorbidityNoYesDiabetesNoYesHypertensionNoYesAnemiaNoYesHIVAIDSNoYesFrequencyPercentage101371journalpone0237837t002 and were underweight and overweight respectively Ninetytwo participants had additional comorbidity Table Type of cancer and treatmentrelated characteristicsBreast cancer was the commonest cancer Whereas cervical cancer colorectal cancer35 and lung cancer are ranked second to fourth Most of thepatients were diagnosed with the disease in the past six months prior to the studyRegarding the clinical stage of the disease the third stage accounts for patients Atotal of participants have taken chemotherapy for less than three months Table Prevalence of depression among cancer patientsIn this study patients had depression making the prevalence CI The prevalence of depression among male cancer patients was CI whereas it was CI among female patients From the totalpatients with depression and had moderate moderatelysevere and severe depression respectively The magnitude of depression has also shown thedifference among different types of cancer Fig PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Type of cancer and treatmentrelated characteristics of study participant at UoGCSH and FHRH VariablesFrequencyPercentageType of cancerBreast cancerLung cancerColorectal cancerGastric cancerCervical cancerHead and Neck cancerEsophageal cancerBlood cancerSkin cancerThyroid cancerBladder cancerLymphomaLiver cancerSarcomaTesticular cancerClinical stageStage Stage Stage Stage UnknownDuration since diagnosis months“ months monthsDuration since start of chemotherapy“ months“ months months101371journalpone0237837t003Factors associated with depression among cancer patientsIn bivariable logistic regression age sex marital status educational status occupation BMISocial support and duration of chemotherapy were found to have Pvalue 02subsequentlythese variables were subjected to multivariable analysis and educational level occupational status BMI status and duration of chemotherapy were statistically associated with depressionamong cancer patientsThe odds of depression among patients who attended college and above was significantlyreduced when compared to those with no education AOR CI Whencompared to government employees patients who are unemployed had less risk of depressionAOR CI Underweight patients had 239AOR CI times higher odds of depression as compared to those with normal body mass indexPatients who took chemotherapy for six months or more had AOR CI times higher odds of depression as compared to their counterparts Table PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaFig Prevalence of depression among cancer patients on chemotherapy in UoGCSH and FHRH northwest Ethiopia101371journalpone0237837g001DiscussionIn this study we have assessed the magnitude of depression among cancer patients and the factors affecting it We have found prevalence and occupation educational status bodymass index and duration of chemotherapy were found to be independent predictors ofdepressionThe magnitude of depression in this study was consistent with other studies conductedamong Chinese cancer patients [] whereas this figure was higher than a study conducted in Addis Ababa [] Iran [] and metaanalysis done by Krebber [] This discrepancy could be attributable to the difference in the study populations in terms of types ofcancer the tool used for screening or other sociodemographic variations and severity ofdepression consideredThe odds of depression was significantly reduced in patients who are unemployed whencompared to government employees This piece of evidence is supported by another multicenter study [] This could be related to workrelated stress which worsens feelings of inadequate control over one™s work frustrated hopes and expectations leading to depression []The odds of depression among patient who attended college and above was reduced whencompared to those who have no education This finding is supported by a study from China[] Atlanta [] and Greece [] The possible reason could be these patients may have a betterunderstanding of the disease and have early screening which increases their recovery A higherproportion of educated people are in the first or second clinical stage of cancer ascompared to of patients without educationUnderweight cancer patients had more than double odds of depression as compared tothose who have a normal body mass index This finding is supported by several single studies[ ] and systematic review and metaanalysis [] showing underweight people at higherrisk of depression This shows malnutrition has a significant role in the mental health of peopleand maintaining a healthy weight is essential to improve health in general and mental healthin particularEven though the chemotherapy duration has shown no significant association with depression in few pieces of literature [ ] The odds of depression among cancer patients whoPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Factors affecting depression among cancer patientsat UoGCSH and FHRH VariablesDepressionCOR CIAOR95 CIYesNoAge mean sd SexMarital statusMale Female Single Married Divorced Widowed Educational levelNo education Primary Secondary College OccupationGovernment Farmer Merchant Unemployed Body mass indexUnderweightNormalOver weight Social supportPoorModerateStrongDuration of chemotherapy� months months� Pvalue � � � �101371journalpone0237837t004took chemotherapy for more than six months was higher than their counterparts in this studyThis could be a side effect of chemotherapy [] or it could be also associated with the staggering cost of chemotherapy which makes these patients stress to buy it for an extended durationThis study assessed the frequently ignored aspect of cancer comorbidity depression Butthe study has some limitations as it was a crosssectional study The causeeffect relationshipsare not guaranteed in these studies therefore we recommend a prospective study Even thoughwe have used a validated tool some of the symptoms used in PHQ“ like weight loss andtiredness might be related to cancer itself and may overestimate depressionConclusionThe burden of depression among cancer patients in this study was high Occupation educational status body mass index and duration of chemotherapy were found to be independentlyassociated to depression We recommend concerned bodies working to curve the problem toPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiaintervene based on the identified risk factors Improving educational status reducing workstress and maintaining normal weight would reduce depression Clinicians shall also provideintegrated care of mental health and cancer treatmentAcknowledgmentsWe would like to thank the University of Gondar Oncology unit staff of FelegeHiwot referralhospital and University of Gondar comprehensive specialized hospitals and all data collectorsAuthor ContributionsConceptualization Adhanom Gebreegziabher Baraki Getahun Mengistu Tessema EyayawAdisu DemekeData curation Eyayaw Adisu DemekeFormal analysis Adhanom Gebreegziabher Baraki Eyayaw Adisu DemekeInvestigation Adhanom Gebreegziabher BarakiMethodology Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaProject administration Eyayaw Adisu DemekeSoftware Adhanom Gebreegziabher BarakiValidation Getahun Mengistu TessemaVisualization Getahun Mengistu TessemaWriting “ original draft Adhanom Gebreegziabher BarakiWriting “ review editing Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaEyayaw Adisu DemekeReferences Bray F Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA a cancer journal for clinicians p “ World Health anization Mental Health [cited August ] wwwwhointmental_healthmanagementdepressionen Hailemariam S The prevalence of depression and associated factors in Ethiopia findings fromthe National Health Survey International journal of mental health systems p 10118617524458623 PMID Nikbakhsh N Prevalence of depression and anxiety among cancer patients Caspian journal ofinternal medicine p “ PMID AlShakhli H Harcourt D and Kenealy J Psychological distress surrounding diagnosis of malignantand nonmalignant skin lesions at a pigmented lesion clinic Journal of Plastic Reconstructive Aesthetic Surgery p “ Sotelo JL Musselman D and Nemeroff C The biology of depression in cancer and the relationshipbetween depression and cancer progression Int Rev Psychiatry p “ PMID Krebber A Prevalence of depression in cancer patients a metaanalysis of diagnostic interviewsand selfreport instruments PsychoOncology p “ 101002pon PMID Hong JS and Tian J Prevalence of anxiety and depression and their risk factors in Chinese cancerpatients Supportive care in cancer p “ 101007s005200131997y PMID Mitchell AJ Prevalence of depression anxiety and adjustment disorder in oncological haematological and palliativecare settings a metaanalysis of interviewbased studies The lancet oncology p “ 101016S147020451170002X PMID PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaLinden W Anxiety and depression after cancer diagnosis prevalence rates by cancer type gender and age J Affect Disord “ p “ 101016jjad201203025PMID JimenezFonseca P Factors associated with anxiety and depression in cancer patients prior toinitiating adjuvant therapy Clin Transl Oncol p “ 101007s1209401818739 PMID Alemayehu M Deyessa N Medihin G Fekadu A A descriptive analysis of depression and pain complaints among patients with cancer in a low income country PloS one Colleoni M Depression and degree of acceptance of adjuvant cytotoxic drugs Lancet p “ 101016S014067360002821X PMID Pitman A Depression and anxiety in patients with cancer Bmj p k1415 101136bmjk1415 PMID Prieto JM Psychiatric morbidity and impact on hospital length of stay among hematologic cancerpatients receiving stemcell transplantation J Clin Oncol p “ 101200JCO200207101 PMID Tian J Chen ZC and Hang LF The effects of psychological status of the patients with digestive systemcancers on prognosis of the disease Europe PMC p “ Yousaf U Suicides among Danish cancer patients “ British journal of cancer p “ 101038sjbjc6602424 PMID Spiegel D and GieseDavis J Depression and cancer mechanisms and disease progression Biologicalpsychiatry p “ 101016s0006322303005663 PMID Ng CG Anxiety depression perceived social support and quality of life in Malaysian breast cancer patients a 1year prospective study Health and quality of life outcomes p Burgess C Depression and anxiety in women with early breast cancer five year observationalcohort study Bmj p 101136bmj38343670868D3 PMID Grassi L Screening for distress in cancer patients a multicenter nationwide study in Italy Cancer p “ 101002cncr27902 PMID Gelaye B Williams MA Lemma S Deyessa N Bahretibeb Y Shibre T Validity of the patienthealth questionnaire9 for depression screening and diagnosis in East Africa Psychiatry research Dec “ 101016jpsychres201307015 PMID Dalgard OS Dowrick C Lehtinen V VazquezBarquero JL Casey P Wilkinson G Negative lifeevents social support and gender difference in depression Social psychiatry and psychiatric epidemiology Jun “ 101007s0012700600515 PMID Iacovides A The relationship between job stress burnout and clinical depression Journal ofaffective disorders p “ 101016s0165032702001015 PMIDTorres MA Predictors of depression in breast cancer patients treated with radiation role of priorchemotherapy and nuclear factor kappa B Cancer p “ 101002cncr28003 PMID Polikandrioti M EVALUATION OF DEPRESSION IN PATIENTS UNDERGOING CHEMOTHERAPY Health Science Journal De Wit LM Depression and body mass index a ushaped association BMC public health p MartinRodriguez E Relationship between body mass index and depression in women a 7yearprospective cohort study The APNA study European Psychiatry p “ 101016jeurpsy201511003 PMID Jung SJ Association between body size weight change and depression systematic review andmetaanalysis The British Journal of Psychiatry p “ 101192bjpbp116186726 PMID Ciaramella A and Poli P Assessment of depression among cancer patients the role of pain cancertype and treatment PsychoOncology Journal of the Psychological Social and Behavioral Dimensionsof Cancer p “ Atag E Prevalence of depressive symptoms in elderly cancer patients receiving chemotherapyand influencing factors Psychogeriatrics p “ 101111psyg12329PMID Thornton LM Delayed emotional recovery after taxanebased chemotherapy Cancer 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Thyroid_Cancer

"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partner™s occupation and economic statusPatient™s occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatient™s marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patient™s hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patient™s family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants™ hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ‰¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ‰¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0c"

Thyroid_Cancer

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

Thyroid_Cancer

Neurosteroids Biosynthesisand Physiological FunctionsShogo Haraguchi and Kazuyoshi Tsutsui Department of Biochemistry Showa University School of Medicine Tokyo Japan Graduate School of Integrated Sciencesfor Life Hiroshima University Hiroshima JapanSimilar to the adrenal glands gonads and placenta vertebrate brains also producevarious steroids which are known as œneurosteroids Neurosteroids are mainlysynthesized in the hippocampus hypothalamus and cerebellum however it has recentlybeen discovered that in birds the pineal gland a photosensitive region in the brainproduces more neurosteroids than other brain regions A series of experiments usingmolecular and biochemical techniques have found that the pineal gland producesvarious neurosteroids including sex steroids de novo from cholesterol For instanceallopregnanolone and 7αhydroxypregnenolone are actively produced in the pinealgland unlike in other brain regions Pineal 7αhydroxypregnenolone an upregulator oflocomotion enhances locomotor activity in response to light stimuli in birds Additionallypineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronalapoptosis within the developing cerebellum in juvenile birds Furthermore exposure tolight during nighttime hours can cause loss of diurnal variations of pineal allopregnanolonesynthesis during early posthatch life eventually leading to cerebellar Purkinje cell deathin juvenile birds In light of these new findings this review summarizes the biosynthesisand physiological functions of pineal neurosteroids Given that the circadian rhythms ofindividuals in modern societies are constantly interrupted by artificial light exposure thesefindings in birds which are excellent model diurnal animals may have direct implicationsfor addressing problems regarding the mental health and brain development of humansKeywords allopregnanolone 7αhydroxypregnenolone neurosteroid pineal gland cerebellum lightINTRODUCTIONSimilar to the gonads and placenta vertebrate brains actively also produce various steroidhormones These steroid hormones produced in the brain are named œneurosteroids Theproduction of neurosteroids was demonstrated firstly in mammals and then in other vertebrates“ Thus neurosteroid production appears to be a universal feature of the brain in vertebratesIt is known that neurosteroids are produced in glial cells and neurons of the centraland peripheral nervous systems However we have demonstrated thatthe pinealgland produces neurosteroids from cholesterol in birds during early posthatch period “Notably allopregnanolone also known as 3α5αtetrahydroprogesterone 3α5αTHP and 7αhydroxypregnenolone are the two major neurosteroids produced in the pineal gland Of thesetwo pineal allopregnanolone prevents the death of developing Purkinje cells and pineal 7αhydroxypregnenolone functions as an upregulator of locomotion regulating locomotor activity inresponse to light stimuli in birds Edited byVance L TrudeauUniversity of Ottawa CanadaReviewed byVincent M CassoneUniversity of Kentucky United StatesMaria Claudia Gonzalez DeniselleCONICET Instituto de Biolog­a yMedicina ExperimentalIBYME ArgentinaCorrespondenceShogo HaraguchishogoharaguchigmailcomSpecialty sectionThis was submitted toNeuroendocrine Sciencea section of the journalFrontiers in EndocrinologyReceived April Accepted July Published August CitationHaraguchi S and Tsutsui K Pineal Neurosteroids Biosynthesisand Physiological FunctionsFront Endocrinol 103389fendo202000549Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsBIOSYNTHESIS OF PINEALNEUROSTEROIDSThe pineal glands of vertebrates respond to light stimuli andfulfillimportant functions in the anization of circadianrhythms The secretion of melatonin a major hormone producedby the pineal gland shows a clear daily rhythm with its peakconcentration occurring at night However it was notknown whether the pineal gland produces neurosteroids untilrecently We have recently demonstrated that the pineal gland isa newly found neurosteroidogenic an producing a variety ofneurosteroids from cholesterol αhydroxypregnenolone and allopregnanolone are activelyreleased Taken together these findings indicate that the pineal gland injuvenile birds produces various neurosteroids from cholesterolAccordingly this is the first demonstration of neurosteroidsynthesis in the pineal gland in a vertebratePHYSIOLOGICAL FUNCTION OF PINEAL7αHYDROXYPREGNENOLONE INLIGHTDEPENDENT LOCOMOTIONthatgene3hydroxysteroidPregnenolone is an anabolic intermediate of most endogenoussteroid hormones and is produced from cholesterol throughthe mitochondrial cholesterol side chain cleavage enzymecytochrome P450scc P450scc encoded by the Cyp11a geneWe have demonstrated by transcriptionpolymerase chainreaction RTPCR thatthe pineal gland in juvenile birdsexpresses P450scc mRNA Figure The proteinproduct of this mRNA is localized in the cells that form thefollicular structures in the pineal glands of birds We havedemonstrated by highperformance liquid chromatography3HHPLC with radioactive flow detector analysischolesterolis converted to radioactive pregnenolone whenincubated with pineal gland extract from juvenile birds This observation has confirmed the presence offunctionalP450scc in the pineal gland Figure which has also beendetected by gas chromatographymass spectrometry GCMS Subsequent RTPCR“based assessment has revealed thatkey steroidogenic enzymes cytochrome P450 7αhydroxylaseP4507α encoded by the Cyp7b gene 3αhydroxysteroiddehydrogenase 01 014isomerase 3αHSD encoded by thedehydrogenase 01 01Hsd3aisomerase 3HSD encoded by the Hsd3b gene 5αreductaseencoded by the Srd5a gene 5reductase encoded by theSrd5b gene cytochrome P450 17αhydroxylasec1720lyaseP45017αlyase encoded by the Cyp17 gene 17hydroxysteroiddehydrogenase 17HSD encoded by the Hsd17b gene andcytochrome P450 aromatase P450arom encoded by the Cyp19gene are expressed in the pineal gland of birds Figure We further demonstrated that steroid hormones are indeedpresent in the pineal gland Incubation of 3Hpregnenolonewith pineal glands from posthatch birds generates 7α andor7hydroxypregnenolone by the action of P4507α foundin the pineal glands Figure In addition to theseneurosteroid isomers progesterone allopregnanolone 3α 5αTHP andor epipregnanolone 3 5THP androstenedionetestosteroneandestradiol17 are also produced Figure These exvivo observations have confirmed that the pineal glands injuvenile birds have the biosynthetic machinery for majorsteroid hormones which have also been verified to be producedas neurosteroidsFigure Although HPLCanalysis has failed to resolve the isomers of these hormonesalloepipregnanolonesuch as 7αhydroxypregnenoloneand 5αdihydrotestosterone several sets ofisomers havebeen successfully isolated by GCMS analysis Especially5dihydrotestosteronein vivo andor5αanalysis hasactivation oftranscriptomicslightinduced transcriptionalfor studiesThe chick pineal gland is used as a modeltheon the lightdependent phaseshifting mechanism ofcircadian clock To search for genes involved in thisa diï¬erential GeneChip analysis has beenmechanismidentifiedperformed Thisthethefullset of genes in the pineal gland involved in cholesterolbiosynthesis When the pineal gland was exposed tolightit produced cholesterol and 7αhydroxypregnenoloneex vivo Interestingly this lightinduced production of 7αhydroxypregnenolone occurred only when the gland wasexposed to light at early night but not atlate night orduring the daytime During early night time the circadianclock is sensitive to light which causes phasedelay of theclock Thusthe lightsensitive pineal production of7αhydroxypregnenolone appearsto be regulated by thecircadian clockactivateslocomotorIn vertebratesan intracerebroventricularinjection of7αhydroxypregnenoloneactivities“ Thusthe intracerebroventricular injection of 7αhydroxypregnenolone was administered in a dosedependentmanner at early night in chicks After the injection chickswere placed individually for locomotor activity measurementin an open field apparatus for min Spontaneous locomotoractivities of chicks were stimulated by the intracerebroventricularinjection of 7αhydroxypregnenolone in a dosedependentmanner Furthermore when chicks are exposed to lightduring early nightreachthe daytime level These results suggest that pineal 7αhydroxypregnenolone reaches the target sites within the brain byvolume transmission upon light exposure at early nightlocomotor activitiestimetheirPHYSIOLOGICAL FUNCTION OF PINEALALLOPREGNANOLONE IN PURKINJECELL SURVIVAL DURING DEVELOPMENT7αHydroxypregnenolone and allopregnanolone are activelyreleased during early posthatch period compared with adulthood Therefore 7αhydroxypregnenolone and allopregnanolonemay play key roles in birds during early posthatch periodIn vertebrates pinealectomy decreases cell number in thedeveloping brain These findings suggest that these majorneurosteroids secreted from the pineal gland are involved in thedevelopment of brain cellsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal NeurosteroidsFIGURE Biosynthetic pathways of pineal neurosteroids Allopregnanolone and 7αhydroxypregnenolone are the major neurosteroids produced in the pineal glandof birds P450scc cytochrome P450 sidechain cleavage enzyme P4507α cytochrome P450 7αhydroxylase 3HSD 3hydroxysteroiddehydrogenase 01 014isomerase 3αHSD 3αhydroxysteroid dehydrogenase 01 014isomerase 5αreductase 5reductase P45017αlyase cytochrome P45017αhydroxylasec1720lyase 17HSD 17hydroxysteroid dehydrogenase and P450arom cytochrome P450 aromataseFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidstheincreasessupplementation oftheIn chicks pinealectomy decreases the concentration ofallopregnanolone and the number of cerebellar Purkinjeallopregnanolonecells whereasto pinealectomized birdsconcentration ofallopregnanolone and recovers the number of Purkinje cells Thus pineal allopregnanolone is considered to be an essentialfactor for the normal development of cerebellar Purkinje cellsIt thus appears that pineal allopregnanolone functions as anessential factor for Purkinje cells during posthatch periodIn addition pinealectomy in juvenile birds increases theexpression of active caspase3 in Purkinje cells whereasallopregnanolone supplementation decreases the expressionof active caspase3 during posthatch period Thus theneuroprotective action of pineal allopregnanolone on cerebellarPurkinje cells is exerted by suppressing the activation of caspase3Figure Allopregnanolone acts mainly as a ligand ofthe γaminobutyric acid type A GABAA receptor and may alsoact as an agonist of the membrane progesterone receptors αmPRα as well as the mPR and mPRγ “ Thereforeeither mPR siRNA orisoallopregnanolone an antagonistof allopregnanolone was delivered into the cerebellum ofposthatched chicks It was found that the silencing of mPRαincreases the number of Purkinje cells that express active caspase in the cerebellum of chicks Furthermore to uncoverthe mechanism of neuroprotective action of allopregnanoloneFIGURE A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a h lightdark cycle or with hlight exposure during the dark period lightatnight condition Left panel The normal cerebellar development under a h lightdark cycle during the first weekafter hatching Pineal allopregnanolone induces the expression of pituitary adenylate cyclaseactivating polypeptide PACAP a neuroprotective factor through themembrane progestin receptor α mPRα receptor binding mechanism in Purkinje cells Subsequently PACAP inhibits the activation of caspase3 that facilitates theapoptosis of cerebellar Purkinje cells Right panel The abnormal cerebellar development under the lightatnight condition during the first week after hatching Thelightatnight condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis Decreased pineal allopregnanolone synthesis leads to decreased expressionof PACAP in Purkinje cells Consequently the active caspase3 level increases inducing the apoptosis of Purkinje cells in the cerebellumFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroidsin cerebellar Purkinje cells allopregnanolone action on theexpression of neuroprotectiveneurotoxic factors “ hasbeen investigated Pinealectomy decreases the mRNA levels ofpituitary adenylate cyclaseactivating polypeptide PACAP aneuroprotective factor in the cerebellum of juvenile birds It has been found that a daily injection of allopregnanolone inpinealectomized juvenile birds upregulates PACAP relative tothe levels in control birds These findings show that PACAPmediates the neuroprotective action of pineal allopregnanolonethrough mPRα receptor binding during cerebellar developmentFigure LIGHTATNIGHT AFFECTS THEDEVELOPMENT OF CEREBELLUMTHROUGH A MECHANISM MEDIATED BYPINEAL ALLOPREGNANOLONE ACTIONIt is known that environmental stimuli aï¬ect the developmentof animals including humans In vertebrate brain development anatural lightdark cycle promotes better brain development thanconstant conditions such as constant light or constant darkness“ Howeverthe molecular mechanisms that controlhow environmental light conditions aï¬ect brain developmentremain unclear The pineal gland is a photosensitive anTo investigate whether light conditions are involved in thesynthesis of allopregnanolone in the pineal gland the birdshave been incubated under either a h lightdark LDcycle or LD cycle with h light exposure during the darkperiod lightatnight Consequently it has been found that theallopregnanolone concentration and synthesis during the darkperiod are higher in the pineal glands of LD birds than in thoseof lightatnight birds Figure Furthermore the numberof cerebellar Purkinje cells is decreased by the lightatnightcondition Figure It is therefore considered that pinealallopregnanolone is a critical metabolite that aï¬ects cerebellardevelopment in vertebrates depending on the environmentallight conditionsREFERENCES Baulieu EE Neurosteroids of the nervous system by the nervous system forthe nervous system Rec Prog Hormone Res “ Tsutsui K Ukena K Takase M Kohchi C Lea RW Neurosteroidbiosynthesis in vertebrate brains Comp Biochem Physiol C “ 101016S0742841399000651 Compagnone NA Mellon SH Neurosteroids biosynthesis and functionthese novel neuromodulators Front Neuroendocrinol “of 101006frne19990188CONCLUSIONSthedatarecentreview summarizedThison pinealneurosteroids Studies have indicated that the pineal glandproduces neurosteroids from cholesterol in birds Pineal 7αhydroxypregnenolone regulates locomotion in response to lightstimuli in birds Pineal allopregnanolone prevents the death ofdeveloping Purkinje cells by suppressing neuronal apoptosisduring development In addition circadian disruption by lightexposure during nighttime leads to cell death of developingPurkinje cellsthrough pineal allopregnanolonedependentmechanisms in juvenile birds These observations suggest thatnighttime artificial light exposure in modern societies may alsoperturb the development of the human brainAlmost all animals have circadian rhythms However modernlife conditions chronically disrupt circadian rhythm throughartificial light exposure The disruption of circadian rhythm isassociated with a decline in mental and physical health “The most potent circadian rhythm disruption is inappropriatelytimed bright light exposure eg lightatnight To investigatethe eï¬ects of chronic circadian disruption in modern societieson mental and physical health which is efficiently modeled bythe lightatnight condition presented here many studies havebeen conducted on mice However it is important for us tobear in mind that laboratory mice are mainly nocturnal animalswhereas humans are diurnal Thus birds are excellent animalmodels to uncover the eï¬ect of lightatnight on diurnal animalsincluding humansAUTHOR CONTRIBUTIONSSH and KT wrote the manuscript All authors contributed to the and approved the submitted versionFUNDINGThis work wasJSPS GrantsinAid forScientific Research KAKENHI Grant Numbers JP15K18571and JP19K09033supported bysterol regulatory elementbinding protein Xboxbinding protein andheat shock factor pathways Proc Natl Acad Sci USA “ 101073pnas1015959108 Haraguchi S Hara S Ubuka T Mita M Tsutsui K Possible role of pinealallopregnanolone in Purkinje cell survival Proc Natl Acad Sci USA “ 101073pnas1210804109al Lightatnight Haraguchi S Kamata M Tokita T Tashiro KI Sato M Nozaki Mthroughaï¬ects brain developmenteLifeetallopregnanolonedependentpineal8e45306 107554eLife45306037mechanismsexposure Tsutsui K Matsunaga M Miyabara H Ukena K Neurosteroid biosynthesis in Reiter RJ Pineal melatonin cell biology of its synthesis and of its physiologicalthe quail brain J Exp Zool 305A733“ 101002jeza302interactions Endocr Rev “ 101210edrv122151 DoRego JL Seong JY Burel D Leprince J LuuThe V Tsutsui Ket al Neurosteroid biosynthesis enzymatic pathways and neuroendocrineregulation by neurotransmitters and neuropeptides Front Neuroendocrinol “ 101016jyfrne200905006 Hatori M Hirota T Iitsuka M Kurabayashi N Haraguchi S KokameK et al Lightdependent and circadian clockregulated activation of Fukada Y Okano T Circadian clock system in the pineal gland Mol Neurobiol “ 101385MN251019 Matsunaga M Ukena K Baulieu EE Tsutsui K 7αHydroxypregnenoloneacts as a neuronal activator to stimulate locomotor activity of breeding newtsby means of the dopaminergic system Proc Natl Acad Sci USA “ 101073pnas0407176101Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cHaraguchi and TsutsuiPineal Neurosteroids Tsutsui KInoue K Miyabara H Suzuki S Ogura Y Haraguchi7αHydroxypregnenolone mediates melatonin action underlying“Sdiurnal 101523JNEUROSCI3562072008locomotorNeuroscirhythmsJ Haraguchi S Koyama T Hasunuma I Vaudry H Tsutsui K Prolactin increasesthe synthesis of 7αhydroxypregnenolone a key factor for induction oflocomotor activity in breeding male newts Endocrinology “ 101210en20091229 Haraguchi S Koyama T Hasunuma I Okuyama S Ubuka T Kikuyama S et alAcute stress increases the synthesis of 7αhydroxypregnenolone a new keyneurosteroid stimulating locomotor activity through corticosterone action innewts Endocrinology “ 101210en20111422 Haraguchi S Yamamoto Y Suzuki Y Hyung Chang J Koyama T Sato Met al 7αHydroxypregnenolone a key neuronal modulator of locomotionstimulates upstream migration by means of the dopaminergic system insalmon Sci Rep 101038srep12546 Fillenz M Volume transmission in the brain Novel mechanisms for neuronaltransmission In Fuxe K Agnati LF editors Advances in Neurosciences Vol New York NY Raven Press p “ Kilic E Hermann DM Isenmann S B¤hr M Eï¬ects of pinealectomy andmelatonin on the retrograde degeneration of retinal ganglion cells in a novelmodel of intraorbital optic nerve transection in mice J Pineal Res “ 101034j1600079x20021823x Tun§ AT Turgut M Aslan H Sahin B Yurtseven ME KaplantheS Neonatalcerebellum of“ 101016jbrainres200510011pinealectomythechick a stereologicalstudy Brain ResPurkinjeinduceslosscellin Pang Y Dong J Thomas P Characterization neurosteroid binding and braindistribution of human membrane progesterone receptors δ and ε mPRδand mPRε and mPRδ involvement in neurosteroid inhibition of apoptosisEndocrinology “ 101210en20121772 Schumacher M Mattern C Ghoumari A Oudinet JP Liere P LabombardaF et al Revisiting the roles of progesterone and allopregnanolone in thenervous system resurgence of the progesterone receptors Prog Neurobiol “ 101016jpneurobio201309004 Belelli D Lambert JJ Neurosteroids endogenous regulators of the GABAAreceptor Nat Rev Neurosci “ 101038nrn1703 Bernal J Thyroid hormone receptors in brain development and function NatClin Pract Endocrinol Metabol “ 101038ncpendmet0424 FalluelMorel A Vaudry D Aubert N Galas L Benard M Basille M et alPituitary adenylate cyclaseactivating polypeptide prevents the eï¬ects ofceramides on migration neurite outgrowth and cytoskeleton remodelingProc Natl Acad Sci USA “ 101073pnas04096 Koibuchi N Chin WW ThyroidEndocrinoldevelopment 101016S1043276000002381TrendshormoneMetabactionandbrain“ Vaudry D FalluelMorel A Leuillet S Vaudry H Gonzalez BJ Regulators ofcerebellar granule cell development act through specific signaling pathwaysScience “ 101126science1085260 Sasahara K Shikimi H Haraguchi S Sakamoto H Honda S Harada N et alMode of action and functional significance of estrogeninducing dendriticgrowth Spinogenesis and synaptogenesis in the developing Purkinje cell JNeurosci “ 101523JNEUROSCI0710072007 Bakkum BW Benevento LA Cohen RS Eï¬ects of lightdark and darkrearing on synaptic morphology in the superior colliculus and visualcortex ofJ Neurosci Res “ 101002jnr490280107the postnatal and adult rat Brooks E Waters E Farrington L Canal MM Diï¬erential hypothalamictyrosine hydroxylase distribution and activation by light in adult mice rearedunder diï¬erent light conditions during the suckling period Brain Struct Funct “ 101007s0042901103189 DulcisDSpitzerNCIlluminationcontrolsdopamineof“ 101038nature07569neuronsregulatingbehaviourdiï¬erentiationNature Li Y Komuro Y Fahrion JK Hu T Ohno N Fenner KB et al Lightstimuli control neuronal migration by altering ofinsulinlike growthfactor IGF1 signaling Proc Natl Acad Sci USA “ 101073pnas1111326109 Ohta H Mitchell AC McMahon DG ConstantPediatrthe“ 10120301pdr00002331141840366developing mousebiologicalclocklight disruptsRes Kantermann T Roenneberg TIsfactor or a health risk predictor Chronobiol lightatnightInthealthriska “ Wu J Dauchy RT Tirrell PC Wu SS Lynch DT Jitawatanarat P et alLight at night activates IGF1RPDK1 signaling and accelerates tumrowth in human breast cancer xenografts Cancer Res “ 10115800085472CAN103837 Smarr BL Grant AD Perez L Zucker I Kriegsfeld LJ Maternal andearlylife circadian disruption have longlasting negative consequenceson oï¬spring development and adult behavior in mice Sci Rep 101038s41598017034064Conflict 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 Haraguchi and Tsutsui This is an openaccess distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer