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" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteam™s experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as œclaysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest œhighly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [“] They havetherefore been a potential alternate medicine for several diseases [“] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof single“single type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [“] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors™ previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells™ interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as œclaysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterials”MoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [“] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately × × microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethods”MolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CED”CED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors™ previous research[“] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvˆ’ygreneBAlraopcilihpordyHˆ’ˆ’ˆ’ˆ’aCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[“] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3”obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study ˆ’ to ˆ’ mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[“] we propose that nanoclays may be injected asœclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors™ ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature “ httpsdoi101038nature02145Li F Structural analysis of major species barriers between humansand palm civets for severe acute respiratory syndrome coronavirusinfections J Virol “ httpsdoi101128jvi0044208 Wu KL Peng GQ Wilken M Geraghty RJ Li F Mechanisms of hostreceptor adaptation by severe acute respiratory syndrome coronavirus JBiol Chem “ httpsdoi101074jbcM111325803 Wang C Li W Drabek D Okba NMA van Haperen R Osterhaus ADME A human monoclonal antibody blocking SARSCoV2 infection NatCommun Jiang S Hillyer C Du L Neutralizing antibodies against SARSCoV2and other human coronaviruses Trends Immunol “Pinto D Park YJ Beltramello M Walls AC Tortorici MA Bianchi S Crossneutralization of SARSCoV2 by a human monoclonal SARSCoV antibody Nature “da Rocha Dias S Salmonson T van ZwietenBoot B Jonsson B Marchetti SSchellens JH Pignatti F The European Medicines Agency review ofvemurafenib Zelboraf® for the treatment of adult patients with BRAF V600mutationpositive unresectable or metastatic melanoma summary of thescientific assessment of the Committee for Medicinal Products for HumanUse Eur J Cancer “Sahel N Abduljauwad and HabiburRehman Ahmed Enhancing cancer celladhesion with clay nanops for countering metastasis Nature ScientificReports April httpsdoi101038s4159801942498y Zhang Y Long M Huang P Yang H Chang S Hu Y Mao L Intercalated 2D nanoclay for emerging drug delivery in cancer therapyNano Res “ Chianelli R R Das S US Patent No Washington DC US Patent and Trademark Office Han S Liu F Wu J Zhang Y Xie Y Wu T Y Targeting of fluorescentpalygorskite polyethyleneimine nanocomposite to cancer cells Appl ClaySci “Sun B Ranganathan B Feng SS Multifunctional poly D Llactidecoglycolide montmorillonite PLGAMMT nanops decorated byTrastuzumab for targeted chemotherapy of breast cancer Biomaterials“Lin FH Lee YH Jian CH Wong JM Shieh MJ Wang CY A study ofpurified montmorillonite intercalated with 5fluorouracil as drug carrierBiomaterials “ Bothiraja C Thorat UH Pawar AP Shaikh KS Chitosan coated layeredclay montmorillonite nanocomposites modulate oral delivery of paclitaxel incolonic cancer Mater Technol 29sup3B120“B126Kevadiya BD Thumbar RP Rajput MM Rajkumar S Brambhatt H Joshi GVBajaj HC Montmorillonitepolyεcaprolactone composites asversatile layered material reservoirs for anticancer drug and controlledrelease property Eur J Pharm Sci “ Guo MY Wang AF Muhammad F Qi WX Ren H Guo YJ Zhu GS Halloysite nanotubes a multifunctional nanovehicle for anticancer drugdelivery Chin J Chem “ Martínez C D Cationic clays upon cancer therapy Virtual MultidisciplinaryConference QUAESTI Konta J Clay and man clay raw materials in the service of man ApplClay Sci “ Murray HH Traditional and new applications for kaolin smectite andpalygorskite a general overview Appl Clay Sci “ Volzone C Retention of pollutant gases comparison between clayminerals and their modified products Appl Clay Sci “ Dong Y Feng SS Poly dllactidecoglycolidemontmorillonitenanops for oral delivery of anticancer drugs Biomaterials “ Clarka KJ Sarrb AB Grantb PG Phillipsb TD Woodea GN In vitrostudies on the use of clay clay minerals and charcoal to adsorb bovinerotavirus and bovine coronavirus Vet Microbiol “ Choi G Huiyan P Alothman Z Vinu A Yun C Choy J Anionic clay asthe drug delivery vehicle tumor targeting function of layered doublehydroxidemethotrexate nanohybrid in C33A orthotopic cervical cancermodel International Journal of nanomedicine DOI httpsdoi102147IJNS95611 Hosseini F Hosseini F Jafari S M and Taheri A Bentonite nanoclaybaseddrugdelivery systems for treating melanoma Clay Minerals DOI httpsdoi101180clm201842018 Inamuddin Asiri A M and Mohammad Ali Applications of nanocompositematerials in drug delivery DOI httpsdoi101016C20160050751 Avolume in Woodhead Publishing Series in Biomaterials Zhang Y Long M Huang P Yang H Chang S Hu Y Tang A and MaoL Emerging integrated nanoclayfacilitated drug delivery system forpapillary thyroid cancer therapy doi 101038srep33335 Sci Rep Li S Xia B Javed B Hasley W D MelendezDavila A Liu M Kerzner MAgarwal S Xiao Q Torre P Bermudez J G Rahimi K Kostina N YMöller M RodriguezEmmenegger C Klein M Percec V and Good M CDirect visualization of vesicle disassembly and reassembly usingphotocleavable dendrimers elucidates cargo release mechanisms ACS 0cAbduljauwad Nanoscale Resear

Thyroid_Cancer

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

Thyroid_Cancer

"MiRNAs play important roles in the development of ovarian cancer activation of primitive folliclesfollicular development oocyte maturation and ovulation In the present study we investigated the specific role ofmiR23a in cov434 cellsResults Downregulation of miR23a was observed in serum of PCOS patients compared with the healthy controlsuggesting the inhibitory effect of miR23a in PCOS MiR23a was positively correlated with Body Mass Index BMI andnegatively correlated with Luteinizing hormone LH Testostrone T Glucose Glu and Insulin INS of PCOS patientsMiR23a mimic inhibited the proliferation and promoted apoptosis of human cov434 cells In addition flow cytometryassay confirmed that miR23a blocked cell cycle on G0G1 phase MiR23a inhibitor showed opposite resultsFurthermore double luciferase reporter assay proved that miR23a could bind to the ™UTR of FGD4 directly throughsites predicted on Target Scan FGD4 level was significantly suppressed by miR23a mimic but was significantlyenhanced by miR23a inhibitor We further proved that miR23a increased the expression of activated CDC42 GTPbround and pPAK1 suggesting that miR23a induced cell cycle arrest through CDC42PAK1 pathwayConclusions In our study reveals that miR23a participates in the regulation of proliferation and apoptosisof cov434 cells through target FGD4 and may play a role in the pathophysiology of PCOSKeywords miR23a Polycystic ovary syndrome FGD4 Binding site Cell cycleBackgroundPolycystic ovary syndrome PCOS is the most common reproductive endocrine and metabolic disorder disease inwomen characterized by ovulation disorders hyperandrogenism and insulin resistance [ ] PCOS affects about “ of women of childbearing age accounting for ofanovulatory infertility and usually a lifelong disease Itscommon clinical manifestations include menstrual disorders subfertility acne vulgaris alopecia seborrheia obesity hirsutism and acanthosis [] Women with PCOS havean increased risk of insulin resistance hypertension type Correspondence linjinet163com3Gynaecology Mindong Hospital in Ningde City No Heshan Road FuanFujian ChinaFull list of author information is available at the end of the diabetes oxidative stress dyslipidemia cardiovasculardisease and endometrial cancer [] Therefore understanding the molecular mechanism of metabolic diseases underlying the pathophysiology of PCOS will help to identify newdiagnostic and therapeutic strategies In addition althoughthe exact etiology of PCOS remains to be understood ithas been clear that the survival and proliferation of granulosa cells are closely related to the pathogenesis of PCOS[]In recent years the role of microRNAs miRNAs inovarian physiology and pathology has attracted muchattention Some studies have shown that miRNAs playimportant roles in the development of ovarian canceractivation of primitive follicles follicular developmentoocyte maturation and ovulation [“] Several studies The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLin Journal of Ovarian Research Page of have found a variety of differentially expressed microRNAs in ovarian granulosa cells of PCOS patients whichare closely related to the proliferation and apoptosis ofovarian granulosa cells and the production of progesterone estradiol and testosterone [ ]responsibleforinducing caspasedependentThe human miR23a gene is located on chromosome of the human genome and transcribed into a part of themiR23a27a242 cluster [] Mi23a27a242 clusterwhich encodes primicroRNA transcripts composed ofthree kinds of miRNAs miR23a miR27a and miR242isandcaspaseindependent apoptosis of embryonic kidney cellsHEK293Tthrough human cJun Nterminal kinasepathway [] In recent years more and more evidencehas shown that miR23a is essential for folliculogenesis Ithas been reported that the expression of circulating miR23a of patients with PCOS was downregulated comparedwith healthy women and proved that miR23a is a betterindicator for evaluation of PCOS than the miR23b []However as far as we know the specific role and mechanism of miR23a in PCOS have not been studiedStudies proved that miR23a issignificantly upregulated in premature ovarian insufficiency POI patients™ serum and poor ovarian response POR patients™ovarian granulosa cells [“] Compared with normalwomen miR23a was significantly upregulated in follicular cells of women receiving assisted reproductive technology ART due to oviduct and endometriosis []More critically miR23a can promote the apoptosis byaffecting the expression of multiple targetsincludingXIAP SMAD5 and Sirt1 [ ]Thereforein the present research we hypothesizedthat miR23a is involved in the development of PCOS byregulating downstream pathways related to cell survivalin ovarian cells The objective of this study was to confirm the regulatory effect and mechanism of miR23a onthe growth of cov434 cells We analyzed the expressionof miR23a in serum samples from PCOS patients andhealthy women and the correlation between miR23alevel and PCOS symptoms We focused on a new molecular mechanism by which miR23a induces apoptosisin granular cellsdisease smoking and using alcohol or drugs The serumof healthy women was collected as the control groupThe volunteers in the control group had normal menstruation normal ovaries and no history of reproductivesystem disease or appendicitis The control and PCOSgroup did nottake any medications in the past months including oral contraceptives or other hormonalmedications with no intrauterine devices or smokingPatients with reproductive system disease or appendicitishistory were excluded from the control group All volunteers had understood the purpose and requirements ofthis study and signed a written informed consent beforeparticipating in the study ml of elbow venous bloodfrom each sample was taken and stored in a refrigeratorat ˆ’ °C All the experiments involved in this studyhave obtained the ethical approval of Mindong hospitalin Ningde CityEvaluation of BMI and sex hormoneThe weight and height of the volunteers were measuredto calculate Body mass index BMIBMI weightheight2 Radioimmunoassay RigorBio Scientific andTechnology Co Beijing was used to measure the levelof total testosterone and other sex hormonesCell line and transfectionCell lines KGN derived from a granulosa cell tumorcov434 derived from a granulosa cell tumor and SVOGderived by immortalization of granulosaluteal cellsusing SV40 large T antigen were purchased from cellresource bank of Chinese Academy of Sciences — cells were seeded into well plates MiR23a micmic nM miR23a inhibitor nM and negative control NC nM mimic NC and nM inhibitor NCRuibo Biotechnology Co Ltd Guangzhou China weretransfected into cov434 cells by Lipofectamine„¢ Normal untreated cov434 cells were cultured as control Thesequence of siRNA used in this study is as follows miR23amimic ²CCTTTAGGGACCGTTACACTA3² mimicNC ²TTCTCCGAACGTGTCACGTTTC3² miR23ainhibitor ²TAGTGTAACGGTCCCTAAAGG3² inhibitor NC ²TTCTCCGAACGTGTCACGTTTC3²Materials and methodsSamplesThe serum of Chinese women with PCOS was collected in Mindong hospital Ningde City Fujian Provincefrom September to December According tothe revised PCOS diagnostic criteria published by theRotterdam consensus [] the PCOS group excluded patients with Cushing™s syndrome delayed congenital adrenal hyperplasia thyroid dysfunction hyperthyroidismhyperprolactinemia or androgen secreting tumor as wellas patients with diabetes hypertension chronic kidneyReal time fluorescence quantitative PCR qPCRTotal RNA were extract from samples or cells using Trizolreagent Related expression of target gene was calculatedusing 2ΔΔCt method This study involves the followingsequences miR23a3p Reverse transcription ² GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAAAT3² miR242 Reverse transcription ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGTGT3² miR27a3P ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGAA3² U6 Reverse transcription ²AAAATATG 0cLin Journal of Ovarian Research Page of GAACGCTTCACGAATTTG3² miR23a3p forward primer ²GCGATCACATTGCCAGGG3² and reverse primer²AGTGCAGGGTCCGAGGTATT3² miR242 forwardprimer ²CGCGTGCCTACTGAGCTGAA3² and reverseprimer ²AGTGCAGGGTCCGAGGTATT3² miR27a3Pforward primer ²GCGCGTTCACAGTGGCTAAG3² andreverse primer ²AGTGCAGGGTCCGAGGTATT3² U6forward primer ²CTCGCTTCGGCAGCACATATACT3²and reverse primer ²ACGCTTCACGAATTTGCGTGTC² FGD4 forward primer ²CCTGCCTCTGCTTCTTGTGTCTC3² and reverse primer ²TGGTTGTCAATCCATGCCTTCCTG3²Cell proliferation assayAfter h of transfection cells were seeded into a well plate at the density of — cells per well Eachgroup of cells was treated with replicates After incubation for the specified time and h μl of CCK8 reagent was added and incubated at °C for h The absorbance of each pore was measured at nm by an enzyme labeling instrumentFlow cytometry analysis for cell cycleAfter h of transfection the cell cycle was detected byflow cytometry The cells were fixed with ethanolovernight at °C The cells were resuspended with μl of binding buffer μl PI was added to the cellsuspension and incubated at room temperature for min The results were analyzed by ModFit and displayedby FL2w and FL2aFlow cytometry analysis for apoptosisAfter h of transfection the apoptotic cells were detected by flow cytometry μl of PI and FITC annein Vwere added into μl cell suspension and incubated atroom temperature for min Cell apoptosis was detected using a flow cytometerWestern blotThe total protein was extracted with RIPA buffer BCAmethod was used to detect the protein concentrationThe extracted protein was electrophoresis by SDSPAGEand transferred to PVDF membrane PVDF membranewas incubated in skimmed milk at room temperaturefor h and then primary antibody overnight at °Cfollowed by the secondary antibody at room temperaturefor h QUANTITY ONE software is used for resultanalysis The following antibodies were used in this research antiFGD4 Abcam ab97785 87KDaantiCDC42 Abcam ab155940 21KDa antiPAK1 Abcam ab223849 60KDa and βactinTransGen Biotech HC201 42KDaDouble luciferase reporting assayThe plasmids of wild type FDG4WT and mutant typeFDG4MUT luciferase reporter genes were constructedusing pcDNA31as the empty vector MiR23a mimicmimic NC FDG4WT and FDG4MUT plasmids were cotransfected into cov434 cells by LipofectamineTM Cells were divided into four groups FGD4WT ²UTR miR23a mimic NC FGD4Mut ²UTR miR23a mimicNC FGD4WT ²UTR miR23a mimic FGD4Mut ²UTR miR23a mimic After h of transfection FirelyLueiferase F and Renilla Luciferase R were detected byGLOMAX \\fluorescence detector and the relativeluciferase activity F R was calculatedStatistical analysesAll data were analyzed with SPSS SPSS Inc Chicago IL software and represented as mean ± SD Spearman method was used to analyze the relationshipbetween miRNA level and other indicators Independentsample ttest was used to evaluate the difference between two groups and Oneway ANOVA was used toanalyze the difference between three and more groupswith post hoc contrasts by Bonferroni test P wasconsidered statistically significantResultsMiR23a was downregulated in serum of PCOS patientsPeripheral blood was collected from local PCOS patients for the detection of miR23a level with healthywomen™s peripheral blood as the control Clinical information on age BMI and sex hormone levels of PCOSpatients and normal control samples are alllisted inTable As shown in Fig 1a the serum miR23a level inPCOS patients was significantly lower than that in thecontrol group P Then we detected the level ofmiR27a and miR242 using qPCR As shown in Fig 1amiR27a and miR242 also downregulated in peripheralblood of PCOS patients compared with healthy sampleTable The clinical information of PCOS and control groupsClinical indexPPCOSn ± Controln ± ± ± ± ± ± ± ± ± ± ± AgeE2 pgmLBMI Kgm2LH mIUmLFSH mIUmLPRL mIULT mIUmL ± ± ± Glu nmolmLINS μUmLE2 Estradiol BMI Body Mass Index LH Luteinizing hormone FSH Folliculestimulatinghormone PRL Prolactin T Testostrone Glu Glucose INS Insulin ± ± ± 0cLin Journal of Ovarian Research Page of Fig MiR23a was downregulated in serum of PCOS patients a qPCR was performed to detect the expression of miR23a in PCOS samplePCOS and healthy control Normal b miR27a and miR242 levels were detected using qPCR in PCOS and normal group Correlation betweenmiR23a level and BMI was analyzed in PCOS c and control d group Correlation between miR23a level and LH was analyzed in PCOS econtrol f group Correlation between miR23a and GLU level was analyzed in PCOS g and control h group Correlation between miR23a andINS level was analyzed in PCOS i control j group Correlation between miR23a and T level was analyzed in PCOS k and control l groupP P P The correlation between the expression of miR23a andclinical index of PCOS patientsWe further analyzed the correlation between the expression of miR23a and clinical index As shown in Table the BMI of PCOS patients were significantly higher thanthat of healthy controls P The correlation analysis showed that there was a positive correlation between serum miR23a level and BMI in PCOS patientsFig 1b P r but no correlation wasfound in healthy control group Fig 1c P r As shown in Table the serum LH concentration in PCOS patients was ± mIUmL whichwas significantly higher than that in healthy women ± mIUmL P Furthermore therewas a negative correlation between serum miR23a leveland LH concentration in PCOS patients Fig 1d P r but no correlation was found in healthy controlgroup Fig 1e P r The serum miR23alevel was also negative correlated with GLU Fig 1fP r INS Fig 1h P r and T Fig 1j P r concentration inPCOS patients but not in healthy control group GLUFig 1g P r INS Fig 1i P r and T Fig 1k P r MiR23a inhibits the proliferation of cov434 cellsIn this study the expression of miR23a in three humangranulosa cell lines was detected by qPCR As shown inFig 2a the expression level of miR23a was lowest incov434 cells and highest in KGN cells Therefore wechose cov434 cell line for subsequent experiments Subsequently miR23aspecificsiRNA or mimic was transfected into cov434 cells to explore the role of miR23aAs shown in Fig 2b the expression of miR23a in cells 0cLin Journal of Ovarian Research Page of Fig MiR23a inhibits the proliferation of human ovarian granulosa cells a The expression of miR23a in three human ovarian granulosa celllines KGN cov434 and SVOG was detected by qPCR b MiR23a was overexpressed by the transfection of miR23a mimics c MiR23a was knockeddown by the transfection of miR23a inhibitor d CCK8 was performed to detect the proliferation of cov434 cells P P was significantly increased by the transfection of miR23a mimic P Similarly the expression of miR23a in cells was significantly knocked down by the transfection of miR23a inhibitor Fig 2c P Then CCK8 assay was performed to detect the effectof miR23a on the proliferation of cov434 cells Asshown in Fig 2d compared with the control group thetransfection of miR23a mimic significantly inhibited theproliferation of cov434 cells P on the contrarythe transfection of miR23a inhibitor significantly promoted the proliferation of cov434 cells P Thesedata proved that the expression level of miR23a was involved in the regulation of cov434 cell proliferationMiR23a induced cell cycle arrest on G0G1 phase ofcov434 cellsNext flow cytometry was used to detect the effect ofmiR23a on the cell cycle of cov434 As shown in Fig cells stagnated in G0G1 phase after transfection ofmiR23a mimic P and the proportion of cells inS phaseand G2M phase decreased significantlyP The results were consistent with the inhibition of cell proliferation by overexpression of miR23asuggesting that miR23a induced cell cycle arrest andthus inhibit cell proliferation in cov434 cells On thecontrary the proportion of G2M phase cells increasedsignificantly in the miR23a inhibitor group P while that of G0G1 and S phase cells decreased P The results showed that low expression of miR23a promoted cell cycle progression and thus cell proliferationMiR23a promotes apoptosis of cov434 cellsFlow cytometry was performed to detect the effect of theexpression of miR23a on the apoptosis of cov434 cellsAs shown in Fig apoptotic cells increased significantlyP after the transfection of miR23a mimic anddecreased significantly P after the transfection ofmiR23a inhibitor These results suggested that overexpression of miR23a promoted apoptosis while low expression of miR23a inhibited apoptosisFGD4 is the bind target of miR23a in cov434 cellsThen we predicted six novel potential target of miR23avia the analysis on bioinformatics software Target ScanSubsequently the results of double luciferase reporterassay proved that only FGD4 could bind to miR23a directly through predicted sites The binding sites areshown in Fig 5a Cotransfection of miR23a mimicinhibited the luciferase activity of FGD4WT plasmidP but had no effect on the luciferase activity ofFGD4Mut plasmid Fig 5b The results showed thatmiR23a and FGD4 bind directly through predictive sitesThe effect of miR23a on the expression of FGD4 incov434 cells was investigated using qPCR and westernblot As shown in Fig 6a the expression of FGD4 wassignificantly decreased by the transfection of miR23amimic P whereas the transfection of miR23a 0cLin Journal of Ovarian Research Page of Fig MiR23a induced cell cycle arrest on G0G1 phase of cov434 cells a Flow cytometry was used to detect the effect of miR23a on the cellcycle of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell cycle P inhibitor significantly increased the mRNA expression ofFGD4 in cov434 cells P As shown in Fig 6b andc the protein level of FGD4 was significantly decreasedby the transfection of miR23a mimic P whereasthe protein level of FGD4 was significantly increasedby miR23a inhibitor P Combining with thedouble Luciferase Report experiment these results indicated that miR23a physically bind to the ²UTRregion of FGD4 thereby regulating the level of FGD4in cov434 cellsMiR23a induces the activation of CDC42PAK1 signalingpathway in cov434 cellsCDC42 is a member of the Rho GTPase protein familyFGD4 is responsible for activating CDC42 through GTPexchange of GDP PAK1 a serinethreonine kinase wasinitially identified as a protein interacting with CDC42[] CDC42PAK1 signaling pathway involved in theregulation of cell proliferation apoptosis and cell cycle[] As shown in Fig 6d the protein expression of activated CDC42 GTP bround was significantly increasedby the transfection of miR23a mimic P and significantly decreased by the transfection of miR23a inhibitoreffect of miR23a on theexpression of pPAK1 protein was similar to that ofCDC42 protein Fig 6fP TheDiscussionIn this study we explored the differences in serum levelsof miR23a between PCOS patients and normal womenas well as the effects of miR23a on biological behaviorsuch as proliferation and apoptosis of cov434 cells andrelated specific molecular mechanisms in order to provide limited theoretical support and experimental datafor the application of miRNA in PCOS treatmentFirstly we found that compared with healthy womenthe serum level of miR23a in PCOS patients decreasedsignificantly According to previous reports the level ofmiR23a in patients with ovarian disease remains uncertain Yang reported that miR23a was highlyexpressed in the plasma from premature ovarian failure POF patients compared with controls with afold change [] However Dang et alfoundthat miR23a is downregulated in the plasma ofChinese patients with premature ovarian failure []This inconsistency may be caused by individual differences and low sample size MiR23a level in patientswith ovarian disease still needs to be verified in alarge number of samplesMoreover miR23a was positively correlated with BMIand negatively correlated with serum LH T Glu andINS concentration Hyperandrogenism and hyperinsulinemia in PCOS patients are the most important physiological changes exacerbating endocrine disorders [] 0cLin Journal of Ovarian Research Page of Fig MiR23a promotes apoptosis of human ovarian granulosa cells a Flow cytometry was used to detect the effect of miR23a on theapoptosis of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell apoptosis P MiR23a is closely related to the changes of hormonelevels suggesting that it may be involved in the progression of PCOS and is a potential clinical treatment targetMurri also reported an inverse relationship betweenBMI and LH concentrations in patients with PCOS []Serum is composed of multiple components from a variety of tissues and ans Therefore the concentration ofmiR23a in serum is regulated by a variety of componentsand factors In addition the results also indicated that thedecrease in miR23a had a negative impact on the occurrence of PCOS and the increase in LHThen we investigated the role of miR23a in cov434cells We have found that miR23a can affect the proliferation of cov434 cells by regulating cell cycle and participate in the regulation of cell apoptosis through aseries of cell functional studies It has been shown thatmiR23a is closely related to apoptosis by inhibiting theexpression of Apaf1 and Bcl2 apoptotic proteins including Noxa Puma and Bax in neurons [] It hasalso been reported that miR23a protects differentiatedembryonic stem cells from apoptosis induced by bonemorphogenetic protein BMP4 by targeting SMAD5[] These data provide strong support for our resultssuggesting that miR23a may be closely related to granulosa cell apoptosis through a variety of pathwaysThese results suggest that miR23a may be closely related to the pathogenesis and development of PCOSTherefore we further study the molecular mechanism ofmiR23a involved in the proliferation and apoptosis ofcov434 cells The biological functions of miRNAs depend mainly on their effects on targets The same microRNAs may have hundreds oftarget proteins thosechange with cell type and cell state MiR23a can promote the apoptosis of cov434 cells by affecting the expression of multiple targets [ ] At presentmany targets have been found including Xlinked inhibitor of apoptosis protein XIAP SMAD5 and Sirt1 [] In this study we found FGD4 as a new target ofmiR23a The direct interaction between the ²UTR region of FGD4 mRNA and the expression of miR23awas demonstrated by double luciferase reporter assayThe results of qPCR and Western blot showed thatoverexpression of miR23a inhibited the expression ofFGD4 at the level of protein and mRNA while low expression of miR23a promoted the expression of FGD4at the level of protein and mRNAFGD4 is a Guanine Nucleotide Exchange Factor GEFspecific to CDC42 Rho GTPase and also an Factinbinding protein which is essential for maintaining myelin formation in Schwann cells [] FGD4 consists of N 0cLin Journal of Ovarian Research Page of Fig FGD4 binds to miR23a via the ™UTR in cov434 cells a the binding site of miR23a to ™UTR of FGD4 b Double luciferase reporter assaywas performed to confirm the binding between miR23a and FGD4™s ™UTR P terminal Factin bindingFAB domain Dbl homologyDH domain two pleckstrin homology PH domainand FYVE domain [] FGD4 has many functions including binding to Factin through FAB domain activating Rho GTPasetransduction pathway byincreasing the concentration of CD42 binding to GTPsignalThe structure domain of FGD4 indicates that it acts as acrosslinker between membrane structure and actincytoskeleton therefore the functional deletion mutationof FGD4 coding gene may result in truncated FGD4 expression and lead to motor sensory neuropathy orCharcotMarieToothCMTtype [ ] TheFig MiR23a induces the activation of CDC42PAK1 signaling pathway in cov434 cells a The expression of FGD4 was detected using qPCR incov434 cells with transfection of miR23a mimics or inhibitor b Western blot was performed to detect the levels of CDC42 and pPAK1 incov434 cells with transfection of miR23a mimics or inhibitor c Column diagram showed the expression level of FGD4 d Column diagramshowed the expression level of CDC42 e Column diagram showed the expression level of pPAK1 0cLin Journal of Ovarian Research Page of mutation is mediated by inhibiting guanine nucleotideexchange leading to the decrease of CDC42 activity andthe demyelination of peripheral nerves ultimately However in this study mir23a expression and function wereonly studied by using patients™ peripheral blood and celllines cultured in vitro The expression and function ofmir23a in vivo and patients™ ovarian cells still need further verificationIn addition recent studies have shown that FGD4 expression in prostate cancer clinical samples is significantly upregulated compared with the normal groupand downregulation expression of FGD4 in prostatecancer cell lines can cause cell cycle arrest and proliferation reduction [] It seems that FGD4 is also involvedin the tumorigenesis of nasopharyngeal carcinoma dueto its activation of CDC42 [] Studies have shown thatactivated CDC42 regulates downstream signals such asPAK1 WASP and ACK PAK1 as a serinethreoninekinase was originally identified as a protein that interacts with CDC42 and was subsequently found to serveas a downstream node for various oncogenic signalingpathways Studies have shown that the CDC42PAK1signaling pathway involved in cell cycle proliferationand apoptosis regulation [] Our study found thatmiR23a affects the expression of FGD4 as well as theprotein levels of activated CDC42 GTP bround and pPAK1 Therefore we hypothesized that miR23 regulated CDC42PAK1 signaling pathway by targetingFGD4 expression ultimately affecting apoptosis ofcov434 cellsIn our study reveals that the serum level ofmiR23a is significantly downregulated in PCOS patients and that miR23a participates in the regulation ofproliferation and apoptosis of cov434 cells through target FGD4 which may have potential for clinical treatment of PCOS patientsAcknowledgementsNot applicableAuthors™ contributionsJL and HH mainly performed the experiments and analyzed the data JL was amajor contributor in writing the manuscript LL helped with the data analysisand carried out the experiment design WL and JH helped with theexperiments and analysis All authors read and approved the final manuscriptFundingThis study was supported by Ningde medical technology improvementprojectAvailability of data and materialsAll data generated or analysed during this study are included in thispublished Ethics approval and consent to participateThis research study was approved by the Institutional Review Board of FujianMedical UniversityConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Graduate School Fujian Medical University Fuzhou China 2The 900thhospital of the Joint Service Support Force of the Chinese People™sLiberation Army Fuzhou China 3Gynaecology Mindong Hospital in NingdeCity No Heshan Road Fuan Fujian ChinaReceived December Accepted July ReferencesUtiger RD Insulin and the polycystic ovary syndrome Diabetes Res ClinPract Polak K Czyzyk A Simoncini T Meczekalski B New markers of insulinresistance in polycystic ovary syndrome J Endocrinol Invest “httpsdoi101007s4061801605238Trikudanathan S Polycystic ovarian syndrome Med Clin N Am “American College of Obstetricians and Gynecologists' Committee onPractice Bulletins”Gynecology ACOG Practice Bulletin No PolycysticOvary Syndrome Obstet Gynecol 20181316e157“ httpsdoi101097AOG0000000000002656Li X The role of androgen in autophagy of granulosa cells from PCOSGynecol Endocrinol “OvidiuLeonard B miRNA expression profiling in formalinfixedparaffinembedded endometriosis and ovarian cancer samples OncoTargets Ther “ Maalouf SW Liu WS Pate JL MicroRNA in ovarian function Cell Tissue Res“Kim SH Paeonol inhibits anaphylactic reaction by regulating histamineand TNFα Int Immunopharmacol “Naji M Differential Expression of miR93 and miR21 in Granulosa Cellsand Follicular Fluid of Polycystic Ovary Syndrome Associating with DifferentPhenotypes Sci Rep Bindu M miR122 Regulates LHR Expression in Rat Granulosa Cells byTargeting Insig1 mRNA Endocrinology Chhabra R Dubey R Saini N Gene expression profiling indicate role of ERstress in miR23a27a24“ cluster induced apoptosis in HEK293T cellsRNA Biol “ Xiong W Circulatory microRNA 23a and microRNA 23b and polycysticovary syndrome PCOS the effects of body mass index and sex hormonesin an Eastern Han Chinese population J Ovarian Res Guo Y Sun J Lai D Role of microRNAs in premature ovarian insufficiencyReprod Biol Endocrinol Yang X Role of microRNAs in premature ovarian insufficiency ReprodBiol Endocrinol “ Dang Y MicroRNA223p is downregulated in the plasma of HanChinese patients with premature ovarian failure Fertil Steril “807e1 Nie M Yu S Peng S Fang Y Wang H Yang X miR23a and miR27apromote human granulosa cell apoptosis by targeting SMAD5 Biol Reprod httpsdoi101095biolreprod115130690 Alford C Toloubeydokhti T AlKatanani Y The expression of microRNAmiRNA mir23a and 23b and their target gene CYP19A1 aromatase infollicular cells obtained from women undergoing ART[J] Fertil Steril 88suppS1 Nie M miR23a and miR27a Promote Human Granulosa CellApoptosis by Targeting SMAD5 Biol Reprod S¸rensen AE MicroRNA Species in Follicular Fluid Associating WithPolycystic Ovary Syndrome and Related Intermediary Phenotypes J ClinEndocrinol Metab “ jc20153588 Wu C Exercise activates the PI3KAKT signal pathway by decreasingthe expression of 5αreductase type in PCOS rats Sci Rep Murri M Effects of polycystic ovary syndrome PCOS sex hormonesand obesity on circulating miRNA21 miRNA27b miRNA103 and miRNA expression J Clin Endocrinol Metab 20139811E1835“Sabirzhanov B Downregulation of miR23a and miR27a followingExperimental Traumatic Brain Injury Induces Neuronal Cell Death throughActivation of Proapoptotic Bcl2 Proteins J Neurosci “ 0cLin Journal of Ovarian Research Page of Musto A miR23a miR24 and miR27a protect differentiating ESCsfrom BMP4induced apoptosis Cell Death Differ “ Nie MY Yang X Physiological and pathological effects of miR23a and miR27a in ovary Horn M Myelin is dependent on the CharcotMarieTooth Type 4Hdisease culprit protein FRABINFGD4 in Schwann cells Brain “Kondo D A novel mutation in FGD4 causes Charcot“Marie“Toothdisease type 4H with cranial nerve involvement Neuromuscul Disord “ Zis P A novel mutation in the FGD4 gene causing CharcotMarietooth disease J Peripher Nerv Syst “Edwards D PRL3 increases the aggressive phenotype of prostatecancer cells inVitro and its expression correlates with highgrade prostatetumors in patients Int J Oncol “Liu HP EpsteinBarr VirusEncoded LMP1 Interacts with FGD4 toActivate Cdc42 and Thereby Promote Migration of NasopharyngealCarcinoma Cells PLoS Pathog 201285e1002690Kumar R Gururaj AE Barnes CJ p21activated kinases in cancer Nat RevCancer “ httpsdoi101038nrc1892Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliat

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

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

EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food This risk assessment coversedible parts of potato plants and other food plants containing GAstomato andaubergine In humans acute toxic effects of potato GAs asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea For these effects the CONTAMPanel identified a lowestobservedadverseeffect level of mg total potato GAskg body weight bwper day as a reference point for the risk characterisation following acute exposure In humans noevidence of health problems associated with repeated or longterm intake of GAs via potatoes hasbeen identified No reference point for chronic exposure could be identified from the experimentalanimal studies Occurrence data were available only for asolanine and achaconine mostly forpotatoes The acute dietary exposure to potato GAs was estimated using a probabilistic approach andapplying processing factors for food Due to the limited data available a margin of exposure MOEapproach was applied The MOEs for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the P95 exposure in all surveysFor adult age groups the MOEs indicate a health concern only for the food consumption surveys withthe highest P95 exposures For tomato and aubergine GAs the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data For horses farm andcompanion animals no risk characterisation for potato GAs could be performed due to insufficient dataon occurrence in feed and on potential adverse effects of GAs in these species European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityKeywords glycoalkaloids GAs solanine chaconine potato margin of exposure MOE food feedRequestor European CommissionQuestion number EFSAQ201600811Correspondence contamefsaeuropaeu Leon Brimer was a member of the Working Group on Glycoalkaloids in food and feed until August wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodPanel members Margherita Bignami Laurent Bodin James Kevin Chipman Jes 13us del Mazo BettinaGraslKraupp Christer Hogstrand Laurentius Ron Hoogenboom JeanCharles Leblanc Carlo StefanoNebbia Elsa Nielsen Evangelia Ntzani Annette Petersen Salomon Sand Dieter Schrenk TanjaSchwerdtle Christiane Vleminckx and Heather WallaceAcknowledgements The Panel wishes to thank the following for the support provided to thisscientific output Kelly Niermans The Panel wishes to acknowledge all European competentinstitutions Member State bodies and other anisations that provided consumption and occurrencedata for this scientific outputSuggested citation EFSA CONTAM Panel EFSA Panel on Contaminants in the Food Chain Schrenk DBignami M Bodin L Chipman JK del Mazo J Hogstrand C Hoogenboom LR Leblanc JC Nebbia CSNielsen E Ntzani E Petersen A Sand S Schwerdtle T Vleminckx C Wallace H Brimer L Cottrill BDusemund B Mulder P Vollmer G Binaglia M Ramos Bordajandi L Riolo F Rold 13anTorres R and GraslKraupp B Scientific Opinion “ Risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products EFSA Journal pp 102903jefsa20206222ISSN European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityThis is an access under the terms of the Creative Commons AttributionNoDerivs Licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodifications or adaptations are madeReproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderFigure Elsevier Figure Springer Figure American Chemical Society SpringerThe EFSA Journal is a publication of the European FoodSafety Authority an agency of the European UnionwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodSummaryThe European Commission asked EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food in particular in potatoes andpotatoderived products This risk assessment covers edible parts of potato plants and other foodplants containing GAs in particular tomato and aubergine Nonedible parts of GA containing plantshave not been considered with the exception of potato sprouts The Panel developed the draftscientific opinion which underwent a public consultation from February to April Thecomments received and how they were taken into account when finalising the scientific opinion werepublished in an EFSA Technical Report EFSA GAs are present in many plants of the family of Solanaceae and contribute to plant resistanceagainst pests and pathogens GAs are composed of a steroidal aglycone and an oligosaccharide sidechain In commercial potato cultivars S tuberosum the main GAs are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively The aubergine fruit S melongena contains primarily the GAs asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively Inlycopersicum atomatine and adehydrotomatine are the major GAs withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelySHuman risk assessmentIn experimental animals the potato GAs asolanine and achaconine show a relatively low oralbioavailability with differences between species Hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats Due to the limited information themetabolic profiles of potato GAs in experimental animals could not be characterisedIn humans asolanine and achaconine are systemically absorbed following ingestion For bothsubstances relatively long serum halflives were reported suggesting a possible accumulation The bloodclearance of the respective aglycone solanidine appears to be slow Accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of GAs Nofurther information is available on metabolism and excretion of potato GAs in humansThere are no toxicokinetic data on tomato and aubergine GAs and their aglycones in experimentalanimals and humansIn acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice Reliable data on other potatoGAs or tomato and aubergine GAs and their aglycones are missingIn repeated oral dose studies on potato GAs rodents showed nonspecific effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine Hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw Solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the GAsThe tomato GA atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day At higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats In mice a to 2weektreatment with the aubergine GA asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per dayDevelopmental studies have been performed mainly in hamsters treated with potato GAs and theiraglycones for only one day or for a short very restricted time period during gestation Outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia These malformations occurred at dosesof mgkg bw per day and above for GAs and of mgkg bw per day and above for theaglycones No noobservedadverseeffectlevelLOAEL could be identified from these studies Reduced postnatal survival of pups due to insufficientmilk production was reported when pregnant Holtzman rats had been exposed to mg of asolaninekg bw per day Studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine Decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectNOAEL orlevelwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodfor month Similar effects were observed in Rhesus monkeys exposed to mgkg bw per day for monthsFrom the limited number of studies available there was no evidence for genotoxicity of the potatoGAs asolanine and achaconine and the aglycone solanidine as well as for the aubergine GA asolamargine However there is not sufficient information to conclude on the genotoxic potential ofthese GAsNo longterm chronic toxicitycarcinogencity study for potato tomato or aubergine GAs or for therespective aglycones could be identifiedIn humans acute toxic effects following ingestion of potato GAs include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato GAs potato TGA intake of mgkg bw or more Further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeaIn severe cases paralysis respiratory insufficiency cardiac failure coma and death have beenreported Doses in the range of “ mg potato TGAskg bw are considered to be potentially lethal forhumans Results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato GAsRegarding the mode of action adverse effects of GAs may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesAfter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeaGAs inhibit acetylcholinesterase AChE and serum butyrylcholinesterase BuChE by a reversiblecompetitive mode of action The relative potency of inhibition of asolanine and achaconine appearsto be similar The aglycones exert weak or no inhibitory effects The excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with GAsAt high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol As a consequence blood cholesterollevels were lowered in rodentsThe CONTAM Panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato GAs in humans The CONTAM Panel selectedthe LOAEL of mg potato TGAkg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers The available data onacute toxicity were considered insufficient to establish a healthbased guidance value Instead thePanel used the margin of exposure MOE approach to assess a possible health concern from acuteexposure to potato TGAs via foodAssuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of AChE activity the Panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics Accordingly anMOE higher than indicates that there is no health concern This MOE of takes into account theextrapolation from a LOAEL to a NOAEL a factor of and the interindividual variability intoxicodynamics a factor of The experimental data available for repeated dose toxicity are not sufficient to identify a referencepoint for chronic exposure to potato GAs In humans no evidence of health problems associated withrepeated or longterm intake of GAs via potatoes has been identifiedRegarding GAs or aglycones occurring in edible parts of food plants other than S tuberosum nosuitable study for determining a reference point for tomato or aubergine GAs or aglycones wasidentifiedOccurrence data were only available for asolanine and achaconine and mostly for ˜Maincroppotatoes™ and ˜New potatoes™ Few data were available for processed food No data on the occurrenceof tomato and aubergine GAs and their aglycones were submitted to EFSASince the occurrence data on potato GAs did not cover all the food categories containing potatoesin the Consumption Database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities RPC maincrop potatoes andnew potatoes and the RPC Consumption Database The Panel decided to combine the occurrence of˜New potatoes™ with that of ˜Maincrop potatoes™ and the mean upper bound UB occurrence sum ofwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the P95 occurrence was mgkg The minimum and maximum reported concentrations were and mgkg respectivelyThe acute dietary exposure to potato TGAs was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes As no occurrence data wereavailable for GAs in tomato and aubergine these foods were not included in the exposure assessmentProcessing of potatoes has been reported to reduce the content of GAs in the final processedproduct In general and according to the literature the peeling of potatoes reduced the GA contentby “ boiling in water and blanching of peeled potatoes by “ and frying in oil of peeledpotatoes by “ Microwave and oven baking of unpeeled potatoes may cause a reduction in theGA content by “ and by “ respectively No information has been found about thechemical nature of the GA degradation products For the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsInformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled Where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedThe mean UB exposure to potato TGAs across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers The 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the confidence intervalComparing the LOAEL for potato TGAs of mgkg bw per day with the acute exposure estimatesthe MOEs for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the P95 exposure in all surveys For adult age groups theMOEs indicate a health concern only for the food consumption surveys with the highest P95exposuresThe CONTAM Panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato TGA intake may be below the MOE of The highestnumber of survey days with intake of potatoes below the MOE of was estimated for toddlers followed by children For the other age groups the estimated TGA intake was below the MOEof in up to “ of the survey daysFor tomato and aubergine GAs the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansThe CONTAM Panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato GAs in food is moderate and that overall the identified uncertainties may eithercause an over or underestimation of the riskFarm animals horses and companion animals risk assessmentInformation on the toxicokinetics of GAs was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows™ milkNo data on the potential adverse effects of potato GAs in horses companion animals cats anddogs or fur animals were identified Due to an insufficient database on the adverse effects of GAs inruminants pigs poultry rabbits and fish an acute reference dose could not be derivedPotatoes are not grown specifically as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs Some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsData on potato GAs in feed were insufficient to perform an exposure assessmentThus no risk characterisation could be performed due to insufficient occurrence data of GAs forfeed and the lack of or limited data on the adverse effects of GAs in farm animals horses orcompanion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodRecommendationsThe following needs have been identified to improve the risk assessment for humans and reducethe uncertaintiescid129Research on the occurrence of GAs and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 Occurrence data on GAs and their aglycones in potato processed products including foods forinfantscid129 Occurrence data on GAs and their aglycones in tomato and aubergine and products thereofcid129 Data on the toxicokinetics of potato tomato and aubergine GAs and aglycones in experimentalanimals and humanscid129 Data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine GAs and aglycones in experimental animalsStudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129The following needs have been identified to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 Occurrence data on potato GAs and their aglycones in feedcid129Studies on the kinetics and the potential adverse effects from feed material containing GAs ofpotato GAs in farm animals horses and companion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodTable of contentsAbstractSummaryIntroduction Background and Terms of Reference as provided by the requestor Interpretation of the Terms of Reference Supporting information for the assessment Chemistry Analytical methods Sources Potatoes Tomatoes Aubergine Previous risk assessments Legislation and other standards Data and methodologies Methodology for data collection selection of evidence and study appraisal Food and feed occurrence data submitted to EFSA Data collection and validation Data analysis Food and feed consumption data Food consumption data Feed consumption data Food classification Methodology for Exposure assessment Methodology for Risk characterisation Assessment Hazard identification and characterisation Toxicokinetics Experimental animals aSolanine aChaconine Humans Mixtures of asolanine and achaconine Solanidine Biomarkers of exposure Farm animals horses and companion animals Summary on toxicokinetics Toxicity in experimental animals Acute toxicity studies GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Summary on acute toxicity studies Repeated dose toxicity studies GAs and aglycones from edible parts of S tuberosum GAs and aglycones from edible parts of food plants other than S tuberosum Developmental and reproductive toxicity studies Developmental effects Reproductive effects Immunotoxicity studies Studies on cardiovascular effects Neurotoxicity studies Genotoxicity GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Carcinogenicity studies Studies on metabolic effects GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Observations in humans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and food GAs from S tuberosum Reports on intoxications Studies in human volunteers Epidemiological studies Summary GAs from food plants other than S tuberosum Case Reports Adverse effects in farm animals horses and companion animals Ruminants Pigs Poultry Rabbits Fish Horses Companion animals cats and dogs Fur animals Reports on intoxications Mode of action Membrane effects with implications for the gastrointestinal tract Inhibition of cholinesterases ChEs Comparative determination of inhibition of ChEs in vitro Determination of inhibitory constants Ki for GAs on inhibition of ChEs in vitro Inhibition of ChEs in vivo Developmental and reproductive effects of GAs and their aglycones Inhibition of cholinesterases and effects in the immune system Interference with metabolism Considerations of critical effects and doseresponse analysis for the human risk assessment GAs from edible parts of S tuberosum Considerations of critical effects and doseresponse analysis Derivation of a healthbased guidance value HBGV or margin of exposure MOE approach GAs from edible parts of food plants other than S tuberosum Considerations of critical effects and doseresponse analysis Consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment Occurrence data Occurrence data submitted to EFSA Previously reported occurrence data in the literature Literature on occurrence data on food Occurrence data on GAs in potatoes Occurrence data on GAs in tomatoes Occurrence data on GAs in aubergines Occurrence data on GAs in other food products Literature occurrence data in feed Influence of storage and processing on the content of GAs GAs from S tuberosum Storage of potatoes Processing of potatoes for food consumption Processing of potatoes for feed GAs from food plants other than S tuberosum Summary on the influence of storage and processing on the levels of GAs Exposure assessment Current acute dietary exposure assessment for humans Previously reported dietary exposure assessments Current dietary exposure assessment for farm animals horses and companion animals Risk characterisation Human health risk characterisation GA from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Farm animals horses and companion animal risk characterisation Uncertainty analysis Assessment objectives Exposure scenarioexposure model wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodHazard identification and characterisation Summary of uncertainties Conclusions Hazard identification and characterisation Toxicokinetics Toxicity in experimental animals Observations in humans Adverse effects in farm animals horses and companion animals Mode of action Margin of exposure MOE approach Occurrence and exposure Food Feed Risk characterisation Human health risk characterisation Farm animals horses and companion animal health risk characterisation Recommendations Documentation provided to EFSA References Abbreviations Appendix A “ Major glycoalkaloids and their aglycones present in Solanum species Appendix B “ Identification and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food Appendix C “ Details of the study design of the toxicokinetic studies Appendix D “ Comparison of developmental toxicity of single dose studies Appendix E “ Inhibition of cholinesterases by GAs Appendix F “ Rapid Alert System for Food and Feed RASFF reports on the presence of Solanum nigrum infood products Appendix G “ Studies on the toxicity of Glycoalkaloids not considered in the risk assessment Appendix H “ Additional scenario for the human risk characterisation Annex A “ Occurrence data in food and feed submitted to EFSA and dietary exposure assessment forhumans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodIntroductionBackground and Terms of Reference as provided by the requestorBackgroundMany plants in the family Solanaceae contain glycoalkaloids and they are considered to be naturaltoxins The plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine Their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcAmongst the most widely cultivated food crops aubergines tomatoes and potatoes are in theSolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowThe glycoalkaloids of most relevance to food safety are those occurring in the potato Thepredominant toxic steroidal glycosides in potato are asolanine and achaconine They occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors Concentrations ofglycoalkaloids are “ times greater in the peel than in the flesh There is considerable variation inglycoalkaloid content among potato cultivars Storage conditions especially light and temperature aremainly responsible for increases in solanine Although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light Increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel Thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsBitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include flulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea More severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision There are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriesPotatoes and potatoderived products are listed in the Catalogue of feed materials1Terms of ReferenceIn accordance with Art of Regulation EC No the European Commission asks theEuropean Food Safety Authority for a scientific opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsInterpretation of the Terms of ReferenceThe CONTAM Panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids GAs eg tomato and aubergine Nonedible parts ofGA containing plants have not been considered with the exception of potato sprouts In particular theCONTAM Panel concluded this Opinion should comprise thea evaluation of the toxicity of GAs in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid profile ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to EFSAc estimation of the dietary exposure of the European population to GAs in food in particular inpotatoes and potatoderived products including the consumption patterns of specific groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to GAs in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the European population including specific groupsof the population if appropriate as the consequence of the estimated dietary exposure Commission Regulation EU No of January on the Catalogue of feed materials OJL p wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in Europe as the consequence ofthe estimated dietary exposure Exposure to GAs from weeds containing GA is only addressedin this Opinion in the context of accidental intake by farm animalsWhen referring to GAs in potatoes the term total GAs TGA refers to a material comprising asolanineand achaconine as major fraction with no specification on the occurrence of minor GAs as well as band cforms of solanine and chaconine Similarly when referring to tomato and aubergine the termTGA refers to the GAs from the corresponding species and forms thereofSupporting information for the assessment ChemistrySolanine is one of the first alkaloids that has been isolated from nature by Desfosses in Friedman et al In Zwenger and Kind reported that solanine contains a glycoside sidechain Zwenger and Kind Only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids GAs asolanine and achaconine that share the same solanidineaglycone Kuhn and L‚¬ow Since then at least different GAs have been isolated and fullystructurally elucidated from over species of the Solanaceae family S 13anchezMata et al AlSinani and Eltayeb The chemical structures and some physical properties of the most importantones are listed in Appendix AGAs are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see Figure Friedman et al Friedman Milner et al The GAs of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone GAs often contain a double bond between C5 and C6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents The stereochemistry at carbons C22 and C25 is well definedtheconfiguration is 22R 25Stheitconfiguration is 22S 25S Friedman et al in solanidineis 22R 25R and in tomatidenoltomatidinein solasodineFurther diversification is generated by the composition of the glycoside sidechain Most GAscontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate In commercial potato cultivars Solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentFigure Wild S tuberosum varieties may contain a much wider range of GAs Friedman et al Distl and Wink The aubergine fruit derived from S melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose Friedman derived from SIn tomato fruitThe prefix alpha a refers to the intact glycoside while the prefixe

Thyroid_Cancer

distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedVon Hippel“Lindau disease is an autosomal dominant inherited syndrome predisposing to a variety of highly vascularised tumorsin diï¬erent ans Although bilateral pheochromocytoma was reported in patients with von Hippel“Lindau disease the coexistence of primary hyperparathyroidism is not a common condition We report an observation of a primary hyperparathyroidism secondary to an ectopic secretion of intact parathyroid hormone in a 17yearold girl with von Hippel“Lindau diseaseand bilateral pheochromocytoma She presented with a newly diagnosed diabetes mellitus and a severe arterial hypertensionBlood tests disclosed hypercalcemia with increased intact PTH level Cervical ultrasound and sestamibi scintigraphy were normalTwentyfourhour urinary normetanephrine level was highly elevated pointing to a catecholaminesecreting tumor e abdominal computed tomography showed bilateral adrenal masses MIBG scintigraphy exhibited a high accumulation of the tracerin both adrenal tumors Genetic testing revealed a mutation of the VHL gene e patient underwent a bilateral adrenalectomye postoperative outcome was marked by normalization of blood pressure blood glucose calcium and PTH levels In our casethe elevation of intact PTH and its spontaneous normalization after surgical treatment of pheochromocytomas confirms itsectopic secretion IntroductionPheochromocytomas are uncommon neuroendocrinetumors that arise from chromaffin cells of the adrenalmedulla and produce excessive amounts of catecholamines epinephrine norepinephrine and dopamine []Although most pheochromocytomas are sporadic morethan are associated with an inherited mutation andthis frequency can be as high as if diagnosed before years of age [] In childhood pheochromocytomasare mostly due to genetic causes including von Hippel“Lindau VHL disease multiple endocrine neoplasiatype MEN2 hereditary pheochromocytoma paraganglioma syndrome and rarely neurofibromatosis type [] Compared to adults VHL disease is reported to bethe most frequent genetic disorder causing pheochromocytomasbilateral[] Whileinchildrenpheochromocytomas were reported in patients with VHLdisease the coexistence of primary hyperparathyroidismis not a common condition []Herein we report a case of a VHL disease with bilateralintactpheochromocytoma and an ectopic secretion ofparathyroid hormone in an adolescent girl Presentation of CaseA 17yearold girl was referred to our department for theinvestigation of a newly diagnosed diabetes mellitus and asevere arterial hypertension She was the first child ofconsanguineous parents Her past medical history wasunremarkable Her family history was notable for type diabetes and dyslipidemia but no history of pheochromocytoma paraganglioma unexplained sudden death or 0cCase Reports in Endocrinologycondition that may lead to thinking about VHL disease wasreportedAs symptoms the patient complained of headachespalpitations diaphoresis and hot flashes since one monthShe reported also asthenia and body weight loss of fivekilograms in two weeksOn examination she had a body weight of kg abody height of cm a body mass index of kgm2 ablood pressure of mmHg without orthostatichypotension a regular pulse of bpm and a largeabdominal caf´eaulait spot Figure yroid abdominal and neurological examinations were normalTwentyfourhour blood pressure monitoring confirmed the diagnosis of hypertension and the presence ofpeaks of mmHg Electrocardiogram showed asinus tachycardia Capillary glucose level was glwithout ketosisFundoscopy showed grade hypertensive retinopathywithout any other abnormalitiese results of biological investigations are shown inTable e diagnosis of clinically suspected pheochromocytomawas confirmed by the dosage of urinary methoxylated derivatives at times the upper limit of normal In addition thediagnosis of diabetes mellitus dyslipidemia and primaryhyperparathyroidism were madethe first on thee abdominal computed tomography CT showed tworight measuringadrenal masses — — mm with a spontaneous density of HUheterogeneously enhanced in the arterial time showing areasof necrosis with an absolute washout of the second onthe left measuring — mm with the same characteristicsas the first one Figure ere were neither other localizations norlymph nodes MetaiodobenzylguanidineMIBG scintigraphy exhibited a high accumulation of thetracer in both adrenal tumors with no other localizationCervical ultrasound and 99mTcsestamibi scintigraphy werenormal Cardiac ultrasound was normale diagnosis of multiple endocrine neoplasia type 2awas highly suspected and the patient underwent a molecularinvestigation DNA analysis did not find a RET protooncogene mutation However it showed a missense mutationc191G C pArg64Pro in exon of the VHL gene onchromosome e diagnosis of bilateral pheochromocytoma in the setting of VHL disease was established Abdominal CT scan and craniospinal magnetic resonanceimaging with contrast did not show any cysts or othertumorse patient was treated with an alpha blocker prasozinea calcium channel blocker a beta blocker and insulin at adaily dose of unitskg She underwent a bilateral adrenalectomy in two steps e pathological examinationconfirmed bilateral adrenal pheochromocytomas Replacementtherapy with hydrocortisone was initiated aftersurgerye postoperative outcome was determined by thespontaneous normalization of blood pressure blood glucose calcium and PTH levels e patient remainedasymptomatic with no evidence oflocal recurrence ordistant metastasis during the months of followup efamily screening for VHL has not been performed yet DiscussionVHL disease is a dominantly inherited familial cancersyndrome caused by a germline mutation in the VHL tumorsuppressor gene and predisposing to a variety of benign andmalignant neoplasms most frequently retinal central nervous system and spinal hemangioblastomas renal cell carcinoma RCC pheochromocytoma and pancreatic tumors[ ] While central nervous system and retinal hemangioblastomas are the earliest expressions of the VHL syndrome pheochromocytoma may be the first manifestationof the disease especially in children and adolescents as it wasthe case of our patient [“] e frequency of pheochromocytoma in VHL syndrome is about to []Families with VHL disease have been divided into twosubtypes VHL type and VHL type based on the likelihood of developing a pheochromocytoma e presence ofpheochromocytoma defines types VHL disease is latteris subdivided based on the risk of developing RCC Type 2Aand 2B families have a low and high incidence of RCCrespectively whereas VHL type 2C kinds are characterizedby the development of pheochromocytoma without anyother manifestations of the disease [] However late onsetof other attacks is possible and a followup even spaced isrequired []Genotypephenotype correlations have been documented for this disorder and specific mutations are associated with the appearance of tumors in certain ansWhile most type families were reported to be more likelycarrying a missense mutation in the VHL gene most type families are aï¬ected by truncating or deletion mutations[] In our case the presence of pheochromocytomas andthe missense mutation in VHL gene suggested type VHLdisease Moreover the mutation found in our patientpArg64Pro has been described in patients with isolatedpheochromocytoma associated with RCC or with pancreaticneuroendocrine tumor [“] However our patient didnot present any sign of RCC or pancreatic neuroendocrinetumor during the months of followup is evaluationmay be early in our case as RCC and pancreatic neuroendocrine tumors in patients with VHL disease generallyappear at more advanced ages around “ years []Pheochromocytoma in VHL disease tends to be seen at ayounger age and is more frequently multifocal as in ourpatient and may be extraadrenal [ ] In most publishedcases the mean age at presentation was about years butvery young cases have been described the youngest before years [ ] In addition VHLassociated pheochromocytomas are less likely to be associated with symptoms orbiochemical evidence of catecholamines production compared with those occurring in patients without VHL [ ]In a report of the National Institute of Health about patients with VHL disease and pheochromocytomas a totalof tumors were identified Of these originatedoutside the adrenal gland and of the patients wereasymptomatic without hypertension orevidence of 0cCase Reports in EndocrinologyFasting glucose level mmollGlycated hemoglobin Total cholesterol mmollTriglycerides mmollNatremia mmollKalemia mmollCreatinine mgLCalcium mgLPhosphate mgLAlbumin glIntact PTHˆ— μgLPTH parathormone TSH thyroid stimulating hormone FT4 freeT4 NMN normetanephrines MN metanephrinesˆ—e intact PTH dosage was madeTSH mUILFT4 ngdlCalcitonin ngL hurine NMN μg24 hurine MN μg24 hurine sodium mmol24 h hurine potassium mmol24 h hurine calcium mg24 h hproteinuria g24““using 3rd generation chemiluminescence immunoassayReference ranges‰¤““““““““““““Figure A large abdominal caf´eaulait spotTable Biological parameters before and after surgeryBefore surgeryAfter surgery””””””””Figure Abdominal computed tomography showing the two adrenal masses two white arrowsincreased catecholamines production [] is was not thecase of our patient who had sustained severe hypertensionassociated with the classic symptoms of pheochromocytomapalpitation sweating and hot flashes hypokalemia and asecondary diabetes mellitus In our case as reported in theliterature [ ] a remarkable remission of diabetes 0cCase Reports in EndocrinologySoahighmellitus and an improvement of lipid profile were noticedafter tumor removal confirming the secondary character ofthese two metabolic disorders In fact catecholamine excessaï¬ects insulin secretion decreased glucose uptake in theperipheral tissues and increased insulin resistance leading toimpaired fasting glucose or overt diabetes mellitus []Moreover the increase in catecholamine production may beresponsible for decreased inhibition of lipolysis by insulinand decreased activity of lecithincholesterol acyltransferasean enzyme which breaks down free cholesterol []e risk of malignancy is low Less than of allpheochromocytomas in VHL disease are malignant [] Inour case neither distant metastasis nor lymph nodes werefound but a long term followup should be carried outMeasurement of plasma or urinary metanephrines andnormetanephrines is the gold standard in diagnosingpheochromocytoma and can also provide important diagnostic information [] In fact in a study carried out byEisenhofer [] comparing the clinical and biochemical characteristics of pheochromocytomas in multipleendocrine neoplasia type versus the VHL syndrome andincluding and patients with these disorders respectively VHL patients almost exclusively produced normetanephrinesnormetanephrinestometanephrines ratio is expected in patients with VHL disease as was found in our patient Furthermore in comparison with MEN2 tumors VHL tumors had lower totaltissue contents of catecholamines and expression of tyrosinehydroxylase TH the ratelimiting enzyme in catecholamine synthesis ey also had much lower expression ofphenylethanolamine Nmethyltransferase PNMT the enzyme that converts norepinephrine to epinephrine andtissue stores of epinephrine [] Regarding the histopathological features VHLassociated pheochromocytomas arecharacterized by a thick vascular tumor capsule and are incontrast to MEN not associated with adrenal medullarhyperplasia []While bilateral pheochromocytomas were reported inpatients with VHL disease the coexistence of primary hyperparathyroidism as in our case is not a common condition[] Hypercalcemia associated with pheochromocytoma hasbeen documented and is thought to be caused by severalmechanisms First elevated catecholamines can activate thePTH receptor resulting in catecholamineinduced osteoclastic bone resorption but in contrast to our case the PTHlevel is not elevated [] Secondly hypercalcemia can be dueto the production of PTHrelated peptide PTHrp by thetumor which was doubtless not the case in our patient as thelevel of intact PTH was elevated [] Furthermore intactPTH in our case was quantified using a 3rd generationchemiluminescence immunoassay which does not recognizePTHrpird parathyroid adenoma can be a part of multipleendocrine neoplasia that was however rarely reported inVHL disease [] e negativity of the topographic investigations and the spontaneous normalization of intact PTHafter surgical treatment are against this hypothesis Finallyalthough the exact physiopathological mechanism is notclear the fact that both serum calcium and PTH levels wereelevated before surgery and became normal after the removal of the pheochromocytomas strongly suggests that thetumor itself was secreting PTH or a substance that stimulatesexcessive PTH secretion by the parathyroid glands Only fewcases of ectopic hormonal secretion by pheochromocytomasuch as adrenocorticotropic hormone ACTH calcitoninparathyroid hormone PTH vasoactive intestinal peptideVIP and growth hormonereleasing hormone GHRHwere reported [ ] Unfortunately immunohistochemistry assay for PTH in the tumor tissue was not available toclarify this question in our case Conclusionis report highlights the rare case of ectopic intact PTHsecretion by a bilateral pheochromocytoma in an adolescentgirl with VHL disease We consider that controlling calciumand PTH after adrenalectomy is useful if the topographicassessment of primary hyperparathyroidism is negativeTo the best of our knowledge our patient is the secondyoungest reported childhood VHL case in the literaturepresenting with a bilateral pheochromocytoma secretingectopic intact PTH Genetic testing and a meticulous followup are necessary for the diagnosis ofthe associatedcomorbidities in VHL diseaseData 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 interestAcknowledgmentse authors thank Professor Anne Barlier Laboratory ofMolecular Biology Hospital La Conception MarseilleFrance for her help in molecular studyReferences[] J W M Lenders QY Duh G Eisenhofer œPheochromocytoma and paraganglioma an endocrine societyclinical practice guideline e Journal of Clinical Endocrinology Metabolism vol no pp “ [] E Sbardella T Cranston A M Isidori œRoutine geneticscreening with a multigene panel in patients with pheochromocytomas Endocrine vol no pp “ [] S G Waguespack T Rich E Grubbs œA current reviewof the etiology diagnosis and treatment of pediatric pheochromocytoma and paraganglioma e Journal of ClinicalEndocrinology Metabolism vol no pp “[] M Barontini G Levin and G Sanso œCharacteristics ofpheochromocytoma in a to 20yearold population Annals of the New York Academy of Sciences vol no pp “ [] T Arao Y Okada T Tanikawa œA case of von HippelLindau disease with bilateral pheochromocytoma renal cell 0cCase Reports in Endocrinology[] O Mete A S Tischler R D Krijger œProtocol for theexamination of specimens from patients with pheochromocytomas and extraadrenal paragangliomas Archives ofPathology Laboratory Medicine vol no pp “[] N O Atuk T McDonald T Wood œFamilial Pheochromocytoma Hypercalcemia and von HippelLindauDisease a ten year study of a large family Medicine vol no pp “ [] K Takeda N Hara M Kawaguchi T Nishiyama andK Takahashi œParathyroid hormonerelated peptideproducing nonfamilial pheochromocytoma in a child International Journal of Urology vol no pp “ [] J KirkbyBott L Brunaud M Mathonet œEctopichormonesecreting pheochromocytoma a francophone observational study World Journal of Surgery vol no pp “ [] L V Neto G F Taboada L L Corrˆea œAcromegalysecondary to growth hormonereleasing hormone secreted byan incidentally discovered pheochromocytoma EndocrinePathology vol no pp “ carcinoma pelvic tumor spinal hemangioblastoma and primary hyperparathyroidism Endocrine Journal vol no pp “ [] A N A V D HorstSchrivers W J Sluiter R C Kruizinga œe incidence of consecutive manifestations in VonHippelLindau disease Familial Cancer vol no pp “ [] R R Lonser G M Glenn M Walther œvon HippelLindau disease e Lancet vol no pp “[] A D ‡akır H Turan A Aykut A Durmaz O Ercan andO Evliyao˘glu œTwo childhood pheochromocytoma cases dueto von HippelLindau disease one associated with pancreaticneuroendocrine tumor a very rare manifestation Journal ofClinical Research in Pediatric Endocrinology vol no pp “ [] S P Rednam A Erez H Druker œVon hippellindauand hereditary pheochromocytomaparagangliomasyndromes clinical features genetics and surveillance recommendations in childhood Clinical Cancer Research vol no pp e68“e75 [] G F C Fagundes J Petenuci D M Lourenco œNewinsights into pheochromocytoma surveillance of young patients with VHL missense mutations Journal of the Endocrine Society vol no pp “ [] F Hes R V D Luijt A Janssen œFrequency of VonHippelLindau germline mutations in classic and nonclassicVon HippelLindau disease identified by DNA sequencingSouthern blot analysis and multiplex ligationdependentprobe amplification Clinical Genetics vol no pp “ [] T Krauss A M Ferrara T P Links œPreventivemedicine of von HippelLindau diseaseassociated pancreaticneuroendocrine tumors EndocrineRelated Cancer vol no pp “ [] E Wittstr¨om M Nordling and S Andr´easson œGenotypephenotype correlations and retinal function and structure invon HippelLindau disease Ophthalmic Genetics vol no pp “ [] G Eisenhofer M M Walther TT Huynh œPheochromocytomas in von HippelLindau syndrome and multiple endocrine neoplasia type display distinct biochemicaland clinical phenotypes e Journal of Clinical Endocrinology Metabolism vol no pp “ [] M M Walther R Reiter H R Keiser œClinical andgenetic characterization of pheochromocytoma in von HippelLindau families comparison with sporadic pheochromocytomaofpheochromocytoma Journal of Urology vol no pp “ naturalhistoryinsight[] J Cha M Khurram L Gellert P Epstein N Baregamian andC Hendrickson œCase of reversible diabetes mellitus in thesetting of benign Pheochromocytoma Journal of Clinical andTranslational Endocrinology Case Reports vol pp “[] M L Good P Malekzadeh S M Ruï¬ œSurgical resection of pheochromocytomas and paragangliomes is associated with lower cholesterol levels World Journal of Surgeryvol no pp “ [] B Mesmar S PoolaKella and R Malek œe physiologybehind diabetes mellitus in patients with pheochromocytomaa review of the literature Endocrine Practice vol no pp “ givesinto 0c'

Thyroid_Cancer

"attention has been paid to whether snoring frequency is associated with body composition inmenopausal women particularly in China This study objected to investigate the association between selfreportedsnoring and body composition in peripost menopausal Chinese women as well as metabolic indicatorsMethods This crosssectional study enrolled participants aged “ years from the Menopause Clinic in theShanghai Sixth People™s Hospital Participants were categorized into four subgroups stratified by selfreportedsnoring frequency never rarely night per week occasionally “ nights per week regularly ‰¥ nights perweek while body composition was measured using bioelectrical impedance analysis BIA Besides blood samplewere collected to test the glycolipid indicatorsResults In our sample of investigation regular snoring ‰¥ nights per week was found to be an independent riskfactor for higher fat mass total upper limbs trunk with the highest risk of times for fat mass of trunk afteradjusting for metabolic confoundersp Meanwhile regular snoring was independently associated withhigher fat mass total and each segment only in menopausal transition p Conclusions We suggested that selfreported regular snoring may be taken as a simple alternative to predict higherfat mass ‰¥ kg upper quartile in menopausal women Similarly body composition should be attached to thegreat importance to those who in menopausal transition in order to help to prevent obstructive sleep apnea OSAKeywords Body composition Snoring Menopausal transitionBackgroundSnoring the manifestation of increased upper airway resistanceis commonly regarded as a reliable proxymarker of obstructive sleep apnea OSA [ ] Moreover regular snoring has been suggested to be correlatedwith obesity [] hypertension [] and diabetes mellitus[] OSA is supposed to be more prevalent in men than Correspondence yctengsjtueducn taomfsjtueducn Yang Zhou and Fei Liu contributed equally to this work1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth People™s Hospital Yishan Road Shanghai ChinaFull list of author information is available at the end of the women however the gap was narrowed when womenapproach menopause [ ] Women in menopause transition are more likely to report perspective poor sleepsnoring [] which largely affected quality life of menopausal women In addition previous studies have reported that menopause was an important risk factor forsnoring mainly due to the declining ovarian hormones[ ] Thus it is important to combat snoring in peripost menopausal womenMeanwhile menopause is a vital window for variationsin the body composition and rising in the body weightcaused by hormonal alterations [] However body The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhou BMC Women's Health Page of in menopausal women [] Changesmass index BMI is not a valid measure of true obesitystatusinmenopauserelated body composition may be coveredand underestimated by stable BMI since the counteractive effect of loss of lean mass and gain of fat masswhen aging Therefore body composition by bioelectrical impedance analysis BIA may be a more representative and precise instrument rather than BMI amongmenopausal Chinese women []So far current studies on the association of snoringand obesity have focused primarily on men and children[ ] while underrepresented women In addition anyassociation between snoring and body composition inmenopausal women has received little attention Sinceit™s possible that glycolipid metabolism may confoundthe association and whether snoring is associated withbody composition in menopausal women independentlyof glycolipid metabolism confounders remains unknownGiven the evidence of the cross interplay among snoringobesity and menopause we aim to explore the association with snoring and body composition in menopausalwomenMethodsStudy participantsThis crosssectional study enrolled participants who visited the Menopause Clinic in the Shanghai Sixth People™s Hospital HanChinese woman aged “ yearspassing through the menopause were recruited Exclusion criteria were with rhinitis having severe internalsuch as myocardialinfarction stroke and cancer current smoking atleast once per week for the previous months excessive alcohol drinking at least one pack per month forthe previous months suffering from thyroid disease having tubercle and cachexy missing dataUltimately715 participants were recruited in this studyillnesses andor diseasesGeneral questionnaireBaseline sociodemographic information was collectedfrom a questionnaire through facetoface interviewwhich has been previously employed [] seen in supplementary file Variables included age marital statusemployment status education level income per monthmenopausal age menopausal status history of chronicdisease ie hypertension diabetes mellitus rhinitisother diseases besideslifestyle ie smoke alcoholconsumption were recorded Guiding by the Stages ofReproductive Aging Workshop STRAW []participants were divided into three different menopausalsubgroups namely menopausal transition group consecutive irregularities for over days of menstrual cycleearly postmenopausal group absence of menstrual periods for months ˆ’ years and late postmenopausalfor ‰¥ yearsgroup absence of menstrual periodsHypertension was defined by any prior diagnosis fromthe questionnaire or by the criteria recommended by theseventh report of the Joint National Committee on Prevention Detection Evaluation and Treatment of HighBlood Pressure JNC7 [] While diabetes mellitus wasidentified by FPG ‰¥ mmolL or received any treatmentfor diabetes according to the WHO criteria []Snoring frequency assessmentParticipants were asked by the question to assess thesleep snoring frequency which was applied previously[ ] œOver the past weeks did you snore And ifdid how many times per week and the options for responses were œnever œrarely œoccasionally and œregularly corresponding to œnever œ night per weekœ“ nights per week and œ‰¥ nights per week respectively seen in supplementary file Anthropometric and lab testsWe measured and recorded participants™ weight heightBody mass index BMI was computed by dividingweight in kilograms by the square of their height in meters We took the blood pressure for all participants onthe right arm three consecutive times after 5min sittingsystolic blood pressure SBP diastolic blood pressureDBP Blood samples were collected for the detectionof serum concentration of triglyceride TG cholesterolTC highdensity lipoprotein HDL lowdensity lipoprotein LDL and fasting blood glucose FBG after anovernight fastBody compositionWe measured the body composition by BIA TBF418Banalyzer TANITA of lean mass LM fat mass FMand fatfree mass FFM and each segment includedupper lower limbs and trunk We also recorded basalmetabolic rate BMR concurrently [] The welltrained staff guided the participants to take off heavyclothes socks and shoes and hold the hand electrodesstanding barefoot in contact with footpad electrodes[] Fat mass total and each segment and lean masstotal and each segment were stated in the dichotomized form with a cutoff of the highest quartile as thehigher one comparing the highest to the lower two tertiles We defined ‰¥ kg ‰¥ kg and ‰¥ kg ashigher total fat mass higher fat mass of upper limbs andhigher fat mass of trunk respectivelyStatistical analysesAll statistical analyses were taken by SPSS IBMCorporation Armonk NY USA Data were tested fornormal distribution bythe Kruskal WallisHtestLevene™s test of homogeneity of variance was also 0cZhou BMC Women's Health Page of performed Variables were presented as mean ± standarddeviation SD when they showed normal distributionswhereas medians inter quartile range or values Oneway ANOVA normal distributions the KruskalWallis Htest skewed continuous variables and χ2 testcategorical variables were carried out to compare thedifferences among the four groups Snoring was analyzedas a categorical variable with never as the referencegroup Relationship between body composition andsnoring frequency was computed by multiple logistic regression analysis Covariates included TG TC HDLLDL FBG SBP DBP age marital status employmentstatus education level income per month menopausalage menopausal status hypertension diabetes mellitusTwosided p was considered significantResultsCharacteristics of the study participants based on snoringfrequencyA total of participants were finally entered into thestudy The basic characteristics among the four groupsdivided by the snoring frequency never rarely occasionally regularly were presented in Table Participants were on average ± years of age with amean weight of ± kg and the average BMIwas ± kgm2 The mean lean mass fat massand fat free mass were ± kg ± kgand ± kg respectively Compared with nonsnorers rare and occasional snorers regular snorerstended to be older showed higher triglyceridelowerHDLC and had less income p Moreover therewas an ascending trend in the incidence of hypertensionin different snoring frequency subgroupswith innonsnorers increasing to in regular snorersp However we did not observe the differenceamong three menopausal status respect to the snoringfrequencyBody composition of the study participants distributed bysnoring frequencyAs presented in Table compared with nonsnorersrare and occasional snorers regular snorers had higherfat mass upper limbs trunk lower limbs In additionwe found that there was an increasing trend in the fatmass of upper limbs trunk and lower limbs and also inlean mass of upper limbs with the increase of the sleepsnoring frequency p Odds ratio of snoring frequency for body composition bymultiple logistic regression analysisWe next investigated the odds ratio of snoring frequencyin predicting for body composition p in univariate analysis after adjusting for potential confounders Asdepicted in Fig compared with nonrare andoccasional snoring regular snoring was the risk predictor for higher total fat mass ‰¥ kg OR 95CI P higher fat mass of upperlimbs ‰¥ kg OR 95CI P higher fat mass of trunk ‰¥ kg OR 95CI P while other segmentsshowed no significance after adjustments In additionregular snoring increased the highest odds ratio OR of for fat mass of trunk among the other statisticallysignificant body compositionsIndependent determinants for regular snoring stratifiedby menopausal statusWe also investigated the independent roles of body composition for predicting regular snoring in multivariate logistic regression analysis in Table however we did notobserve any significance of body composition in predicting for regular snoring after adjusting confoundersInterestingly when the participants were stratified bydifferent menopausaltransitionearly postmenopause late postmenopause we observedthat fat mass segments were independently associatedwith regular snoring in menopausal transition but notpostmenopause TotalOR 95CI P fat mass of upper limbs OR 95CI P fat mass of trunkOR CI P fat mass oflower limbs OR 95CI P were independent indicators for regular snoring afteradjusting for confounders in menopausal transitionmenopausalstatusfat massDiscussionTo our knowledge this is the first study to document associations of snoring and body composition as well asmetabolic indicators in women with regard to menopausal status The main finding was that regular snoring‰¥ nights per week was an independent risk factor forhigher fat mass total trunk upper limbs in menopausalwomen after adjusting for wellestablished metabolicvariables Of special concern was that regular snoringhad a times significantly higher odds of higher fatmass of trunk which was the highest among other significant body composition This finding was in concordant with the previous study that OSA was more inclinedto a centralobesity phenotype than a wholeobesity pattern []Several mechanisms can interpretthis associationUpper airway resistance and collapsibility caused byregular snoring could result in intermittent hypoxia andsympathetic activation thus leading to the aggravationof obesity especially for abdominal fat [] In additionprotective role of progesterone and estrogen in respiratory control vanished after menopause which was associated with continuum from increased airway resistance 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequencyVariablesRarelyn ± ± ± ± Occasionallyn ± ± ± ± Regularlyn ± ± ± ± Totaln ± ± ± ± ± “ ± ± “ ± ± “ ± ± “ ± ± ± ± ± ± ± “ ± ± “ ± ± “ ± ± “ ± ± “ ± ± ± ± ± ± Snoring FrequencyNevern ± ± ± ± ± “ ± Age yearsWeight KgHeight cmBMI Kgm2BMRTG mmollTC mmollHDLC mmollLDLC mmollFPG mmolLSBP mmHgDBP mmHgChronic disease n HypertensionDiabetesMarital status n MarriedSingleSeparatedDivorcedWidowedMenopausal status n Perimenopause Early postmenopause Late postmenopause Employment n Education n Junior or belowSenior highCollege or aboveIncome RMBmonth n “““ Lean mass kgUpperTrunkLowerFat mass kgUpperTrunkLower ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fatfree kg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P value 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequency ContinuedVariablesRarelyn ± ± ± Occasionallyn ± ± ± Regularlyn ± ± ± Totaln ± ± ± P valueSnoring FrequencyNevern ± ± ± Upper limbsTrunkLower limbsHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Higher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsRare snoringOR OR OR OR OR OR Occasional snoringHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Fig Odds ratios 95CI of snoring frequency for body composition in women analyzed by multivariate logistic regression Covariates age BMITG TC HDL LDL FBG SBP DBP hypertension diabetes mellitus menopause income education employment status means p0005Regular snoring 0cZhou BMC Women's Health Page of Table Odds ratio of body composition for regular snoring stratified by menopausal status by logistic regressionFat mass kgRegular snoringPerimenopause n Odds ratio CI Pvalue Fat mass of upper limbs kg Fat mass of trunk kg Fat mass of lower limbs kg manifested as snoring [“] Taken together menopause make women lose protective effects against snoring and further augmentsnoreobesity associationespecially snorecentralobesity associationInterests in obesity and OSA as regards to œwhich isthe chicken or the egg has existed since the dawn ofhistory [ ] Thus to identify the mutual effect ofsnoring and obesity we also assessed the role of bodycomposition in predicting the snoring We found thatfat mass was an independent risk factor for regular snoring only in menopausal transition not postmenopause ina multivariable model Taken together we suggestedthat the rise in obesity may serve as a key contributor tothe burgeoning prevalence of snoring in women whilemenopausal transition not postmenopause period maymark this relationshipThe reason can be explained by the fact that menopausal transition is more concerned with fluctuation ofsex hormone than postmenopause which predisposed tomodulate sleep regulation and breathing thus leading tothe snoringBesides other independent factors for regular snoringsuch as higher TG lower LDL were compatible with onestudy [] Although selfreported snoring was closelyrelated with hypertension and diabetes Unexpectedlywe did not find that hypertension was related with regular snoring after multiple adjustments These divergentfindings may be attributed to difference in sample sizeethnicity culture and the definition of hypertension anddiabetes etc Another possible explanation is that manyprevious studies did not consider menopause status in toaccount which may aggravate the snoreobesity association thus overshadow the snorehypertensiondiabetesassociationHowever our study should be interpreted in light ofthe following limitations First one limitation of thepresent study is that the crosssectional design does notpermit conclusion of causality further prospective studies are needed to verify the association between snoringfrequency and body composition Second selfreportedsnoring frequency but not polysomnography the goldstandard for diagnosing OSA which could bring aboutthe statistical error However precious study has suggestthat selfreport is a reliable measure []Early postmenopause n Odds ratio CI Pvalue Late postmenopause n Odds ratio CI Pvalue ConclusionsRegular snoring ‰¥ times per week may be an independent strong predictor for fat mass of trunk in menopausal women while fat mass in turn serves as a strongpredictor for regular snoring only in menopausal transition Taken together early detection and interventionsof participant showing regular snoring and higher fatmass in menopause could have important preventiveimplicationsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12905020010252Additional file AbbreviationsBIA Bioelectrical impedance analysis OSA Obstructive sleep apneaTG Triglyceride TC Cholesterol HDL Highdensity lipoprotein LDL Lowdensity lipoprotein FBG Fasting blood glucose BMI Body mass indexFM Fat mass LM Lean mass FM Fat mass FFM Fatfree mass BMR Basalmetabolic rateAcknowledgementsThe authors would like to acknowledge all women who consented to takepart of this study We are also thankful for the support and cooperation fromstaff members of obstetrics and gynecology in Shanghai Jiao TongUniversity Affiliated Sixth People™s HospitalAuthors™ contributionsMT conceived the study and YT designed the study [YZ] drafted andcritically revised the manuscript FL designed the questionnaire and analyzedthe data CL [YZ] JH [YZ] LG and SJ administered the questionnairesurvey and managed the data All authors read and approved the finalmanuscriptFundingThis study was supported by grants from the Science and TechnologyCommission of Shanghai Municipality The role of thefunding body in the design of the study and collection analysis andinterpretation of data and in writing the manuscript should be declaredAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateAll participants had provided verbal informed consent after full explanationbecause the study posed no threat to the health of patients This study wassubmitted to and approved by the ethics committees of Institutional ReviewBoard in Shanghai Sixth People™s Hospital affiliated for Shanghai JiaotongUniversity No2016R07 0cZhou BMC Women's Health Page of Consent for publicationNot applicableCompeting interestsThe authors declare that they have no conflict of interestAuthor details1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth People™s Hospital Yishan Road Shanghai China2Reproductive medicine center Shanghai Jiao Tong University Affiliated SixthPeople™s Hospital Yishan Road Shanghai ChinaReceived July Accepted July ReferencesLee YH Kweon SS Choi JS A GenderSpecific Association betweenSelfReported Snoring and Hemoglobin A1c Levels in a General Populationwithout Type Diabetes Mellitus Yonsei Med J “ httpsdoi103349ymj20175861152 PubMed PMID PubMedCentral PMCID PMCPMC5653480Song J Wang C Ma A Selfreported snoring is associated with chronickidney disease independent of metabolic syndrome in middleaged and elderlyChinese J Diabetes Investig “ httpsdoi101111jdi12855PubMed PMID PubMed Central PMCID PMCPMC6319474 engBiggs SN Tamanyan K Walter LM Overweight and obesity add to behavioralproblems in children with sleepdisordered breathing Sleep Med “httpsdoi101016jsleep201709001 PubMed PMID Shivashankar R Kondal D Ali MK Associations of Sleep Duration andDisturbances With 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PMID PubMed CentralPMCID PMCPMC5432489Young T Finn L Austin D Menopausal status and sleepdisorderedbreathing in the Wisconsin Sleep Cohort Study Am J Respir Crit Care Med“ httpsdoi101164rccm2002091055OC PubMedPMID engLi QY Huang SG Li M BMI is an independent risk factor for snoring inChinese women aged over years Sleep Breath “httpsdoi101007s1132500802360 PubMed PMID engStenholm S Harris TB Rantanen T Sarc ic obesity definitioncause and consequences Curr Opin Clin Nutr Metab Care “ httpsdoi101097MCO0b013e328312c37d PubMed PMID PubMed Central PMCID PMCPMC2633408 eng Banack HR WactawskiWende J Hovey KM Is BMI a valid measure ofobesity in postmenopausal women Menopause “ httpsdoi101097gme0000000000000989 PubMed PMID PubMedCentral PMCID PMCPMC5821529 engThomson R Brinkworth GD Buckley JD Good agreement betweenbioelectrical impedance and dualenergy Xray absorptiometry forestimating changes in body composition during weight loss in overweightyoung women Clin Nutr “ httpsdoi101016jclnu PubMed PMID eng Marin JM Carrizo SJ Vicente E Longterm cardiovascular outcomes inmen with obstructive sleep apnoeahypopnoea with or without treatmentwith continuous positive airway pressure an observational study LancetLondon England “ httpsdoi101016s0140 PubMed PMID eng Harlow SD Gass M Hall JE Executive summary of the Stages ofReproductive Aging Workshop addressing the unfinished agenda ofstaging reproductive aging Fertil Steril “ httpsdoi101016jfertnstert201201128 PubMed PMID PubMed CentralPMCID PMCPMC3340904 engLenfant C Chobanian AV Jones DW Seventh report of the JointNational Committee on the Prevention Detection Evaluation andTreatment of High Blood Pressure JNC resetting the hypertension sailsHypertension “ httpsdoi10116101hyp000007579033892ae PubMed PMID engLu B Yang Y Song X An evaluation of the International DiabetesFederation definition of metabolic syndrome in Chinese patients older than years and diagnosed with type diabetes mellitus Metabolism “ httpsdoi101016jmetabol200604003 PubMed PMID engEndeshaw Y Rice TB Schwartz AV Snoring daytime sleepiness andincident cardiovascular disease in the health aging and body compositionstudy Sleep “ httpsdoi105665sleep3140PubMed PMID PubMed Central PMCID PMCPMC3792392 eng Wang HB Yan WH Dou JT Association between Selfreported Snoringand Prediabetes among Adults Aged Years and Older without DiabetesChin Med J “ httpsdoi10410303666999202741PubMed PMID PubMed Central PMCID PMCPMC5381312 eng Zhou Y Zheng Y Li C Association of body composition with menopausalsymptoms in perimenopausal women Climacteric “ httpsdoi1010801369713720181428295 PubMed PMID engTanaka NI Hanawa S Murakami H Accuracy of segmental bioelectricalimpedance analysis for predicting body composition in pre andpostmenopausal women J Clin Densitometry “ httpsdoi101016jjocd201407002 PubMed PMID eng Gasa M LopezPadros C Monasterio C Anthropometrical phenotypesare important when explaining obstructive sleep apnea in female bariatriccohorts J Sleep Res 2019e12830 httpsdoi101111jsr12830 PubMedPMID eng Pillar G Shehadeh N Abdominal fat and sleep apnea the chicken or the eggDiabetes Care 200831Suppl 2S303“ httpsdoi102337dc08s272PubMed PMID PubMed Central PMCID PMCPMC2453667 eng Behan M Kinkead R Neuronal control of breathing sex and stresshormones Comprehensive Physiology “ httpsdoi101002cphyc100027 PubMed PMID eng Behan M Zabka AG Thomas CF Sex steroid hormones and the neuralcontrol of breathing Respir Physiol Neurobiol ““PubMed PMID eng Bixler EO Vgontzas AN Lin HM Prevalence of sleepdisordered breathing inwomen effects of gender Am J Respir Crit Care Med Pt “httpsdoi101164ajrccm16339911064 PubMed PMID eng Carneiro G Zanella MT Obesity metabolic and hormonal disordersassociated with obstructive sleep apnea and their impact on the risk ofcardiovascular events Metabolism “ httpsdoi101016jmetabol201803008 PubMed PMID eng Zhang N Chen Y Chen S SelfReported Snoring Is Associated withDyslipidemia High Total Cholesterol and High LowDensity 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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

Breast cancer is a common malignancy in women Among breast cancer types triplenegative breast cancer TNBC tends to affect younger women is prone to axillary lymph node lung and bone metastases and has a high recurrence rate Due to a lack of classic biomarkers the currently available treatments are surgery and chemotherapy no targeted standard treatment options are available Therefore it is urgent to find a novel and effective therapeutic target As alteration of ion channels and transporters in normal mammary cells may affect cell growth resulting in the development and progression of TNBC ion channels and transporters may be promising new therapeutic targets for TNBC This review summarizes ion channels and transporters related to TNBC and may provide new tumor biomarkers and help in the development of novel targeted therapiesKeywords Triplenegative breast cancer Ion channels Ion transporters Pathological roles Targeted therapyBackgroundBreast cancer BC is the common malignancy in women its incidence is increased each year [] and it has become a significant threat to women™s health [] BC is a heterogeneous disease that can be divided into multiple molecular subtypes based on estrogen receptor ER progesterone receptor PR and human epidermal growth factor receptor HER2 expression providing important prognostic and predictive information [] There are four BC subtypes depending on receptor status luminal A luminal B HER2overexpressing and triplenegative breast cancer TNBC Among them TNBC is defined as ER PR and HER2 negative and it tends to affect younger women a0years of age it is Correspondence onlyoneliuxuemei163com 0078029sinacom Chengli Lu and Zhiyuan Ma contributed equally and share first authorship Department of Thyroid and Breast Surgery Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province China Department of Gastroenterology Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province ChinaFull list of author information is available at the end of the prone to axillary lymph node lung bone metastases and has a high recurrence rate [ ] Lehmann et a0al classified TNBC into six subtypes based on gene cluster sequence expression basallike basallike immunomodulatory mesenchymal mesenchymal stemlike and luminal androgen receptor subtypes [] After analyzing the RNA and DNA profile of TNBC tumors Matthew et a0 al classified TNBC into four subtypes including luminal androgen receptor mesenchymal basallike immunesuppressed and basallike immuneactivated subtypes [] The two classification methods have similarities and both provide theoretical bases for exploring targeted therapies for TNBCAlthough TNBC is the BC subtype that responds best to chemotherapy its recurrence and metastasis rates are higher than those of other BC subtypes [] Furthermore due to the lack of classic biomarkers TNBC lacks standard treatments guided by tumor biology and only surgery and chemotherapy are currently available as treatments [] Previous studies have shown that ion channels and transporters play important regulatory roles in mammary physiology and the initiation and progression of BC 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 0cLu a0et a0al Cancer Cell Int Page of [] However the detailed functional role of ion channels and transporters in TNBC has not been clarified and summarized In recent studies upregulation of NaH exchanger has been shown to promote the proliferation migration and invasion of the TNBC cell line MDAMB231 [ ] In addition Ca2 channels such as mitochondrial calcium uniporter MCU can promote TNBC cell migration invasion and lung metastasis [] and Alvarez et a0al [] reported that the twopore domain potassium channel KCNK5 is associated with a poor prognosis in TNBC Therefore ion channels and transporters play important regulatory roles in the pathophysiology of TNBC but there is currently no relevant review on this topic Here we review the pathological roles of ion channels and transporters including AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters in the initiation and progression of TNBCAQP channelsAQPs which compose a family of transmembrane water channel proteins modulate the movement of water and small solutes into and out of cells and maintain suitable concentrations of water and solutes for cell survival [] At least AQP subtypes AQP012 have been identified in mammals and are divided into two families based on transfer specificity namely the classic watertransporting AQP family and the solute water and glyceroltransporting glycoprotein family [] AQP02 AQP4 to AQP68 are mainly waterselective AQP3 AQP7 AQP9 AQP10 and AQP12 also transport glycerol and possibly other small solutes AQPs also play roles in the transport of ammonia urea carbon dioxide metalloids nitric oxide and certain ions [] Expression of AQP1 AQP35 and AQP1012 has been detected in normal human mammary tissue and is closely related to milk secretion [ ] In addition deletion of œCCAATenhancer binding protein a family of transcription factors isoforms results in changes in mammary ductal morphogenesis and changes in expression of transport proteins such as AQP5 suggesting that AQP5 may be involved in mammary development [] Recent studies have shown that AQPs play carcinogenic roles by promoting angiogenesis enhancing invasive and metastatic potential and enhancing the transport of reactive oxygen species ROS [ ] In femalespecific cancers such as BC AQP1 and are the most important AQPs and they are been reported to be upregulated []AQP1 the membrane protein was the first reported mammalian AQP and plays a significant role in tumor cell migration proliferation and angiogenesis [] Clinical studies have shown that patients with TNBC have higher levels of AQP1 expression and that upregulation correlates with a poor prognosis [ ] AQP1 expression is induced by hypoxia through the EBoxChoRE transcription element which is affected by increased glucose consumption and metabolism [] AQP1 expression has been detected only in a subgroup of CK14positive basallike breast cancer BLBC cases [] CK14 has been used as a marker of basal mammary epithelial cells with in vivo regenerative ability in studies on mammary gland progenitor and stem cells [] Therefore it is speculated that expression of AQP1 is related to the stem cell characteristics of BLBC cells Hu et a0al demonstrated that AQP1 upregulation promotes extravasation and increases migration in a0vivo and in a0vitro in the mouse TNBC cell line 4T1 suggesting that this aquaporin enhances the rate of cell migration by promoting water permeability in cell protrusions [] Thus upregulation of AQP1 can promote the proliferation migration and invasion in TNBC cells Moreover in a0 vivo experiments have shown that AQP1 deficiency can reduce tumor mass volume vessel density and lung metastases in MMTVPyVT mouse mammary tumor virusdriven polyoma virus middle T oncogene mice and inhibition of AQP1 function andor expression is predicted to attenuate angiogenesis via reduced migration and invasion of endothelial cells [] Recently Irene AbreuRodriguez et a0al [] revealed that AQP1 expression is also responsive to hypoxiainducible factor HIF which may play a role in the VEGFindependent signaling mechanism inducing angiogenesis in a hypoxic environment Helen et a0al [] also reported that the triterpenoid saponins bacopaside I and bacopaside II can synergistically reduce the transcriptional expression of AQP1 and inhibit proliferation migration and invasion in MDAMB231 cells Similarly ginsenoside Rg3 a compound with anticancer activity isolated from ginseng inhibits AQP1 to attenuate cell proliferation through a mechanism that involves downregulation of AQP1 to induce cell cycle arrest in G0G1 phase by inhibiting cyclin D and E and inhibition of chemoattractantinduced cell migration and invasion by blocking AQP1mediated water flux in MDAMB231 cells [] These findings indicate that AQP1 plays an important role in the development and progression of TNBCOverexpression of AQP3 has been detected in the membranes and cytoplasm of TNBC tumor cells and is significantly associated with poor prognosis [] XuChen Cao et a0 al [] found that the presence of fibroblast growth factor2 FGF2 induced cell migration and metastasis in MDAMB231 cells by increasing AQP3 expression Moreover FGF receptor kinase FGFRK inhibitors PI3K inhibitors and MEK12 inhibitors all inhibit AQP3 expression suggesting that FGF receptor kinases increase AQP3 expression and promote FGF2induced cell migration by initiating downstream PI3K and ERK pathways In addition CuSO4 a water transport 0cLu a0et a0al Cancer Cell Int Page of inhibitor of AQP3 inhibits migration in MDAMB231 cells AQP3 downregulation reduces the proliferation invasion and migration of MDAMB231 cells while increasing sensitivity to 5fluorouracil chemotherapy The mechanism may be related to a decrease in glycerol permeability caused by AQP3 downregulation [] Overall these findings demonstrate that AQP3 plays a pivotal role in the initiation and progression of TNBC and specific inhibitors of AQP3 in clinical applications may improve the therapeutic effect of TNBC patientsSimilarly overexpression of AQP5 in the membrane and cytoplasm of TNBC cells has been detected and is significantly associated with poor prognosis [] Moreover patients with higher Ki67 expression are more likely to have abnormal AQP5 protein expression than patients with lower Ki67 expression [] Ki67 is a widely accepted proliferation marker [ ] and it is speculated that upregulation of AQP5 may promote proliferation in TNBC cellsIn summary AQP1 AQP3 and AQP5 are significantly upregulated in TNBC this upregulation is related to a poor prognosis and can promote the proliferation migration and invasion of TNBC cells These AQPs are promising new targets for the diagnosis and treatment of TNBCClˆ’ channelsCFTRCFTR is a member of the ATPbinding cassette transporter family that localizes at the apical membranes of normal epithelial cells CFTR is mainly responsible for conducting HCO3ˆ’ and Clˆ’ and promoting HCO3ˆ’ secretion in many tissues including the airway intestines and pancreas [] However when the extracellular concentration of Clˆ’ is higher than a0mmolL the permeability of CFTR to Clˆ’ is much greater than that of CFTR to HCO3ˆ’ thus CFTR mainly conducts Clˆ’ under physiological conditions [] CFTR can also transport two other anions glutathione and thiocyanate which are involved in airway inflammation and oxidative stress [ ] Interestingly Pierre et a0al [] reported that CFTR is required for the tightly connected functions of normal epithelial tissues loss of CFTR reduces epithelial resistance and epithelial integrity and this effect is not related to the anion channel function of CFTRCFTR has been reported to be associated with several cancers such as cervical cancer [] colorectal cancer [] prostate cancer [] and BC [] Significant downregulation of CFTR expression is observed in BC tissue compared to normal mammary tissue [] Zhang et a0al demonstrated that overexpression of CFTR inhibits EMT invasion and migration in MDAMB231 cells via a mechanism that involves CFTR inhibition of NFκB targeting of urokinasetype plasminogen activator [] In addition CFTR overexpression inhibits the EMT and the invasiveness of MDAMB231 cells and reduces lung metastasis in xenograft models Increasing evidence reveals that downregulation of CFTR occurs after treatment with EMTinducing factors such as TGF suggesting that as a downstream effector CFTR plays important roles in mediating various EMT effects [ ] Moreover hypermethylation of the cancer genome leads to activation of oncogenes or suppression of tumorsuppressor genes thereby resulting in tumorigenesis [] It has also been observed that the methylation level of CFTR in BC tissues is much higher than that in normal tissues and treatment with DNA methylation inhibitors in TNBC cell lines MDAMB231 and MDAMB435 can rescue CFTR mRNA indicating that CFTR methylation plays an important role in TNBC [] ΔF508 is the most common mutation in CFTR causing the protein to be retained and degraded in the endoplasmic reticulum due to misfolding [] It is worth noting that although there is no difference in the incidence of BC between ΔF508 carriers and noncarriers patients with ΔF508 CFTR mutations all have grade III cancer indicating that CFTR defects are associated with BC progression [] Therefore CFTR methylation or mutation need to be further investigated in the future which may provide novel therapeutic intervention for TNBCChloride channel The chloride channel ClC family also plays an indispensable role in the transport of Clˆ’ [] There are nine family members in humans which are divided into two categories based on their distribution and physiological function Clˆ’ channel proteins ClC1 ClC2 ClCKa and ClCKb which mainly exist in the plasma membrane and play roles in stabilizing membrane electric potential or mediating epithelial transport and Clˆ’H reverse transporter proteins ClC37 which mainly exist in the vascular intima of the endosomelysosomal pathway and are localized at the plasma membrane only to a limited extent due to protein degradation and hydrolase activity [ ] In recent years it has been discovered that ClC3 can transport one hydrogen ion in exchange for two chloride ions [] with important roles in cancers such as nasopharyngeal carcinoma [] and BCClC3 overexpression is observed in tissues and the TNBC cell line MDAMB231 [ ] Studies by Zhou et a0 al revealed that knockdown of ClC3 downregulates expression of cyclin D1 and cyclin E and increases levels of p21 indicating that knockdown of ClC3 can block the cell cycle of MDAMB231 cells at G0G1 phase inhibiting cell proliferation Moreover knockdown of ClC3 suppresses tumor growth in xenograft models and significantly reduces levels of pERK12 in MDAMB231 cells 0cLu a0et a0al Cancer Cell Int Page of This indicates that ClC3 can promote the progression of TNBC by acting on the ERK12 signaling pathway [] Nevertheless relative research on ClC3 in TNBC is still very limited and extensive work is needed in the furtherCa2 channelsCa2 is a key nutrient in milk that plays a vital role in the mineralization of bones and teeth and as a second messenger ionized Ca2 is a key regulator of proliferation migration cell cycle progression and apoptosis [] The level of Ca2 is very low in the cytoplasm ˆ’ a0molL whereas it is somewhat higher in anelles ˆ’ a0 molL and highest in the extracellular level milieu ˆ’ a0 molL Hence a small amount of Ca2 can significantly change intracellular levels to activate downstream signaling molecules including calmodulin nuclear factor of activated Tcells NFAT NFκB calmodulindependent protein kinase II calpain and others [ ] In nonexcitatory mammary cells calcium channels play important roles in lactation and the maintenance of normal physiological functions [ ]Continuous increases in intracellular Ca2 levels will drive expression of oncogenes resulting in tumor growth and development especially the metastatic behavior of cancer cells and conferring tumor cells with resistance to apoptosis [] Abnormal expression of several Ca2 transporters and ion channels such as calcium releaseactivated calcium modulator Orai1 has been observed in TNBC and may lead to oncogenic Ca2 signaling [] Interestingly specific changes in the expression and function of Ca2 channels are related to hormone receptor status and differ significantly among BC subtypes []Calcium modulator Ca2 influx mainly depends on storeoperated calcium channels SOCCs When the Ca2 concentration in the endoplasmic reticulum declines to a certain level the STIM stromal interaction molecule which is located on the endoplasmic reticulum membrane moves to a position close to the highly selective calcium channel protein Orai on the cell membrane Subsequently Orai is activated to cause Ca2 influx and storeoperated calcium entry SOCE is initiated thereby replenishing the calcium store Some researchers have proposed that the canonical transient receptor potential TRPC also participates in the above process though the mechanism remains controversial There are two different claims that both Orai and TRPC form independent channels activated by the STIM protein and that Orai and TRPC subunits form heterochannels triggered by STIM [] There are three Orai1 isomers Orai1 to Orai3 and two STIM homologs STIM1 and STIM2 SOCE has been found to be primarily mediated by Orai1 and STIM1 in TNBC [] Compared with that in nonmalignant breast epithelial cells expression of Orai1 and STIM1 is significantly higher in TNBC cell lines and is associated with a poor prognosis [ ] Liu et a0 al [] reported that hypoxia can induce expression of Orai1 Notch1 and Jagged1 and Orai1 is significantly downregulated after blockade of Notch signaling suggesting that hypoxia can increase Orai1 expression in TNBC by activating Notch signaling Notch1Orai1SOCENFAT4 axis Similarly Mognol et a0al [] found that Orai1 promotes the invasion and angiogenesis of TNBC cell lines and activates NFAT4 which can regulate genes involved in the cell cycle apoptosis angiogenesis and metastasis In addition Yang et a0 al [] demonstrated that Orai1 and STIM1 promote the migration and invasion of MDAMB231 cells both in a0vivo and in a0vitro and the authors proposed that these proteins may at least partially control cell migration by regulating focal adhesion turnover Furthermore treatment with TGF can reduce expression of STIM1 whereas blockade of SOCE can impair TGFinduced G0G1 cell cycle arrest and inhibit the proliferation of MDAMB231 cells [] Based on the above research Orai1 and STIM1 may be new therapeutic targets for TNBC Indeed some selective SOCE inhibitors have shown encouraging inhibitory effects on TNBC but they are still only in the preclinical trial stage For example phemindole a diindole derivative reduces SOCE by downregulating STIM1 expression significantly inhibits the proliferation and migration of MDAMB231 cells reduces the growth of solid tumors in mouse models and produces a targeted antitumor effect in TNBC [] In addition Miroslava Didiasova et a0al [] revealed that elevated cell surfaceassociated enolase1 ENO1 expression correlates with augmented MDAMB231 cell migratory and invasive properties Pharmacological blockade a selective SOCC inhibitor NS1643 or knockdown of STIM1 or Orai1 reduces ENO1dependent migration of MDAMB231 cells These results demonstrate the pivotal role of SOCE in the regulation of ENO1 exteriorization and thus in the modulation of TNBC cell migratory and invasive properties indicated that Orai1 and STIM1 might be promising threptic targets for TNBCSecretory pathway Ca2‘ATPaseThe secretory pathway Ca2ATPase SPCA can direct Ca2 and Mn2 from the cytoplasm to the Golgi and postGolgi vesicles Two isotypes SPCA1 and SPCA2 are known and the distribution and function of the two differ SPCA1 is commonly expressed in mammalian tissues expression of SPCA2 is limited to highly absorptive and secretory epithelial cells including mammary 0cLu a0et a0al Cancer Cell Int Page of and salivary gland cells [] SPCA1 is highly expressed in TNBC cell lines and SPCA2 is highly expressed in cell lines of other subtypes [] Interestingly based on clinical samples Desma et a0al reported SPCA1 levels to be significantly elevated in the basal subtype of BC compared with all other subtypes and it is worth noting that changes in its expression affect posttranslational modification and transport of certain proteins important for tumor progression without significantly changing cytosolic calcium signaling SPCA1 inhibition also decreased MDAMB231 cell proliferation [] Moreover SPCA1 is a key regulator of insulinlike growth factor receptor IGF1R processing in TNBC cells and promotes the production of functional IGF1R IGF1R activity is associated with poor prognosis suggesting that targeting SPCA1 is an alternative IGF1Rinhibiting strategy [ ] Overall upregulation of SPCA1 may promote the initiation and progression of TNBC The main mechanism reported to date involves SPCA1mediated increase in functional IGF1R expressionMitochondrial calcium uniporterUpregulation of MCU expression on the mitochondrial membrane is closely related to a poor prognosis in BC [] miR340 correlates negatively with the metastatic potential of TNBC cells [] it may directly inhibit MCU expression to reduce glycolysis and exercise capacity and knockdown or inhibition of MCU inhibits the growth invasion and metastasis of MDAMB231 cells [] Interestingly Anna et a0 al [] demonstrated that mitochondrial Ca2 uptake is required for TNBC progression in a0vivo and that absorption of Ca2 by mitochondria promotes the production of sustained mitochondrial reactive oxygen species activating the HIF1α signaling pathway and promoting tumor growth and metastasis In addition inhibiting or silencing MCU also block seruminduced migration of MDAMB231 cells and reduce serum or thapsigargininduced SOCE suggesting that MCU promotes TNBC cell migration by regulating SOCE [] The above results indicate that overexpression of MCU may play an important oncogenic role in the growth invasion and metastasis of TNBC cells However the precise mechanism is unclearOther promising calcium channel targets in TNBC include TRPV6 [] Overall calcium channels are promising targets for TNBC treatment but most compounds targeting these channels are only in the preclinical trial stage Thus further research is neededK channelsThrough the action of NaKATPase two K molecules are transported into a cell in exchange for three sodium molecules which increases the intracellular K concentration K channels on the cell membrane are numerous and in humans more than genes encode major K channel subunits [] K channels play key roles in maintaining acidbase balance by functioning in concert with the NaH exchanger and NaKATPase [] controlling electrical excitability of nerves and muscles and participating in energy metabolism and other physiological processes In addition K channels can help regulate cell proliferation and cell cycle progression and are involved in tumorigenesis [] Many studies have reported dysregulated K channel expression in human cancers including BC astrocytictype brain cancer and prostate cancer [ ] Tumorrelated K channels can be divided into four main categories according to their domain structures and activation mechanisms voltagegated potassium channels which are controlled by changes in membrane potential calciumactivated potassium channels which are activated by intracellular calcium inwardly rectifying potassium channels and twoporedomain potassium channels K2P KCNK [] However the carcinogenic mechanism of K channels remains rather clear Nuria et a0 al [] proposed that K channels may participate in and regulate tumor progression through permeationrelated mechanisms including changes in membrane potential Ca2 driving forces and cell volume regulation and nonconductive mechanisms dependent on proteinprotein interactionsThe Kv111 channel also known as human etheragogorelated gene hERG1 is not expressed in normal breast cells but is expressed in BC with a relationship with subtype Indeed TNBC exhibits lower expression of Kv111 compared with other subtypes [] Olivia Crociani et a0al [] showed that the mRNA levels of Kv111 change throughout the cell cycle peaking in G0G1 phase Moreover Lansu et a0al [] reported that stimulation of Kv111 led to inhibition of proliferation in MDAMB231 cells and that an agonist the diphenylurea derivative NS1643 caused a significant inhibition of cell proliferation This phenomenon can be linked to a rapid decrease in the cyclin E2 protein level which causes accumulation of cells in G0G1 phase and an increase in tumor suppressor proteins and markers for cellular senescence including p21 p16INK4a and galactosidase activity Therefore Kv111 inhibits TNBC cell proliferation by activating a cellular senescence program [] Breuer et a0 al confirmed that NS1643 reprograms the EMT by attenuating the Wntcatenin signaling pathway inhibits cancer cell stemness and significantly reduces the metastatic spread of breast tumors in a MDAMB231 mouse model [] Regardless cardiotoxicity is an important limiting factor for potential therapeutic molecules acting on Kv111 Although the activator is well tolerated 0cLu a0et a0al Cancer Cell Int Page of in BC potential effects include tachycardia [] Overall the potential benefits of Kv111 activators as anticancer drugs outweigh their side effectsIn addition many other channels are altered in TNBC For example some K2P channels with differential expression may serve as novel molecular markers associated with TNBC RNASeq analysis of K2P channels has shown that overexpression of KCNK5 KCNK9 and KCNK12 and low expression of KCNK6 and KCNK15 are related to TNBC [] The above findings indicate that K channels play an important role in TNBC and are expected to be diagnosis markersAcid‘base transportersThe pH of milk is significantly lower than that of plasma indicating that there may be some acidbase transporters in the mammary gland that regulate the pH between the extracellular fluid and milk [] A uniform feature among solid tumors with high metabolic and proliferative rates is a significantly different pH from that of normal tissue [] Cancer cells can maintain a weakly acidic intracellular pH that is even more alkaline than that of normal cells suggesting that tumor cells have a powerful pH regulation system a0[]The NaH exchanger NHE a membrane transporter mainly catalyzes the exchange of intracellular H for extracellular Na in mammals thereby maintaining the pH balance inside and outside the cell [ ] There are subtypes of NHE with tissue and membranespecific expression patterns NHE15 are located on the plasma membrane and NHE69 are on intracellular anelle membranes NHE10 is only expressed in osteoclasts [] In addition NHE plays indispensable roles in maintaining normal mammary structure and physiological functions [] NHE1 SLC9A1 is universally expressed in epithelial cells and upregulated in BC tissues compared to normal tissues [] Studies have shown that hypoxia various growth factors and hormones among others can activate NHE1 and enhanced NHE1 activity can reduce extracellular pH and promote metastasis of MDAMB231 cells [] Furthermore it has been proposed that NHE1 promotes metastasis and remodeling of the extracellular matrix by acidifying the extracellular microenvironment [] In addition NHE1 knockdown reduces the migration invasion and growth of xenograft tumors of MDAMB231 cells increasing the susceptibility of these cells to paclitaxel [ ] Moreover knockdown of NHE1 or NBCn1 SLC4A7 in the MDAMB231 cell line significantly reduced the steadystate intracellular pH value after acid load the ability to restore pHi and the primary tumor growth of xenografts in a0 vivo but NBCn1 knockdown prolonged tumorfree survival and reduced cell proliferation [ ] It has been confirmed that NHE1 and NBCn1 promote the development of TNBC through different mechanisms There are two main NHE1 inhibitors amiloride and cariporide which are more effective than amiloride and highly selective [] Amiloride is a potassiumsparing diuretic and has blocking effects on a variety of ion channels such as NHE and the NaCa2 exchanger Cariporide is a highly specific and powerful NHE1 inhibitor that is relatively well tolerated in humans with heart disease [] Moreover a study has suggested that KR33028 a novel small molecule inhibitor of NHE1 produces a cellular phenotype comparable to that of NHE1 knockout cells and significantly decreases rates of migration invasion and colony growth in TNBC cell lines MDAMB231 MDAMB468 and Hs578T [] The above findings suggest that NHE1 may play an important role in the progression TNBCAdditionally other acidbase transporters are also altered in TNBC and are expected to emerge as new targets for TNBC treatment For instance NBCe1 SLC4A4 knockdown reduces cell proliferation invasion and migration in TNBC cells expressing high levels of NBCe1 [] The above findings all suggest that the acidbase transporters have essential functions in the occurrence and development of TNBC but further research is neededConclusionsDysfunction of ion channels and transporters in the mammary resulted in development and progression of TNBC Despite extensive work has been performed to investigate the expression pattern functional diversity regulatory mechanism and pathophysiology of different ion transporters in TNBC the systematic review is rare in this field Therefore this review focuses on different pathological function of multiple families in the development and progression of TBNC including the AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters Fig a0 Table a0 We hope that we can provide a basic systemics and summarised knowledge to this field advocating researchers play more attention on the pathophysiological role of ion channels and transporters in the development and progression of TNBC which may provide novel targets for the clinical diagnosis and treatment of TNBC 0cLu a0et a0al Cancer Cell Int Page of Fig Pathological roles of ion channels and transporters in triplenegative breast cancer cells Alteration and dysfunction of AQPs Clˆ’ channels Ca2 channels K channels NaHCO3ˆ’ transporter and NaH exchanger results in abnormality of ion transport and disorder of multiple signaling pathway including WNT PI3K TGF Notch and VEGF etc eventually promoting TNBC cell proliferation migration and invasion but inhibiting apoptosis

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

"RNA molecules with a unique closed continuous loop structure CircRNAs areabundant in eukaryotic cells have unique stability and tissue specificity and can play a biological regulatory role atvarious levels such as transcriptional and posttranscriptional levels Numerous studies have indicated that circRNAsserve a crucial purpose in cancer biology CircRNAs regulate tumor behavioral phenotypes such as proliferation andmigration through various molecular mechanisms such as miRNA sponging transcriptional regulation and proteininteraction Recently several reports have demonstrated that they are also deeply involved in resistance toanticancer drugs from traditional chemotherapeutic drugs to targeted and immunotherapeutic drugs This review isthe first to summarize the latest research on circRNAs in anticancer drug resistance based on drug classification andto discuss their potential clinical applicationsKeywords Circular RNA Cancer Drug ResistanceIntroductionCancer has become the most serious public health problem worldwide [] As the early diagnosis of cancer urgently needs to be improved many patients are alreadysuffering from advanced cancer at the first clinical visit[] These patients miss the opportunity for surgery andanticancer drugs are their major treatment option Sincethe first clinical report of chemotherapy for advanced cancers multiple anticancer drugs have been developed andhave effectively improved the clinical outcomes of patientswith advanced cancers [] In addition targeted and immunotherapeutic drugs have recently ushered in a newera in medical therapy for cancer [ ] However anticancer drug resistance still cannot be avoided resulting incancer relapse The mechanisms underlying anticancerdrug resistance are multifaceted Different hallmarks such Correspondence wangzhaoxianjmueducn weichenchen1990126com Tianwei Xu Mengwei Wang Lihua Jiang and Li Ma contributed equally tothis work1Cancer Medical Center The Second Affiliated Hospital of Nanjing MedicalUniversity Jiangjiayuan road Nanjing Jiangsu PR ChinaFull list of author information is available at the end of the as tumor growth selective therapeutic pressure immunesystem characteristics and the tumor microenvironmentdetermine the biological behaviors of anticancer drug resistance [] These hallmarks are driven by complexunderlying molecular regulatory mechanisms [] Identifying the key molecules in these processes could help us tounderstand the occurrence of resistance and these molecules play considerable roles in the prediction and reversion of anticancer drug resistance []With the development of sequencing studies haveshown that approximately of the human genome istranscribed but only encodes proteins [] Theremaining RNA which does not encode proteins wasinitially thought to be transcriptional junk [] Accumulating evidence has revealed that these noncodingRNAs ncRNAs also exert a great influence on physiological activities and pathological changes especially incancer [ ] Importantly ncRNAs are classified bysize into small RNAs such as miRNAs nt and longnoncoding RNAs lncRNAs nt Dysregulation ofin the molecular and cellularncRNAs participates 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 0cXu Molecular Cancer Page of processes related to cancer The roles of ncRNAs are divided into oncogenes and tumor suppressors based ontheir effect on the cancer For example miR21 was reported to promote tumor growth and metastasis by targeting PTEN in lung cancer and other cancers such asmelanoma and B cell lymphoma [ ] As shown inour previous study high serum miR21 levels indicate apoor prognosis for patients with nonsmall cell lung cancer NSCLC [] In contrast miR34a functions as adirect downstream target of p53 to block cancerprogression [] HOTAIR a wellstudied oncogeniclncRNA is generally upregulated in cancers and epigenetically silences tumor suppressors such as p21 []However lncRNA MEG3 modulates the expression ofp53 and some other tumor suppressors to increases chemotherapeutic sensitivity [] Several ncRNAs may alsofunction as either tumor suppressors or oncogenesdepending on the context For example the lncRNANKILA reduces cancer metastasis by negatively regulating NFkB signaling while it also promotes tumor immune evasion [] Since ncRNAs such as miRNAs andlncRNAs have been confirmed to play an important rolein cancer studies of new ncRNAs will continue to improve our understanding of cancerIn recent years a novel class of lncRNAs designated circular RNAs circRNAs has also been identified and extensively studied in cancer biology [] CircRNAs are derivedfrom premRNAs through a noncanonical alternative splicing event called backsplicing This process endows circRNAs with a unique fundamental structural feature thatdiffers from common linear lncRNAs namely a covalentlyclosed continuous loop structure without a polyadenylatedtail CircRNAs are very stable because their unique structure is resistant to exonucleasemediated degradation []Fig Biogenesis of circRNAs a Classification of circRNAs CircRNAs can be divided into ecircRNAs EIciRNAs and ciRNAs by their composition bFunctions of circRNAs CircRNAs can regulate gene transcription as their parental genes do in the nucleus CircRNAs can act as miRNA spongesinteract with proteins as protein scaffolds or decoys and under certain circumstances be translated in the cytoplasm 0cXu Molecular Cancer Page of Although backsplicing is much less efficient than canonicalsplicing in linear RNAs circRNAs are still enriched in tissues serum and even urine [] Although circRNAs arewidely expressed the expression pattern of circRNAs displays tissue specificity and cell type specificity For exampleplatelets express more circRNAs than neutrophils whichare also blood cells vs circRNAs [ ]CircRNAs can be divided into subtypes exonic circRNAsEcircRNAs which contain only exon sequences intronic circRNAs ciRNAs which contain only intron sequences and exonintron circRNAs EIciRNAs whichcontain both exon and intron sequences [] Fig 1aStudies have demonstrated that circRNAs may exert biologicalfunctions by transcriptional regulation miRNAsponging protein interactions or under certain circumstances selftranslation [ ] Fig 1b For exampleciRS7 is a wellknown circRNA that is enriched in neurexhibitonalknockout miceCiRS7tissuesneuropsychiatric disorders and the underlying mechanismmay be attributed to the ciRS7miR7Fos axis []CircRNAs also participate in various pathological proincluding the development of cancer [] Accessescording to the classification ofthe mechanism intumors we present some representative circRNAs withclear mechanisms biological functions and clinical significance Table First circRNAs are differentiallyexpressed in almost all cancers including lung cancergastrointestinal cancers and urological cancers SecondcircRNAs are differentially expressed rather than simplyupregulated or downregulated in the same tumor Forexample circPVT1 and circFADS2 are upregulated [] and circNOL10 and circPTPRA are downregulatedin lung cancer [ ] Third the same circRNAs arealso differentially expressed in a variety of tumors butthe upregulated or downregulated expression patternsare not the same in different tumors CircHIPK3 isTable Representative circRNAs with clear mechanisms biological functions and clinical significance in cancersMechanismTranscriptionalregulationInhibit cell proliferation and promote cellapoptosisClinical significanceLung cancer differentiationCircRNAcircNOL10 DownCancerLung cancerExpression Biological functionsColorectalCancercircITGA7 DownInhibit cell growth and metastasisTumor sizeLymph metastasis Distant metastasisand TNM stageBreast CancercircFECR1 UpPromotes tumor metastasisMetastases Advanced stagesMiRNAspongingLung cancercircPVT1UpPromote cell proliferation and Inhibit cellapoptosisTumor size TNM stage Overall survivalcircFADS2 UpPromote proliferation and invasionTNM LNM Overall survivalcircPTPRA DownInhibit proliferation and migrationMetastasis Overall survivalGastric CancercircHIPK3 UpPromote proliferation and migrationOverall survival Infiltrative type GC cellAdvanced TNM stagecircPVT1UpPromote proliferationOverall survival Diseasefree survivalcircLARP4 DownInhibit proliferation and invasionOverall survivalColorectalcancercircHIPK3 UpPromote proliferation migration invasionmetastasis autophagy and inhibitapoptosisAdvanced TNM stage Lymph nodemetastasis Distant metastasis Advancedtumorcirc_DownInhibit proliferation migration andinvasionTNM stageOverall survivalHepatocellularcarcinomacircHIPK3 UpProliferation migrationcirc_DownInhibit proliferation and migrationTumor differentiation Advanced TNMstage HBVDNA copy numbers LivercirrhosisDifferentiation and tumorSatelliteBladder cancer circHIPK3 DownInhibit Migration invasion andangiogenesisAdvanced tumor Lymph nodemetastasisProteininteractionGastric cancerSelftranslation GlioblastomacircDONSONUpcircAGO2 UpcircSHPRHDowncircFBXW7 DownRef ReferenceFacilitate cancer growth and invasionTNM stage Lymph node metastasisOverall survival and Diseasefree survivalPromote growth invasion and metastasis Metastasis Overall survivalSuppress tumor progression andtumorigenesisInhibit proliferation and cell cycleaccelerationOverall survivalOverall survivalRef[][][][][][][][][][][][][][][][][][] 0cXu Molecular Cancer Page of upregulated in most types of cancers such as gastriccancer colorectal cancer and hepatocellular carcinoma[ ] However it is downregulated in bladdercancer [] Based on these findings the expression pattern of circRNAs in malignant tumors is very complexand thus a highthroughput detection method is neededto determine the differential expression profile of circRNAs to further understand the importance of the differential expression patterns of circRNAs in cancertumorssuggestsThe complex expression pattern of circRNAs also implies complex biological functions and clinical significance in cancer CircRNAs modulate multiple biologicalfunctions of cancer such as proliferation metastasis andangiogenesis [ ] For example circPVT1 promotescell proliferation and inhibits cell apoptosis in lung cancer [] and gastric cancer [] while circHIPK3 inhibitsangiogenesis in bladder cancer [] CircRNAs have important tumorrelated clinical significance and are associated with various clinical features of cancer and patientoutcomes As shown in Table the clinical features ofcancer mainly include cancer differentiation lymph nodemetastasis distant metastasis and the TNM stage [] The association between circRNAs and the clinicalcharacteristics ofthe potentialdiagnostic value of circRNAs For examplelowcircNOL10 expression may indicate a low level ofdifferentiation of[] High circDONSON expression suggests that clinicians shouldclosely monitor lymph node metastasis in patientswith gastric cancer [] The main circRNArelatedindicators of outcomes in patients with cancer areoverall survival and diseasefree survival [ ] Inpatients with gastric cancer high circPVT1 expression indicates poor overall survival and diseasefreesurvival [] while high circLARP4 expression is related to longer overall survival [] CircFBXW7 andcircSHPRH are good prognostic biomarkers for glioblastoma because their high expression results inlonger overall survivaltheimportant role of circRNAs in tumors must not beignored Moreover the crucial role of circRNAs inmediating anticancer drug resistance is emerging[] Here we are the first to summarize the role ofcircRNAs in anticancer drug resistance from the perspective of drug classification[ ] In conclusioncancerlungNonplatinum cytotoxic drugsTraditional anticancer drugs can be divided into cellcyclespecific anticancer drugs and cell cyclenonspecificanticancer drugs according to their cellular kinetics [] Classical cell cyclespecific drugs include topoisomerase inhibitors antimetabolite drugs and some drugs derived from plants [“] Typical cell cyclenonspecificanticancer drugs are anticancer antibiotics []isindistributedabundantlyTopoisomerase inhibitorsTopoisomerasealleukaryotic nuclei and resolves topological problems during bioprocesses such as DNA replication and DNAtranscription [] In the 1970s camptothecin was foundto have anticancer activity and topoisomerase I wasidentified as its major target [] Irinotecan is an Sphasespecific camptothecin derivative that exerts efficient anticancer activity against metastatic colorectalcancer CRC [] However clinical resistance to irinotecan is common [] Jian [] found that circ_ increases migration and is involved in irinotecanresistance in CRC Circ_001680 acts as an oncogene bysponging miR340 to upregulate BMI1 in CRC BMI1has been recognized as a positive regulator that inducescancer stem celllike properties [ ] which aredeeply involved in irinotecan resistance [ ] Sphereformation assays showed that SW480 and HCT116 cellswith circ_001680 overexpression formed more stem cellspheres after treatment with irinotecan and had a morerobust cell growth ability than control cells In vivo thesizes and weights of tumors in the control group weremarkedly decreased after treatment with irinotecan butwere not significantly changed in the circ_001680overexpressing group These results indicate that the circ_001680miR340BMI1 axis may contribute to irinotecanresistance by regulating cancer stem celllike propertiesAntimetabolite drugsPemetrexed is a folate antagonist that disrupts folatedependent metabolic processes [ ] Itfunctionsmainly during Sphase Xu [] reported a novelcircRNA circMTHFD2 related to pemetrexed resistancein gastric cancer Overexpression of circMTHFD2 wasconfirmed in pemetrexedresistant gastric cancer cellsFurther investigation showed that circMTHFD2 couldbind with miR124 and promote the protein expressionof FDZ5 and MDR1 to induce pemetrexed resistance5Fluorouracil 5FU a fluoropyrimidine can inhibitthymidylate synthase to induce thymineless cell deathand functions mainly as an Sphase antimetabolite [] 5FU has been the standard treatment for manysolid tumors including CRC for decades [ ] Thebenefit of 5FU is always limited by the development ofresistance and the specific molecular mechanisms arecomplex [] Xiong [] identified differentiallyexpressed circRNAs in 5FUresistant CRC cells bymicroarray hybridization Fortyseven of these circRNAswere upregulated and were downregulated ThesecircRNAs were distributed on each chromosome butmost were located on chromosomes and and respectively suggesting that these chromosomes are more strongly correlated with 5FU resistancethan other chromosomes The top most strongly 0cXu Molecular Cancer Page of upregulated circRNAs were predicted to be capable ofregulating the Wnt signaling pathway which is deeplyinvolved in 5FU resistance [] In addition several FUresistancerelated miRNAs such as miR8853p []may be targets of these circRNAsGemcitabine is another widely used Sphase antimetabolite drug [] Yan [] reported that circ_0035483is significantly increased in TK10 and UO31 renal cancercell lines after treatment with gemcitabine The results ofan MTT assay showed that high expression of circ_ in gemcitabinetreated TK10 cells could enhancecell viability to induce gemcitabine resistance The resultsof RNA pulldown experiments revealed that circ_0035483can bind with miR335 as a sponge Further dualluciferase assay results identified CCNB1 as a target ofmiR335 CCNB1 is an important checkpoint molecule inthe cell cycle and is deeply involved in cell cyclespecificgemcitabine resistance [ ] Promotion of circ_ expression increased the expression of CCNB1and cell apoptosis was inhibited Thus circ_0035483 promotes gemcitabine resistance in human renal cancer cellsby sponging miR335 to upregulate CCNB1found to be differentiallyline and the parental cellGemcitabine resistance can also be developed by selection under exposure to an increasing gradient of gemcitabine in pancreatic cancer PC celllines [] Theline was develgemcitabineresistant SWl990GZ cellline and ciroped from the parental SWl990 cellexpressedcRNAs werebetween this cellline []Twentysix of these circRNAs were upregulated and were downregulated Four circRNAs were validated byqRTPCR Pathway analysis indicated that these dysregulated circRNAs are deeply involved in gemcitabine resistance in PC via modulation of MAPK and mTORsignaling pathways [ ]etShao[]alIn additionestablished theline and comgemcitabineresistant PANC1GR cellpared the differential circRNA profiles between thePANC1GR cell line and the parental PANC1 cell lineA total of circRNAs were identified by highthroughputtranscriptome sequencing as significantlydifferentially expressed in these two celllines ofwhich were upregulated and downregulated Moreover gene ontology GO term and pathway analysis results indicated the functions of these circRNAs in PCprogressionrelated signaling pathways such as the ErbBpathway [] The two most significantly upregulatedchr14101402109“ and chr4circRNAs“ were further validatedandfunctional experiments showed that silencing these twocircRNAs restored gemcitabine sensitivity in PANC1GR cells while overexpression of these circRNAs promoted gemcitabine resistance in both the PANC1 andMIA PACA2 cellIdentification of potentiallinesbinding miRNAs of these two circRNAs by sequenceanalysis suggested that miR1455p a tumor suppressorinhibiting PC progression [] could bind both circRNAs and act as a downstream target Moreover theplasma expression levels of the two circRNAs and miR1455p in gemcitabinetreated PC patients were verifiedvia qRTPCR Consistent with previous results the twocircRNAs were upregulated but miR1455p was downregulated in the plasma of gemcitabineresistant patientsThis finding confirms their potentialfor monitoringgemcitabine resistance via liquid biopsyDrugs derived from plantsTaxol is an Mphase specific plant drug that was originally derived from the bark of the Pacific yew and has become one of the most widely used agents [] Unlikeantimetabolite drugs Taxol can bind tubulin to arrestmitosis and induce apoptosis resulting in cell death []The mechanism underlying Taxol resistance remainspoorly understood [] however ncRNA dysregulationplays an important role in this process [ ] Taxol isan effective chemotherapeutic drug in firstline therapyfor gastric cancer [] Liu [] reported thatcircPVT1 a circRNA derived from the oncogeniclncRNA PVT1 locus [] can mediate Taxol resistancein gastric cancer Overexpression of circPVT1 was confirmed both in Taxolresistant gastric cancer tissues andcells and circPVT1 knockdown promoted apoptosistriggered by Taxol ZEB1 is a wellknown promoter ofTaxol resistance [ ] Mechanistically circPVT1 canpromote ZEB1 expression by sponging miR1243p Inaddition Taxol resistance remains problematic in thetreatment of ovarian cancer [ ] Expression profilesof dysregulated circRNAs were identified in Taxolresistant ovarian cancer tissues A total of aberrantlyexpressed circRNAs were found were upregulatedand downregulated [] Among these circRNAscirc_0063809 was further confirmed by qPCR to be upregulated in both Taxolresistant tissues and cells Invitro and vivo experiments demonstrated that silencingcirc_0063809 promotes Taxolinduced cytotoxicity inTaxolresistant ovarian cancer cells Moreover the results of mechanistic experiments revealed that circ_ can upregulate FOXR2 expression by spongingmiR1252 to contribute to Taxol resistance in ovariancancer Taxol is also recommended for firstline treatment of HER2negative metastatic breast cancer BC[] CircAMOTL1 was found to contribute to Taxol resistance by activating the AKT pathway []Xu [] reported the differentially expressed circRNA profile in the Taxolresistant A549Taxol cell linecompared to the parental A549 cell line A total of circRNAs were identified among which circRNAs were upregulated and were downregulated 0cXu Molecular Cancer Page of GO analysis results showed that the most significantlyenriched terms were linked to Taxol resistancerelatedterms such as GTPase binding and alterations in cell adhesion [ ] In addition Cytoscape was used tovisualize the circRNAmiRNA interaction network Several miRNAs previously reported to drive Taxol resistance were predicted For example miR141 has beensuggested to induce Taxol resistance in ovarian cancer[] and miR34c5p can downregulate p53 to induceTaxol resistance in lung cancer [] Circ_0071799which was upregulated is related to miR141 Downregulated circ_0091931 can interact with miR34c5p Inaddition Li [] reported that circ_0002483 overexpression can enhance Taxol sensitivity in nonsmallcell lung cancer NSCLC Circ_0002483 has been confirmed by a dualluciferase reporter assay to spongemiR1825p and pathway analysis revealed that the circ_0002483miR1825p interaction may be deeply involvedin the FoxO signaling pathway Further analysis indicated that the ² untranslated regions ²UTRs ofGRB2 FOXO1 and FOXO2 contain miR1825p comcirc_plementary0002483miR1825pGRB2FOXO1FOXO3 axiscanreverse Taxol resistance in NSCLCsequences MechanisticallytheYu [] evaluated the overexpression of circ_ in the docetaxelresistant lung adenocarcinomaA549DTX and H1299DTX cell lines Knockdown ofcirc_0003998 in A549DTX and H1299DTX cells partially restored docetaxel sensitivity via promotion of cellapoptosis In addition miR326 was predicted by theCircular RNA Interactome CircInteractome database[] as a potential target of circ_0003998 MiR326 is awellrecognized tumor suppressor and its downregulation is deeply involved in multidrug resistance in tumors[] and a significant negative correlation betweencirc_0003998 expression and miR326 expression wasfound in lung adenocarcinoma tissues Further dualluciferase reporter assays confirmed the direct bindingbetween circ_0003998 and miR326 In addition miR inhibitors were found to significantly reverse theincreased sensitivity to docetaxel caused by circ_ siRNA In summary overexpression of circ_ induces docetaxel resistance in lung adenocarcinoma partially by sponging of miR326 Docetaxelcan also be used in firstline chemotherapy for patientswith metastatic nasopharyngeal carcinoma NPC []Hong [] reported that circCRIM1 binds tomiR422a to upregulate FOXQ1 in NPC FOXQ1 is intimately involved in chemotherapeutic resistance byregulating the epithelialmesenchymal transition EMT[] In vivo and in vitro functional experiments haveconfirmed that FOXQ1 overexpression induced by circCRIM1 also contributes to metastasis and docetaxelresistance in NPC []Anticancer antibioticsAs a cell cyclenonspecific anticancer drug doxorubicinAdriamycin is an anthracycline antibiotic antineoplastic drug widely used in the clinic [] Its underlyinganticancer mechanisms are inhibition of DNA synthesisinterference with topoisomerase II activity and inductionof free radical damage to cells [] CircRNAs have alsobeen reported to play a role in doxorubicin resistanceThe differential expression profile of circRNAs in adoxorubicinresistant acute myeloid leukemia AMLcell line THP1ADM was identified by analysis of ahuman circRNA array Fortynine circRNAs were foundto be differentially expressed with a fold change of inTHP1ADM cells compared to THP1 cells Thirtyfivewere upregulated and were downregulated Amongthese circRNAs circPAN3 was considered a candidatemediator of doxorubicin resistance due to its substantialdifferential expression and the important role of its hostgene PAN3 in AML [] In addition circPAN3 expression was higher in the bone marrow of patients with refractoryrecurrent AML than in the bone marrow ofdoxorubicinsensitive patients Moreover knockdown ofcircPAN3 reversed doxorubicin resistance in THP1ADM cells TargetScan showed that miR1535p andmiR1835p could be target genes of circPAN3 XIAP awellknown chemoresistancerelated gene in AML []was found to be a downstream target of both miR1535pand miR1835p Further mechanistic experiments alsoconfirmed the circPAN3miR1535pmiR1835pXIAPaxis which contributes to doxorubicin resistance in AMLDoxorubicin has also been approved by the FDA for clinical use in patients with BC [] The results of a microarray screen also indicated the important role of circRNAsin doxorubicin resistance in BC [] Eighteen circRNAswere identified as significantly differentially expressed betweenanddoxorubicinsensitive MCF7 cells were upregulatedwhile were downregulated Kyoto Encyclopedia of Genesand Genomes KEGG analysis revealed that the most relevant signaling pathways included the MAPK and PI3KAktsignaling pathways which are closely related to doxorubicinresistance [ ] In addition circ_0006528 expressionwas measured by qRTPCR The results confirmed circ_ overexpression in doxorubicinresistant cell linesand tissues Downregulation of circ_0006528 restored thesensitivity of MCF7ADM and MDAMB231ADM cellsto doxorubicin Moreover miR75p which has been reported to be involved in PI3KAkt signaling pathway activation and chemoresistance in BC [] was predicted to bea target of circ_0006528 while Raf1 a MAPK signalingpathway activator is a direct target of miR75p [] Further experimental results proved that the circ_0006528miR75pRaf1 axis may be responsible for doxorubicin resistance in BCdoxorubicinresistantMCF7ADM 0cXu Molecular Cancer Page of resistanceinreverseddoxorubicinCircKDM4C was reported by Liang to be atumor suppressor in BC [] In vitro experimentsidentified decreased expression of circKDM4C in MDAMB231DOX cells compared with the parental MDAMB231 cells CircKDM4C knockdown in MDAMB cells promoted resistance to doxorubicin whileoverexpression of circKDM4C in MDAMB231DOXcells inhibited cell proliferation enhanced apoptosis andfinallydosedependent and timedependent manners MiR548p waspredicted by CircInteractome database analysis to be atarget of circKDM4C and upregulation of miR548p wasfound to partially abolish circKDM4C overexpressioninduced inhibition of doxorubicin resistance FurthermiRNA target prediction indicated that the ²UTR ofphenazine biosynthesislike domaincontaining proteinPBLD is considered a putative target of miR548pPBLD is associated with various tumor progressionrelated signaling pathways such as the MAPK pathway[] Luciferase reporter assay results confirmed thecircKDM4CmiR548pPBLD axis and the contributionof this axis to doxorubicin resistance was proven bothin vitro and in vivoPlatinum drugsPlatinum drugs such as cisplatin and oxaliplatin arewidely used in the treatment of human cancers and haveachieved clinical success as standard therapies []Oxaliplatin is a platinum complex with oxalate and 1R2R12diaminocyclohexane DACH ligands Oxaliplatin uptake has been reported to be mediated by the anic cation transporters OCT1 and OCT2 which areoverexpressed in CRC cells [] This overexpressionpartially explains why oxaliplatin is an efficient component in the adjuvant FOLFOX treatment regimen inmetastatic CRC Oxaliplatin resistance has become anissue in CRC Drugresistant HCT116 HCT116R cellswere developed by exposure to FOLFOX and circRNAmicroarray analysis was performed to compare circRNAexpression profiles between HCT116R and parentalHCT116 cells [] A total of upregulated and downregulated circRNAs were identified The presenceof two upregulated circRNAs circ_32883 and circ_0338in extracellular vesicles was further validated Circ_ was found to be significantly upregulated in CRCtissues Several microRNAs such as miR5015p werepredicted to interact with circ_32883 to bind to theirtarget genes Hon [] isolated exosomes fromthe cell culture medium of HCT116R and parentalHCT116 cells and used by circRNA microarray analysisto identify significantly upregulated and downregulated circRNAs Consistent with a previous reportcirc_32883 and circ_0338 were upregulated in HCT116R cellderived exosomes In addition the resistance ofHCT116R cells to FOLFOX was partially reversed byknockdown of circ_0338 indicating a potential role forcirc_0338 as a biomarker for FOLFOX resistance inCRC Wang [] identified a novel circRNA circ_ in exosomes derived from oxaliplatinresistantCRC cells This circRNA could be transferred intooxaliplatinsensitive CRC cells and promote glycolysisand oxaliplatin resistance in the recipient cells Furtherexperiments showed that circ_0005963 can sponge miR to upregulate PKM2 In addition sicirc_0005963transferred by exosomes inhibited glycolysis and reversed resistance to oxaliplatin Thus this exosomallyinitiated circ_0005963miR122PKM2 signaling axis hasgreat therapeutic potential for oxaliplatinresistant CRCCisplatin was first synthesized by Michele Peyrone andwas approved by the US FDA for use in testicular andovarian cancer in [] Cisplatin often leads to aninitial therapeutic response but many originally sensitivetumors gradually develop resistance [] Huang et al[] screened circRNAs derived from EIF3a the largest subunit of the translation initiation factor EIF3 incisplatinresistant A549DDP cells Two circRNAs transcribed from EIF3a circ_0004350 and circ_0092857were validated to exhibit differential expression betweenA549DDP and parental A549 cells Moreover downregulation of circ_0004350 and circ_0092857 reversed cisplatin resistancecells Functionalenrichment analysis revealed that these two circEIF3asmay have a synergistic functional effect with their parental EIF3a gene on cisplatin resistancein lung cancerCirc_0076305 is another circRNA identified to promote cisplatin resistance in lung cancer Significantly elevated expression of circ_0076305 was proven incisplatinresistant tissues and cells [] Moreover cisplatin treatment was found to effectively upregulate theexpression of circ_0076305 in A549 and H1650 cellsFurther gain and lossoffunction experiments indicatedthat circ_0076305 can regulate cisplatin resistance inlung cancer cells Bioinformatic and mechanistic experiments such as circRNA immunoprecipitation circRIPand luciferase reporter assays were used to validate thebinding of circ_0076305 to miR2965p as well as tomiR2965p and STAT3 Reports have indicated thatmiR2965p acts as a tumor suppressor in lung cancerand that STAT3 can contribute to cisplatin resistance[ ] Pearson correlation analysis showed that theexpression levels of circ_0076305 are negatively correlated with those of miR2965p and positively correlatedwith those of STAT3 These results suggest that circ_ increases cisplatin resistance by acting as amiR2965p sponge to promote STAT3 expression inlung cancer Zheng [] reported that circPVT1contributes to cisplatin resistance in lung adenocarcinomaaxis Howeverthe miR1455pABCC1via 0cXu Molecular Cancer Page of circRNAs can also act as suppressors of cisplatin resistance in lung cancer Circ_0001946 expression was significantly lower in A549DDP cells than in parentalA549 cells [] The nt long circ_0001946 is derived from cerebellar degenerationrelated protein CDR1 Knockdown of this circRNA was found to increase cisplatin resistance in A549 cells and the resultsof a host cell reactivation HCR assay and Western blotanalysis demonstrated that silencing circ_0001946 activates the nucleotide excision repair NER signalingpathway which increases the ability for DNA damage repair [] to reduce cisplatin sensitivity in lung cancerTreatments for osteosarcoma OS the most commonmalignant bone tumor in teenagers are limited []Cisplatinbased neoadjuvant chemotherapy has greatly impro

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

"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

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

Thyroid_Cancer

"preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesfibrocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[“] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a specific gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identified as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identified and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly defined [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGF𝛽P SMAD3 SMAD3Testosterone𝛽catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBP𝛽MAPKG3K3𝛽P2CEBP𝛽CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis [] Forced expression ofPPARÎ is sufficient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincfinger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function efficientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARΓ“ï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeficient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] fibroblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the first KLF family members which were identified to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic fibroblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBP𝛽CEBP𝛿GATA23PPARá¿«CEBP𝛼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory protein“protein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to specificallyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown [] Histone Modification in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding affinity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [“] Lysinespecific histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modification of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespecific histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a specific proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage specificity [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the ‚uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 “ mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBP𝛽 geneEBF1 geneKLF4EGR2CEBP𝛽CytosolCEBP𝛿 geneCEBP𝛿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBP𝛼 geneZNF638ZNF467CEBP𝛼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGF𝛽1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA

Thyroid_Cancer

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

Thyroid_Cancer

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

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

Thyroid_Cancer

"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

"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

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

Thyroid_Cancer

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

Thyroid_Cancer

Patients with resected oral cavity squamous cell carcinoma OCSCC are often treated with adjuvantradiation RT ± concomitant chemotherapy based on pathological findings Standard RT volumes include all surgicallydissected areas including the tumour bed and dissected neck RT has significant acute and longterm toxicitiesincluding odynophagia dysphagia dermatitis and fibrosisThe goal of this study is to assess the rate of regional failure with omission of radiation to the surgically dissectedpathologically node negative pN0 heminecks compared to historical control and to compare oncologic outcomestoxicity and quality of life QoL profiles between standard RT volumes and omission of RT to the pN0 neckMethods This is a multicentre phase II study randomizing patients with T1“ N0“ OCSCC with at least one pN0hemineck in a ratio between standard RT volumes and omission of RT to the pN0 heminecks Patients will bestratified based on overall nodal status nodal involvement vs no nodal involvement and use of concurrentchemotherapy The primary endpoint is regional failure in the pN0 heminecks we hypothesize that a 2year regionalrecurrence of or less will be achieved Secondary endpoints include overall and progressionfree survival localrecurrence rate of salvage therapy toxicity and QoLDiscussion This study will provide an assessment of omission of RT to the dissected pN0 heminecks on oncologicoutcomes QoL and toxicity Results will inform the design of future definitive phase III trialsTrial registration Clinicaltrialsgov identifier NCT03997643 Date of registration June Current version onJuly Keywords Head and neck cancer Oral cavity Radiotherapy Recurrence Survival Quality of life Randomized controlledtrial Deescalation Correspondence pencillalanglhsconca1Division of Radiation Oncology London Health Sciences Centre Commissioners Rd E London ON N6A 5W9 CanadaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLang Radiation Oncology Page of BackgroundPatients with resected squamous cell carcinoma of the oralcavity OCSCC are at risk of locoregional failure recurrence LRR at either the tumour surgical bed or in theneck Postoperative radiotherapy PORT is often addedafter surgical resection to reduce the risk of local and regional recurrence in patients with highrisk featuresGuidelines generally recommend PORT in patients withmore than one lymph node involved pT3 or pT4 primarydisease lymphovascular invasion LVI perineural invasion PNI close or positive margins extranodal extensionENE and sometimes for patients with lymph node involvement of neck levels IV or V [“]ENE and positive surgical margins are considered thehighestrisk pathological features whereas the other adverse features are considered ˜intermediate risk™ Patientswith highrisk features are generally offered concurrentchemotherapy with PORT based on a posthoc analysistwo of randomized trials [“] In patients with otheradverse features there is limited randomized evidence ofthe benefits of PORT alone [“] Retrospective seriesand comparisons with historical controls have shown reductions in LRR and improvement in overall survivalOS with PORT [“]RT treatment volumes after surgery generally includethe entire surgical bedincluding the dissected heminecks and may include the contralateral undissectedneck at the discretion of the treating physician []However PORT is associated with significant acute andincluding dysphagia mucositis xerostolate toxicitiesmiavoicechanges ototoxicity and hypothyroidism [ ] LargerRT treatment volumes are associated with increasingtoxicity These large treatment volumes are based onhistorical practice without guidance from randomizedevidence The benefit of treating the nodal regions in thepathologically node negative pN0 neck is unknownosteoradionecrosisdermatitisfibrosisRetrospective studies looking at series of oral cavitycancer patients in whom PORT was omitted altogetherhave also demonstrated low rates of isolated nodal recurrence ranging from to [“] In their seriesof patients with mostly T1T2 oral tongue tumourswith a pN0 neck treated with surgery alone PORTto the primary site or PORT to the primary siteand neck So reported a rate of isolatednodal recurrence of “ in all groups [] Mizrachi described a series of patients with T1T4 disease and pN0 neck with not receiving PORT with aneck recurrence rate of [] In Ganly patients with pT1T2 pN0 oral tongue cancer withoutPORT had an isolated regional recurrence rate of with an additional having a simultaneous locoregional recurrence In this study depth of invasion was predictive of neck recurrence []In a series of patients with a pN1 neck the neck recurrencefree survival was in those receiving PORT vs in thosewithout PORT [] It is difficult to draw comparisonsbetween groups receiving or not receiving PORT inthese retrospective studies since those receiving PORThad more risk featuresRetrospective data from a small number of PORT volume studies also demonstrate good oncologic outcomesin patients where radiation was omitted to the contralateral clinically or pathologically N0 neck [ ] In Vegeer patients with welllateralized oral cavity ororopharynx squamousSCC weretreated with unilateral PORT with of patients N0and N1 or N2 Contralateral metastases developedin only of patients with most successfully receivingsalvage therapy []carcinomacellA recent nonrandomized prospective phase II trialeliminated PORT to the pN0 neck in patients withhead and neck squamous cell carcinoma HNSCCdemonstrating excellent results with no isolated failuresand control in the unirradiated pN0 neck Twentypercent of patients included in the study had oral cavitytumours []Taken together the existing retrospective and prospective data suggests that omitting PORT in patientswith a pN0 neck likely has a recurrence rate less than“ However no randomized studies have directlyexamined the omission of PORT in the pN0 neck Thereis also a paucity of data examining the effects of omission of PORT on QoL We hypothesize that omittingPORT to the heminecks that have been dissected andshown to be pN0 will be associated with acceptable ratesof regional recurrence and will improve quality of lifeQoL The goal of this randomized phase II study is toassess oncologic outcomesfunctional outcomes andQoL in patients treated with PORT to the historicallystandard volumes usually including the primary site andall dissected neck areas vs PORT only to the primarysite and pathologically involved hemineck omitting radiation to the pN0 heminecksMethods designObjectivesThe objectives of this trial are to Compare regional recurrence rate to historicalcontrols with omission of PORT to the pN0 neck Compare oncologic outcomes toxicity and QoL forPORT ± chemotherapy based on standardtreatment volumes including the primary sitetumour bed dissected neck ± elective nodalregions vs PORT [± chemotherapy] that avoidstreating the dissected pN0 neck 0cLang Radiation Oncology Page of anisation for Research and Treatment of CancerEORTC Quality of Life Cancer Patients generalQLQC30 [] and head neck HN35 scales[] the EuroQOL 5Dimension 5Level EQ5D5L [] and the Neck Dissection Impairment IndexNDII [] 0f QoL measured at treatment completion and and monthsmeasured with MDADI EORTC QLQC30HN35 EQ5D5L and NDII 0f Rate of feeding tube insertion after start of radiationeither gastric gastrojejeunal or nasogastric andrate of feeding tube use at 1year postrandomization 0f Swallowing function at 1year assessed by ModifiedBarium Swallow MBS study and measured by theModified Barium Swallow Impairment ProfileMBSImP„¢ score [] the Dynamic ImagingGrade of Swallowing Toxicity DIGEST„¢ score []and the Functional Oral Intake Scale FOIS [] 0f Toxicity assessed using the National CancerInstitute Common Toxicity Criteria NCICTCversion 0f Rate of failure in the clinically node negative neck ifapplicable ie the undissected nodenegative neckfor welllateralized tumoursInclusion criteria 0f Age years or older 0f Willing to provide informed consent 0f Eastern Cooperative Oncology Group ECOG 0f Resected OCSCC with at least an ipsilateral selectiveneck dissection The oral cavity includes lips buccalperformance status “It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin Our hypothesis is that for patients with SCC ofthe oral cavity T1“ N0“ as per AJCC 8th editionwith at least one surgically dissected pN0 hemineck theregional failure rate will be or less at years whentreated with PORT omitting the pN0 neckStudy designThis is an label phase II multicentre randomizedtrial Patients will be randomized between current standard of care treatment Arm vs omission of radiationto the pN0 dissected heminecks Arm in a ratioFig The required sample size is patients Stratification factors include neck nodal status pN0 vs pNand use of chemotherapy Patients will be recruited fromtertiary care centres full list of participating sites available on clinicaltrialsgov NCT03997643Primary endpoint 0f Regional failure in the pN0 heminecksSecondary endpoints 0f OS 0f Diseasefree survival DFS 0f Local recurrence 0f Regional recurrence 0f Locoregional recurrence 0f Rate of salvage treatment surgery ± radiotherapy inthe pN0 neck and freedom from unsalvageable neckrecurrence 0f QoL at year assessed with the MD AndersonDysphagia Inventory MDADI [] the EuropeanFig Study schema 0cLang Radiation Oncology Page of mucosa oral tongue floor of mouth gingivaretromolar trigone and hard palate 0f Patient has at least one pathological feature that isan indication for PORT positive or close ‰¤ mmmargin presence of LVI or PNI pT3 or pT4 diseasepositive lymph nodes or ENE 0f PORT is recommended by the treating physician 0f Pathologically lymph node negative in at least onedissected hemineck with at least nodes recovered in each pN0 hemineck after a dissection thatat minimum includes nodal levels “ in the pN0heminecks 0f Radiation contours have been peerreviewed andapprovedExclusion criteria 0f Patients with an ipsilateral neck dissection only withpositive lymph nodes unless they undergo acontralateral neck dissection that is pN0 0f Patients with bilaterally involved neck nodes 0f Patients with pT3T4 tumours involving midlinewho undergo an ipsilateral neck dissection unless acontralateral neck dissection is performedcontraindications to radiotherapy 0f Serious medical comorbidities or other 0f Prior history of head and neck cancer within years 0f Any other active invasive malignancy except noncavity or neckmelanotic skin cancers lowrisk prostate cancer andStage IIVA papillary or follicular thyroid cancer 0f Prior head and neck radiation at any time 0f Prior oncologic head and neck surgery in the oral 0f Known metastatic disease 0f Locoregional disease recurrence identified followingsurgical resection but prior to start of radiotherapy 0f Inability to attend full course of radiotherapy or 0f Unable or unwilling to complete QoL questionnaires 0f Pregnant or lactating womenfollowup visitsPretreatment evaluation 0f History and physical examination by a radiationoncologist within weeks prior to randomization 0f Staging prior to randomizationCT or MRI of the neck with contrast unlesscontraindicated within weeks ofrandomization This can include the preoperativeCT or the radiation therapy CT or MRI simulation if reviewed by a radiologist–ª In some instances suspicious lymph nodes arevisible on the scan after surgery In suchinstances recurrence must be ruled outpathologically before enrollment either with aneedle biopsy or resection of these nodesCT of the chest or whole body PETCT usually prior to surgery must be within weeks ofrandomizationedentulous patientswithin weeks of randomization 0f Histological confirmation of SCC 0f Pregnancy test for women of childbearing age 0f Dental evaluation prior to starting treatment except 0f Assessment of dysphagia using NCICTC version 0f Completion of QoL scoring prior to initiation of 0f Prior to randomization radiation contours are to be 0f MBS at baseline prior to initiation of treatment with within weeks of treatment initiationpeerreviewed and approvedtreatmentdocumentation of the MBSIimP„¢ DIGEST„¢ andthe FOIS scoresresectionTreatment planSurgicalandchemotherapy will be delivered in accordance with National Comprehensive Cancer Network NCCN ClinicalGuidelines []and adjuvantradiotherapyPrimary tumours should be resected en bloc wheneverpossible with the goal of achieving clear margins Patients with midline involvement of the primary tumourshould receive a bilateral neck dissectionAdjuvant cisplatinbased chemotherapy concurrentwith radiotherapy is at the discretion of the treatingmedical oncologist and is recommended for patientswith positive margins or ENE for patients who can tolerate chemotherapy For patients who are deemed unfitor too elderly years of agefor cisplatinbasedchemotherapy the standard dose andor schedule canbe modified alternative systemic therapy regimensmaybe used eg weekly carboplatin Calais regimen orsystemic therapy can be omitted at the discretion of thetreating physicians It is strongly recommended that radiation starts within weeks of the date of surgery andit is mandatory to start no more than weeks after thedate of surgeryDuring treatment supportive care should be in accordance with local standard of care which often includes speech language pathology SLP assessment AnySLP interventions eg providing swallowing exercisesshould be the same in both arms and conform to localstandard of careDosefractionation In both arms a dose of Gy in fractions will be delivered to the operative bed targetvolumes Centres that normally treat dissected nodenegative levels to Gy in fractions will be permitted 0cLang Radiation Oncology Page of to do so if used consistently for all patients on trialAreas of positive margins or ENE should receive Gyin fractions if those areas can be localized Undissected areas that require coverage in the opinion of thetreating radiation oncologist eg low neck retrostyloidspace should receive Gy in fractions Table Immobilization and localization All patients will beimmobilized in a custom thermoplastic shell and willundergo a planning CT simulation with or withoutIV contrast encompassing the head and neck tobelow the clavicles The planning CT will be fusedwith other diagnostic imaging eg MRI scans or preoperative CT where necessary Bite blocks tonguedepressors jaw separators may be used as per institutional protocolthetime of planning CT prior to contour generation andpatient randomizationthese must be determined atRadiotherapy volume definitions A randomization volume will be defined as the nodal volume in the dissectedpN0 heminecks The randomization volume will depend on the laterality of the neck dissection performedipsilateral vs bilateral and whether pathologically involved nodes are present in the neck dissection definedin Fig Patients with bilaterally involved neck nodesare ineligible Patients with a unilateral neck dissectionwith positive lymph nodes are ineligible unless theyundergo a staged neck dissection of the opposite sidethat is pN0Standard treatment volumes Arm If randomizedincluding thecontoured volumesto Arm randomization volume will be treatedallOmission of pN0 neck Arm If randomized toArm the randomization volume will be omitted fromtreatment planningClinical Target Volumes CTVThe following radiation volumes will be contoured forall patients prior to randomization Table The suffixœpos denotes CTVs in the nodepositive hemineck andthe suffix œneg denotes the CTVs in the nodenegativehemineck Highrisk volume CTV64 regions correspondingto positive margins or ENE if present and if thoseareas can be localized Primary tumour operative bed CTVp60surgically dissected areas corresponding to theresected primary tumour typically including thepreoperative tumour area and any flap reconstructions and clips with a margin Involved necka Surgically dissected involved neck nodalvolume CTVn60pos surgically dissected necklevels in the nodepositive hemineck if applicable In the dissected neck some centres routinely treat only involved nodal areas to Gyand the remainder of the dissected neck to Gy For centres where this is standard this approach is acceptable but all patients enrolledfrom these centres must be treated with this approach The areas treated to Gy would be included in a CTVn54posb Optional uninvolved lowrisk neck on the involved side CTVn54pos lowrisk undissectedneck nodal volume on the involved side if applicable at the discretion of the radiation oncologist This may include the standardlymphatic drainage sites not dissected at thetime of surgery in the node positive heminecksuch as the nodal levels below or above the dissected areas eg level IVb retrostyloid space œRandomization Volume corresponding to theUninvolved necka Surgically dissected involved neck nodalvolume CTVn60neg surgically dissectedneck levels in the node negative hemineck Incentres that routinely treat the pN0 neck to Gy that dose is acceptable but all patients mustbe treated with that approach In such centresthese areas treated to Gy would be includedin a CTVn54negb Optional uninvolved lowrisk neck on theuninvolved side CTVn54neg lowrisk undissected neck nodal volume on the uninvolvedside if applicable at the discretion of theTable Radiation treatment volumes and dosesPTV VolumePTV64Areas of positive margin or ENEPTV60Dissected neckPTV54optionalNot surgically dissected elective nodal regionsCTV Volumes IncludedArm CTV64CTVp60 CTVn60posCTVn60negCTVn54posCTVn54negArm CTV64CTVp60 CTVn60posCTVn54posDose infractions Gy Gy Gy 0cLang Radiation Oncology Page of Fig The œRandomization Volume corresponds to the pN0 heminecks The neck volumes included in the œRandomization Volume dependon whether the patient had an ipsilateral vs bilateral neck dissection and the pathological findings in each heminecks Patients with bilaterallyinvolved neck nodes are ineligible Patients with an ipsilateral neck dissection with positive lymph nodes are ineligible unless they undergo acontralateral neck dissection that is pN0 If randomized to standard treatment volumes Arm all contoured volumes including theœRandomization Volume will be treated If randomized to omission of the pN0 neck Arm the œRandomization Volume will be omitted fromtreatment planningradiation oncologist In a patient who has had aunilateral neck dissection that was pN0 thismay include the contralateral clinically nodenegative cN0 neck if that is standard institutional practiceIf randomized to omission of PORT to pN0 neckArm the CTVn60neg and CTV54neg contours aredeleted after randomization prior to treatment planningTo prevent bias contours cannot be changed afterrandomization Local peer review of contours must takeplace before randomization occurs An overview of theprotocol timeline is shown in Fig Planning Target Volumes PTVA “ mm expansion is used around the combinedCTVs Table to create the PTV as per institutionalsetup and protocolRadiotherapy planning Intensity modulated radiotherapy IMRT photon therapy or proton therapy will beused for all patients in this study IMRT can be deliveredusing staticbeam techniques or rotational techniqueseg Tomotherapy or Volumetric Modulated Arc Therapy [VMAT] If protons are used the dose will be reported in Gy relative biological effectiveness [RBE]where the proton dose is multiplied by an RBE of All reported doses in Gy are considered equivalent Centres with proton therapy will use their institutionalstandard planning and delivery techniquesAll plans will be normalized to ensure that ofeach PTV is covered by of the prescription dosefor that volume An exception will be allowed forcentres that normally prescribe in such a manner that of the PTV be covered by of the prescription dose and such centres must prespecify this before enrolling the first patient and all subsequentpatients must be planned in the same manner Amodified PTV cropped mm from the externalcontour for dose evaluation may be used as per institutional guidelines 0cLang Radiation Oncology Page of 0f All dose delivery for intensitymodulated plans including arcbased treatments will be confirmed before treatment by physics staffConebeam CT andor orthogonal xrays will be usedon a daily basis to verify treatment positioning as per institutional standard practiceCredentialling Prior to enrolling patients each centrewill be given a sample CT dataset through secure filetransfer protocol FTP for contouring planning andphysics QA Enrollment can begin once the plan andQA have been approved at the London Regional CancerProgram Centres who have been accredited for ORATOR [] or ORATOR2 [] are exempt from thisrequirementParticipant discontinuation withdrawalParticipants may voluntarily discontinue participation inthe study at any time If a participant is removed fromthe study the clinical and laboratory evaluations thatwould have been performed at the end of the studyshould be obtained If a participant is removed becauseof an adverse event they should remain under medicalobservation as long as deemed appropriate by the treating physicianFollowup evaluationDay of followup will be the first day of radiotherapyFollowup will consist of history and physical examination with laryngopharyngoscopy CT imaging of theneck ± thorax QoL assessments and MBS The followup schedule is shown in Additional file Additional imaging or laboratory investigations should be carried outat the discretion of the oncologist based on findings inthe history or physical examination Additional treatment eg salvage treatment with surgery or furtherradiotherapy is at the discretion of the treating physicians but will be recorded in the case report formMeasurement of outcomes 0f pN0 neck failure measured as time fromrandomization until disease recurrence in theinitially pN0 heminecks Patients with prior orsimultaneous recurrence at the primary site or inthe initially pN hemineck will be censored for thisoutcome as of that timepoint The primary endpointis a comparison of pN0 neck failure in Arm vshistorical controls this endpoint will also be compared between the two arms as a secondaryendpoint 0f OS measured as time from randomization untildeath from any causeFig Flowchart showing timing of randomization with respect topeer review and treatment planning Contours must be finalizedbefore randomization and may not be changed after randomizationThe maximum dose to PTV64 or PTV60 if no PTV64present should not exceed of the prescribed doseand no volume cc outside of these PTVs should receive of the prescription doseans at Risk OAR definitions dose constraints andplanning priorities are adapted from the following protocols RTOG protocols [] Arm and RTOG [] Arm NCICCTG HN6 ORATOR [ ] andORATOR2 [] and are described in Additional file Quality assurance QAIn order to ensure patientsafety and effective treatment delivery a robust QAprotocolis incorporated The following requirementsmust be completed for each patient 0f Prior to randomization each set of contours will bepeerreviewed either by another individual radiationoncologist or at a team head and neck QA rounds 0cLang Radiation Oncology Page of 0f DFS measured as time from randomization toeither recurrence at any location or deathwhichever occurs first New primary tumours willnot count as DFS events 0f Local recurrence measured as time fromrandomization until disease recurrence at theprimary site 0f Regional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck 0f Locoregional recurrence measured as time fromrandomization until disease recurrence anywhere inthe neck or at the primary site whichever occursfirst 0f Recurrence in the pN0 neck without otherlocoregional recurrence measured as time fromrandomization until disease recurrence in the pN0neck alone without recurrence at the primary site orpN neck 0f Rate of salvage surgery andor radiation in pN0neck measured as time from randomization tosalvage intervention surgery ± radiation in the pN0neck Freedom from unsalvageable neck recurrencewill be reported as the time from randomization todevelopment of a neck recurrence in the pN0 neckthat could not be salvaged 0f Feeding Tube Insertion Rate of feeding tubeinsertion after start of radiation either gastricgastrojejeunal or nasogastric and rate of feedingtube use at 1year postrandomization Patients withfeeding tubes inserted prior to randomization will becensored for this endpoint 0f Rate of failure in the clinically node negativeneck for patients who have unilateral neckdissections the cN0 neck may be treated withradiation or observed at the discretion of thetreating oncologist see section This is measuredas the time from randomization to failure in the cN0neck and will be reported for the whole group ofpatients who had unilateral dissections and alsostratified by whether radiation was delivered to thatareaEnrollment randomization and allocationPatients will be enrolled by dedicated clinical trials staffandor the investigator at each participating institutionPatients will randomized in a ratio to standard radiation volumes Arm vs omission to the pN0 neckArm A permuted block design with two stratification factors will used with the size of the blocks knownonly to the statistician stratified based upon overall necknodal status pN0 vs pN and use of concurrentchemotherapy yes vs no Randomization sequences aregenerated for each strata separately with a randomnumber generator based on permuted block design Thisgets formatted as a CVS file which gets uploaded intoREDCap []Statistical considerationsSample size The primary endpoint is the rate of relapsein the pN0 neck ie the regional relapse rate in the pN0neck in Arm compared to historical controls Allother endpoints are a comparison between Arm andArm It is generally accepted that a risk of nodal recurrenceof “ is sufficient to warrant radiation to a nodalbasin and therefore we wish to exclude a risk of regionalrecurrence in the pN0 neck of at 2years Using aonesided onesample binomial test allowing for dropout a sample size in Arm of patients provides power at a significance level to detect a regional control rate in the pN0 neck of compared toan unacceptable level of This sample size in Arm and in Arm alsoallows for power to detect a 10point difference inthe total MDADI score at year a secondary endpointassuming the scores are normally distributed with astandard deviation of in each arm It is generally believed that a 10point difference in standardized QoLscores represents a clinically significant difference inQoL [] It is assumed that the QoL scores will be normally distributed with a standard deviation of in eacharm The MDADI will be calculated as the compositescore but will also be reported for each of the subscalesAnalysis planPatients will be analyzed in the groups to which they areassigned intentiontotreat Oncologic outcomes andOS will be calculated from date of randomization usingthe KaplanMeier method A onesided onesample binomial test will be used for the primary endpoint as described above For actuarial comparisons between armsthe stratified logrank test will be used stratified bystratification factors An twosample Ttest will be usedto compare QoL scores at 1year The percentage of patients in each arm who experience a clinically significantQoL decline on the MDADI points will also bereportedMBSImP„¢ scores will be compared at 1year including the total score and the scores on the oral andpharyngeal subscales using a twosample Ttest or aWilcoxon rank sum test as appropriate DIGEST„¢ andFOIS scores will be compared using the Chisquare testPreplanned subgroup analysis will occur based on thestratification variables nodal status [pN0 vs pN] anduse of concurrent chemotherapy as well as based onthe neck dissection performed unilateral vs bilateral 0cLang Radiation Oncology Page of and Tstage T1“ vs T3“ There will also be a preplanned analysis based on the extent of nodes harvestedin the pN0 neck vs or more depth of invasionof the primary tumour mm vs mm or more andphoton versus proton treatmentMultivariable Cox proportional hazards or logistic regression analysis as appropriate will be used to determine baseline and pathologic factors predictive of pN0neck failure DFS OS locoregional recurrence and salvage therapy For the secondary endpoints involvingQoL scales linear mixed effects models will be used forthe MDADI the total scores will be compared betweenthe two arms whereas for the EORTC QLQC30 andHN35 scales each of the subscales eg pain swallowing etc will be compared between the two armsRates of grade ‰¥ toxicity and use of feeding tubes willbe compared between arms using the Chisquare orFisher™s exact test as appropriateUtilities will be calculated from the EQ5D5L whichwill be administered at baseline and at month intervalsQuality adjusted life years QALYs will be assessed asthe area under the preferenceweighted survival curveOverall costs of each treatment strategy will be abstracted from the available literature The incrementalcost effectiveness ratios ICERs between treatment armswill be compared through the standard method of ratiobetween differences in costs and QALYs Point estimatesfor these differences can be derived from multivariablegeneralized estimating equations GEE or generalizedlinear model GLM analysesData safety monitoring committeeThe Data Safety Monitoring Committee DSMC consisting of at least one radiation oncologist and one medical oncologist not involved in the study will meet every months after study initiation Toxicity outcomes willbe monitored but since the experimental arm involvessmaller radiation volumes it is extremely unlikely thattoxicity would be higher in the experimental arm andtherefore no stopping rules for toxicity are includedInterim analysis The DSMC will conduct one interimanalysis once patients have been accrued and completed the 6month QoL questionnaires For this analysis the DSMC will be blinded to the identity of eachtreatment arm but QoL recurrence in the pN0 neckOS and DFS estimates at 2years will be presented foreach armThe DSMC will recommend stopping the trial if thereis an OS difference that is statistically significant with athreshold of p using the stratified logrank testbased on the HaybittlePeto stopping rule this retainsan overall alpha of Ethical considerationsThe Principal Investigator will obtain ethical approvaland clinical trial authorization by competent authoritiesaccording to locallaws and regulations The WorldHealth anization WHO Trial Registration Data Setis shown in Additional file Institutional review board IRB research ethicsboard REB The protocol and any

Thyroid_Cancer

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

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

"Effects of lowdose computed tomography LDCT screening on lung cancercontains a that is not consistent with the data presented With reference to the National Lung ScreeningTrial NLST there are several flaws in the methodology overlooked Also there is no significant reduction in deathsfrom all causes following the screening Therefore any claim that the LDCT screening is superior to usual care isinvalidKeywords Lung cancer screening Low dose computed tomography MethodologyMain textYou recently published a paper by Huang KL entitled œEffects of lowdose computed tomography on lungcancer screening a systematic review metaanalysis andtrial sequential analysis [] In that paper the authorsstate in their Conclusion that œLDCT screening hasshown statistically significant mortality benefits in highquality trials In the they further state thatœLDCT screening is superiority over usual care in lungcancer survivalYet in the Section Benefits and adverse outcomesthey state On the contrary LDCT screening demonstrated no statistically significant difference in allcausemortality RR CI “The authors need to explain how a screening technique that produces no statistically significant differencein allcause mortality between LDCT screening andusual care can be superior to usual careThis comment refers to the available at 101186s128900190883xCorrespondence donbenjaminbigpondcomCancer Information Support Society Chandos St St Leonards NSW AustraliaThe authors also assess the risk for the NLST trial asincludingbeing Good Green on allMethodologycriteriaPotential flaws in methodologyIn fact the NLST trial had several methodological flawsrelated to the randomisation process overlooked by theauthors of the paper The NLST trial compared LDCT screening of highrisk smokers with Chest Xray CXR screening andassumed that Chest Xray screening produced thesame outcome as usual care [] as suggested in theProstate Lung Colorectal and Ovarian PLCOTrial [] despite earlier trials showing it resulted inan increase in allcause mortality [] Anticipating the shortcoming in above theauthors of the NLST trial ensured that the PLCOtrial had in addition to comparing average risksmokers selected high risk smokers who wereoffered Chest Xray screening for comparison withhigh risk smokers offered usual care to validatethe assumption referred to in Yet this selection ofhigh risk smokers was done after randomisation so The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenjamin BMC Pulmonary Medicine Page of the comparison of deaths of high risk smokers afterChest Xray screening with deaths of those receiving usual care was invalid In addition the PLCOtrial published only lung cancer deaths for theNLSTeligible high risk smokers not deaths fromall causes This means the assumption in Point that Chest Xray screening of high risk smokersproduced the same outcome as usual care in termsof allcause mortality was invalidOther irregularitiesReich and Kim observed that the distribution of deathsover time from the NLSTeligible groups selected fromthe PLCO trial showed irregularities suggesting thatthere were some reporting errors in the PLCO trialThey also observed that there were no extra tumoursfound by the screening in the NLSTeligible groups selected from the PLCO trial [] casting further doubt onthis selection process suggesting another flaw in themethodology The PLCO trial identified less than more tumours by screening compared with about more in previous chest Xray trialsThe above potential flaws and irregularities suggestthat a ˜Red “˜ should be applied to the Randomizationprocess the Missing outcome data and the Overall riskrather than a ˜Green ™ On this basis a lower weightingshould be applied to the NLST trial for the purposes ofthe metaanalysisThe main shortcoming of the current metaanalysislike that of many other randomised controlled trialsRCTs is that the authors ignore the most importantoutcome Allcause Mortality and focus on the Deathsfrom Lung cancer If there is no reduction in overalldeaths following the screening it is not valid to claimthat LDCT screening is superior to usual careAs pointed out by Black WC Allcause Mortalityin Randomized Trials of Cancer Screening both trials ofChest XRay screening they reported on in showedan increase in allcause mortality following Chest XRayscreening that they attributed to the harm caused bypostscreening treatments of higher risk smokers Theypointed out that as œdiseasespecific mortality may missimportant harms or benefits of cancer screening because of misclassification in the cause of death this endpoint should only be interpreted in conjunction with allcause mortality In particular a reduction in diseasespecific mortality should not be cited as strong evidenceof efficacy when the allcause mortality is the same orhigher in the screened group []Other issuesThe NLST trial reported major complication rates following invasive procedures for the LDCT and CXRgroups The risk was higher among persons whounderwent LDCT compared with Chest Xray screening vs per screened The earlier CXR screening trials had shown an increase in deaths among thoseoffered screening compared to those not offered screening usual care This is strong evidence in support ofthe suggestion that some of the reduction in deaths fromlung cancer following LDCT screening could have beendue to deaths from other causes resulting from the treatment that as suggested by Black above shouldhave been classified as deaths from lung cancer Thereshould therefore be strong reservations made about anyclaim that the LDCT screening was superior to usualcareFrom the above one possible explanation for the apparently positive result claimed in the NLST trial is thatthe Chest Xray screening had in fact increased thenumber of deaths among those offered screening as hadbeen observed in previous trials [] the LDCT screeninghad reduced the number of deaths by a similar amountcompared to Chest Xray screening the net result beingthat there was no significant reduction in overall deathsas observed Some of the reduction in lung cancerdeaths could have been due to the methodological flawsoutlined aboveFinally the NLST trial is the only large trial to claimbenefits for cancer screening which would make lungcancer screening the only type of cancer screening toproduce significant benefits Randomised trials of breastbowel prostate and ovarian cancer screening have notproduced significant reductions in allcause mortality []and thyroid cancer screening has largely been discontinued due to much evidence suggesting no benefits butsignificant harm from overdiagnosis and overtreatmentAbbreviationsCXR Chest Xray LDCT Low dose computed tomography NLST NationalLung Screening Trial PLCO Prostate Lung Colorectal and Ovarian TrialRCT Randomised Controlled Trial RR Relative Risk CI Confidence IntervalAcknowledgementsNot applicableAuthor™s contributionsThe above letter is completely the work of the author DB The authors readand approved the final manuscriptAuthors™ informationDon Benjamin has previously published papers on the subject of evaluatingthe efficacy of cancer surgery and cancer screeningFundingThe research giving rise to the above letter is being funded by the author™semployer The Cancer Information Support Society Incorporated based ona recommendation from the Society™s Research Director the author Thisresearch is part of an ongoing fouryear project that has identified a flaw inclaims of benefits from lung cancer and other cancer screening The by Huang [] had supported the claim that LDCT lung cancer screeningproduces benefits contrary to the Society™s research findings The current letter commenting on this therefore uses data produced from the original research and funds for writing this letter come from the same project 0cBenjamin BMC Pulmonary Medicine Page of Availability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe author declares that he has no competing interestsReceived October Accepted July ReferencesHuang KL Effects of lowdose computed tomography on lung cancerscreening a systematic review metaanalysis and trial sequential analysisBMC Pulm Med National Lung Screening Trial Research Team Reduced lungcancermortality with lowdose computed tomographic screening N Engl J Med“ 101056NEJMoa1102873Oken MM for the PLCO Project Team Screening by chest radiographand lung cancer mortality The Prostate Lung Colorectal and OvarianPLCO Randomized Trial JAMA “ 101001jama20111591Black W Haggerstrom D Welch HG Allcause mortality in randomized trialsof cancer screening J Natl Cancer Inst “ Author™s responseto discussion June “Reich JM Kim JS The National Lung Screening Trial premise of null andchest radiography equivalence is to question Am J Roentgenol “Benjamin DJ The efficacy of surgical treatment of cancer “ years laterMed Hypotheses “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

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

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

Thyroid_Cancer

"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstraŸe Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT “ Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting “ of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities [“]attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients™ antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ‰¥ GrayGy in line with a radiation dosedependent abscopal[“] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ‰¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing œbiased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patient™s participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ‰¥ or iii age ‰¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis œevent within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup œStop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients œGo Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ‰¤ event in n patients œGoany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the inˆ’ exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of œbothlungs minus GTV should receive Gy in the CONarm no more than of œboth lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ‰¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoˆ’radiotherapy as determined by the site™s multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria—‹ ECOG —‹ ECOG and CCI ‰¥ —‹ Age ‰¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ‰¥ of predictedcid129 DLCO ‰¥ of predictedcid129 FVC or VC ‰¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ‰¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigator™s judgement will substantially increase the risk associated with the patient™s participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ‰¤ event in n patientsconforms to a nontoxicity scenario with œevent beingdefined as the occurrence of pneumonitis grade ‰¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of — Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of — Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the œbiasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ‰¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ‰¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ‰¥ in thiscohort is ‰¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ‰¤ and H0CON Ï CON ‰¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simon™s twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageœOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients™ antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [“] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot

Thyroid_Cancer

distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedVon Hippel“Lindau disease is an autosomal dominant inherited syndrome predisposing to a variety of highly vascularised tumorsin diï¬erent ans Although bilateral pheochromocytoma was reported in patients with von Hippel“Lindau disease the coexistence of primary hyperparathyroidism is not a common condition We report an observation of a primary hyperparathyroidism secondary to an ectopic secretion of intact parathyroid hormone in a 17yearold girl with von Hippel“Lindau diseaseand bilateral pheochromocytoma She presented with a newly diagnosed diabetes mellitus and a severe arterial hypertensionBlood tests disclosed hypercalcemia with increased intact PTH level Cervical ultrasound and sestamibi scintigraphy were normalTwentyfourhour urinary normetanephrine level was highly elevated pointing to a catecholaminesecreting tumor e abdominal computed tomography showed bilateral adrenal masses MIBG scintigraphy exhibited a high accumulation of the tracerin both adrenal tumors Genetic testing revealed a mutation of the VHL gene e patient underwent a bilateral adrenalectomye postoperative outcome was marked by normalization of blood pressure blood glucose calcium and PTH levels In our casethe elevation of intact PTH and its spontaneous normalization after surgical treatment of pheochromocytomas confirms itsectopic secretion IntroductionPheochromocytomas are uncommon neuroendocrinetumors that arise from chromaffin cells of the adrenalmedulla and produce excessive amounts of catecholamines epinephrine norepinephrine and dopamine []Although most pheochromocytomas are sporadic morethan are associated with an inherited mutation andthis frequency can be as high as if diagnosed before years of age [] In childhood pheochromocytomasare mostly due to genetic causes including von Hippel“Lindau VHL disease multiple endocrine neoplasiatype MEN2 hereditary pheochromocytoma paraganglioma syndrome and rarely neurofibromatosis type [] Compared to adults VHL disease is reported to bethe most frequent genetic disorder causing pheochromocytomasbilateral[] Whileinchildrenpheochromocytomas were reported in patients with VHLdisease the coexistence of primary hyperparathyroidismis not a common condition []Herein we report a case of a VHL disease with bilateralintactpheochromocytoma and an ectopic secretion ofparathyroid hormone in an adolescent girl Presentation of CaseA 17yearold girl was referred to our department for theinvestigation of a newly diagnosed diabetes mellitus and asevere arterial hypertension She was the first child ofconsanguineous parents Her past medical history wasunremarkable Her family history was notable for type diabetes and dyslipidemia but no history of pheochromocytoma paraganglioma unexplained sudden death or 0cCase Reports in Endocrinologycondition that may lead to thinking about VHL disease wasreportedAs symptoms the patient complained of headachespalpitations diaphoresis and hot flashes since one monthShe reported also asthenia and body weight loss of fivekilograms in two weeksOn examination she had a body weight of kg abody height of cm a body mass index of kgm2 ablood pressure of mmHg without orthostatichypotension a regular pulse of bpm and a largeabdominal caf´eaulait spot Figure yroid abdominal and neurological examinations were normalTwentyfourhour blood pressure monitoring confirmed the diagnosis of hypertension and the presence ofpeaks of mmHg Electrocardiogram showed asinus tachycardia Capillary glucose level was glwithout ketosisFundoscopy showed grade hypertensive retinopathywithout any other abnormalitiese results of biological investigations are shown inTable e diagnosis of clinically suspected pheochromocytomawas confirmed by the dosage of urinary methoxylated derivatives at times the upper limit of normal In addition thediagnosis of diabetes mellitus dyslipidemia and primaryhyperparathyroidism were madethe first on thee abdominal computed tomography CT showed tworight measuringadrenal masses — — mm with a spontaneous density of HUheterogeneously enhanced in the arterial time showing areasof necrosis with an absolute washout of the second onthe left measuring — mm with the same characteristicsas the first one Figure ere were neither other localizations norlymph nodes MetaiodobenzylguanidineMIBG scintigraphy exhibited a high accumulation of thetracer in both adrenal tumors with no other localizationCervical ultrasound and 99mTcsestamibi scintigraphy werenormal Cardiac ultrasound was normale diagnosis of multiple endocrine neoplasia type 2awas highly suspected and the patient underwent a molecularinvestigation DNA analysis did not find a RET protooncogene mutation However it showed a missense mutationc191G C pArg64Pro in exon of the VHL gene onchromosome e diagnosis of bilateral pheochromocytoma in the setting of VHL disease was established Abdominal CT scan and craniospinal magnetic resonanceimaging with contrast did not show any cysts or othertumorse patient was treated with an alpha blocker prasozinea calcium channel blocker a beta blocker and insulin at adaily dose of unitskg She underwent a bilateral adrenalectomy in two steps e pathological examinationconfirmed bilateral adrenal pheochromocytomas Replacementtherapy with hydrocortisone was initiated aftersurgerye postoperative outcome was determined by thespontaneous normalization of blood pressure blood glucose calcium and PTH levels e patient remainedasymptomatic with no evidence oflocal recurrence ordistant metastasis during the months of followup efamily screening for VHL has not been performed yet DiscussionVHL disease is a dominantly inherited familial cancersyndrome caused by a germline mutation in the VHL tumorsuppressor gene and predisposing to a variety of benign andmalignant neoplasms most frequently retinal central nervous system and spinal hemangioblastomas renal cell carcinoma RCC pheochromocytoma and pancreatic tumors[ ] While central nervous system and retinal hemangioblastomas are the earliest expressions of the VHL syndrome pheochromocytoma may be the first manifestationof the disease especially in children and adolescents as it wasthe case of our patient [“] e frequency of pheochromocytoma in VHL syndrome is about to []Families with VHL disease have been divided into twosubtypes VHL type and VHL type based on the likelihood of developing a pheochromocytoma e presence ofpheochromocytoma defines types VHL disease is latteris subdivided based on the risk of developing RCC Type 2Aand 2B families have a low and high incidence of RCCrespectively whereas VHL type 2C kinds are characterizedby the development of pheochromocytoma without anyother manifestations of the disease [] However late onsetof other attacks is possible and a followup even spaced isrequired []Genotypephenotype correlations have been documented for this disorder and specific mutations are associated with the appearance of tumors in certain ansWhile most type families were reported to be more likelycarrying a missense mutation in the VHL gene most type families are aï¬ected by truncating or deletion mutations[] In our case the presence of pheochromocytomas andthe missense mutation in VHL gene suggested type VHLdisease Moreover the mutation found in our patientpArg64Pro has been described in patients with isolatedpheochromocytoma associated with RCC or with pancreaticneuroendocrine tumor [“] However our patient didnot present any sign of RCC or pancreatic neuroendocrinetumor during the months of followup is evaluationmay be early in our case as RCC and pancreatic neuroendocrine tumors in patients with VHL disease generallyappear at more advanced ages around “ years []Pheochromocytoma in VHL disease tends to be seen at ayounger age and is more frequently multifocal as in ourpatient and may be extraadrenal [ ] In most publishedcases the mean age at presentation was about years butvery young cases have been described the youngest before years [ ] In addition VHLassociated pheochromocytomas are less likely to be associated with symptoms orbiochemical evidence of catecholamines production compared with those occurring in patients without VHL [ ]In a report of the National Institute of Health about patients with VHL disease and pheochromocytomas a totalof tumors were identified Of these originatedoutside the adrenal gland and of the patients wereasymptomatic without hypertension orevidence of 0cCase Reports in EndocrinologyFasting glucose level mmollGlycated hemoglobin Total cholesterol mmollTriglycerides mmollNatremia mmollKalemia mmollCreatinine mgLCalcium mgLPhosphate mgLAlbumin glIntact PTHˆ— μgLPTH parathormone TSH thyroid stimulating hormone FT4 freeT4 NMN normetanephrines MN metanephrinesˆ—e intact PTH dosage was madeTSH mUILFT4 ngdlCalcitonin ngL hurine NMN μg24 hurine MN μg24 hurine sodium mmol24 h hurine potassium mmol24 h hurine calcium mg24 h hproteinuria g24““using 3rd generation chemiluminescence immunoassayReference ranges‰¤““““““““““““Figure A large abdominal caf´eaulait spotTable Biological parameters before and after surgeryBefore surgeryAfter surgery””””””””Figure Abdominal computed tomography showing the two adrenal masses two white arrowsincreased catecholamines production [] is was not thecase of our patient who had sustained severe hypertensionassociated with the classic symptoms of pheochromocytomapalpitation sweating and hot flashes hypokalemia and asecondary diabetes mellitus In our case as reported in theliterature [ ] a remarkable remission of diabetes 0cCase Reports in EndocrinologySoahighmellitus and an improvement of lipid profile were noticedafter tumor removal confirming the secondary character ofthese two metabolic disorders In fact catecholamine excessaï¬ects insulin secretion decreased glucose uptake in theperipheral tissues and increased insulin resistance leading toimpaired fasting glucose or overt diabetes mellitus []Moreover the increase in catecholamine production may beresponsible for decreased inhibition of lipolysis by insulinand decreased activity of lecithincholesterol acyltransferasean enzyme which breaks down free cholesterol []e risk of malignancy is low Less than of allpheochromocytomas in VHL disease are malignant [] Inour case neither distant metastasis nor lymph nodes werefound but a long term followup should be carried outMeasurement of plasma or urinary metanephrines andnormetanephrines is the gold standard in diagnosingpheochromocytoma and can also provide important diagnostic information [] In fact in a study carried out byEisenhofer [] comparing the clinical and biochemical characteristics of pheochromocytomas in multipleendocrine neoplasia type versus the VHL syndrome andincluding and patients with these disorders respectively VHL patients almost exclusively produced normetanephrinesnormetanephrinestometanephrines ratio is expected in patients with VHL disease as was found in our patient Furthermore in comparison with MEN2 tumors VHL tumors had lower totaltissue contents of catecholamines and expression of tyrosinehydroxylase TH the ratelimiting enzyme in catecholamine synthesis ey also had much lower expression ofphenylethanolamine Nmethyltransferase PNMT the enzyme that converts norepinephrine to epinephrine andtissue stores of epinephrine [] Regarding the histopathological features VHLassociated pheochromocytomas arecharacterized by a thick vascular tumor capsule and are incontrast to MEN not associated with adrenal medullarhyperplasia []While bilateral pheochromocytomas were reported inpatients with VHL disease the coexistence of primary hyperparathyroidism as in our case is not a common condition[] Hypercalcemia associated with pheochromocytoma hasbeen documented and is thought to be caused by severalmechanisms First elevated catecholamines can activate thePTH receptor resulting in catecholamineinduced osteoclastic bone resorption but in contrast to our case the PTHlevel is not elevated [] Secondly hypercalcemia can be dueto the production of PTHrelated peptide PTHrp by thetumor which was doubtless not the case in our patient as thelevel of intact PTH was elevated [] Furthermore intactPTH in our case was quantified using a 3rd generationchemiluminescence immunoassay which does not recognizePTHrpird parathyroid adenoma can be a part of multipleendocrine neoplasia that was however rarely reported inVHL disease [] e negativity of the topographic investigations and the spontaneous normalization of intact PTHafter surgical treatment are against this hypothesis Finallyalthough the exact physiopathological mechanism is notclear the fact that both serum calcium and PTH levels wereelevated before surgery and became normal after the removal of the pheochromocytomas strongly suggests that thetumor itself was secreting PTH or a substance that stimulatesexcessive PTH secretion by the parathyroid glands Only fewcases of ectopic hormonal secretion by pheochromocytomasuch as adrenocorticotropic hormone ACTH calcitoninparathyroid hormone PTH vasoactive intestinal peptideVIP and growth hormonereleasing hormone GHRHwere reported [ ] Unfortunately immunohistochemistry assay for PTH in the tumor tissue was not available toclarify this question in our case Conclusionis report highlights the rare case of ectopic intact PTHsecretion by a bilateral pheochromocytoma in an adolescentgirl with VHL disease We consider that controlling calciumand PTH after adrenalectomy is useful if the topographicassessment of primary hyperparathyroidism is negativeTo the best of our knowledge our patient is the secondyoungest reported childhood VHL case in the literaturepresenting with a bilateral pheochromocytoma secretingectopic intact PTH Genetic testing and a meticulous followup are necessary for the diagnosis ofthe associatedcomorbidities in VHL diseaseData 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 interestAcknowledgmentse authors thank Professor Anne Barlier Laboratory ofMolecular Biology Hospital La Conception MarseilleFrance for her help in molecular studyReferences[] J W M Lenders QY Duh G Eisenhofer œPheochromocytoma and paraganglioma an endocrine societyclinical practice guideline e Journal of Clinical Endocrinology Metabolism vol no pp “ [] E Sbardella T Cranston A M Isidori œRoutine geneticscreening with a multigene panel in patients with pheochromocytomas Endocrine vol no pp “ [] S G Waguespack T Rich E Grubbs œA current reviewof the etiology diagnosis and treatment of pediatric pheochromocytoma and paraganglioma e Journal of ClinicalEndocrinology Metabolism vol no pp “[] M Barontini G Levin and G Sanso œCharacteristics ofpheochromocytoma in a to 20yearold population Annals of the New York Academy of Sciences vol no pp “ [] T Arao Y Okada T Tanikawa œA case of von HippelLindau disease with bilateral pheochromocytoma renal cell 0cCase Reports in Endocrinology[] O Mete A S Tischler R D Krijger œProtocol for theexamination of specimens from patients with pheochromocytomas and extraadrenal paragangliomas Archives ofPathology Laboratory Medicine vol no pp “[] N O Atuk T McDonald T Wood œFamilial Pheochromocytoma Hypercalcemia and von HippelLindauDisease a ten year study of a large family Medicine vol no pp “ [] K Takeda N Hara M Kawaguchi T Nishiyama andK Takahashi œParathyroid hormonerelated peptideproducing nonfamilial pheochromocytoma in a child International Journal of Urology vol no pp “ [] J KirkbyBott L Brunaud M Mathonet œEctopichormonesecreting pheochromocytoma a francophone observational study World Journal of Surgery vol no pp “ [] L V Neto G F Taboada L L Corrˆea œAcromegalysecondary to growth hormonereleasing hormone secreted byan incidentally discovered pheochromocytoma EndocrinePathology vol no pp “ carcinoma pelvic tumor spinal hemangioblastoma and primary hyperparathyroidism Endocrine Journal vol no pp “ [] A N A V D HorstSchrivers W J Sluiter R C Kruizinga œe incidence of consecutive manifestations in VonHippelLindau disease Familial Cancer vol no pp “ [] R R Lonser G M Glenn M Walther œvon HippelLindau disease e Lancet vol no pp “[] A D ‡akır H Turan A Aykut A Durmaz O Ercan andO Evliyao˘glu œTwo childhood pheochromocytoma cases dueto von HippelLindau disease one associated with pancreaticneuroendocrine tumor a very rare manifestation Journal ofClinical Research in Pediatric Endocrinology vol no pp “ [] S P Rednam A Erez H Druker œVon hippellindauand hereditary pheochromocytomaparagangliomasyndromes clinical features genetics and surveillance recommendations in childhood Clinical Cancer Research vol no pp e68“e75 [] G F C Fagundes J Petenuci D M Lourenco œNewinsights into pheochromocytoma surveillance of young patients with VHL missense mutations Journal of the Endocrine Society vol no pp “ [] F Hes R V D Luijt A Janssen œFrequency of VonHippelLindau germline mutations in classic and nonclassicVon HippelLindau disease identified by DNA sequencingSouthern blot analysis and multiplex ligationdependentprobe amplification Clinical Genetics vol no pp “ [] T Krauss A M Ferrara T P Links œPreventivemedicine of von HippelLindau diseaseassociated pancreaticneuroendocrine tumors EndocrineRelated Cancer vol no pp “ [] E Wittstr¨om M Nordling and S Andr´easson œGenotypephenotype correlations and retinal function and structure invon HippelLindau disease Ophthalmic Genetics vol no pp “ [] G Eisenhofer M M Walther TT Huynh œPheochromocytomas in von HippelLindau syndrome and multiple endocrine neoplasia type display distinct biochemicaland clinical phenotypes e Journal of Clinical Endocrinology Metabolism vol no pp “ [] M M Walther R Reiter H R Keiser œClinical andgenetic characterization of pheochromocytoma in von HippelLindau families comparison with sporadic pheochromocytomaofpheochromocytoma Journal of Urology vol no pp “ naturalhistoryinsight[] J Cha M Khurram L Gellert P Epstein N Baregamian andC Hendrickson œCase of reversible diabetes mellitus in thesetting of benign Pheochromocytoma Journal of Clinical andTranslational Endocrinology Case Reports vol pp “[] M L Good P Malekzadeh S M Ruï¬ œSurgical resection of pheochromocytomas and paragangliomes is associated with lower cholesterol levels World Journal of Surgeryvol no pp “ [] B Mesmar S PoolaKella and R Malek œe physiologybehind diabetes mellitus in patients with pheochromocytomaa review of the literature Endocrine Practice vol no pp “ givesinto 0c'

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

Sequential Use of aYeastCEA Therapeutic CancerVaccine and AntiPDL1 Inhibitor inMetastatic Medullary Thyroid CancerJaydira Del Rivero Renee N Donahue Jennifer L Mart Ann W Gramza Marijo Bilusic Myrna Rauckhorst Lisa Cordes Maria J Merino William L Dahut Jeffrey Schlom James L Gulley and Ravi A Madan Genitourinary Malignancies Branch Center for Cancer Research National Cancer Institute Bethesda MD United States Developmental Therapeutics Branch Center for Cancer Research National Cancer Institute Bethesda MD United States Laboratory of Tumor Immunology and Biology Center for Cancer Research National Cancer Institute Bethesda MDUnited States Medstar Geetown Lombardi Comprehensive Cancer Center Geetown University Medical CenterWashington DC United States Laboratory of Pathology National Cancer Institute Bethesda MD United StatesMedullary thyroid cancer MTC accounts for ˆ¼ of all thyroid malignancies MTCderives from the neural crest and secretes calcitonin CTN and carcinoembryonic antigenCEA Unlike differentiated thyroid cancer MTC does not uptake iodine and I131RAI radioactive iodine treatment is ineffective Patients with metastatic disease arecandidates for FDAapproved agents with either vandetanib or cabozantinib howeveradverse effects limit their use There are ongoing trials exploring the role of less toxicimmunotherapies in patients with MTC We present a 61yearold male with the diagnosisof MTC and persistent local recurrence despite multiple surgeries He was startedon sunitinib but ultimately its use was limited by toxicity He then presented to theNational Cancer Institute NCI and was enrolled on a clinical trial with heatkilledyeastCEA vaccine NCT01856920 and his calcitonin doubling time improved in months He then came off vaccine for elective surgery After surgery his calcitoninwas rising and he enrolled on a phase I trial of avelumab a programmed deathligand PDL1inhibitor NCT01772004 Thereafter his calcitonin decreased on consecutive evaluations His tumor was subsequently found to express PDL1CEAspecific T cells were increased following vaccination and a number of potentialimmuneenhancing changes were noted in the peripheral immunome over the course ofsequential immunotherapy treatment Although calcitonin declines do not always directlycorrelate with clinical responses this response is noteworthy and highlights the potentialfor immunotherapy or sequential immunotherapy in metastatic or unresectable MTCKeywords medullary thyroid cancer CEA calcitonin immunotherapy PDL1 inhibitorINTRODUCTIONMedullary thyroid cancer MTC accounts for ˆ¼ of allis aneuroendocrine tumor deriving from the neural crestderived parafollicular or C cells of the thyroidgland About of MTC cases are sporadic and the remaining present as part of anautosomal dominant inherited disorder Activating mutations of the RET Rearranged duringthyroid malignancies ItEdited byEnzo LalliUMR7275 Institut de PharmacologieMolculaire et CellulaireIPMC FranceReviewed byMatthias KroissJulius Maximilian University ofW¼rzburg GermanyMouhammed Amir HabraUniversity of Texas MD AndersonCancer Center United StatesCorrespondenceJaydira Del RiverojaydiradelriveronihgovSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived April Accepted June Published August CitationDel Rivero J Donahue RN Mart JLGramza AW Bilusic M Rauckhorst MCordes L Merino MJ Dahut WLSchlom J Gulley JL and Madan RA A Case Report of SequentialUse of a YeastCEA TherapeuticCancer Vaccine and AntiPDL1Inhibitor in Metastatic MedullaryThyroid CancerFront Endocrinol 103389fendo202000490Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCTransfection protooncogene are characteristic with germlineactivating RET mutations seen in fMTC familial MTC andMEN multiple endocrine neoplasia 2aMEN2b “ MTCmost often produces both immunoreactive calcitonin CTNand carcinoembryonic antigen CEA which are used as tumormarkers The growth rate of MTC is estimated by usingRECIST v11 Response Evaluation Criteria in Solid Tumorshowever it can also be determined by measuring serum levelsof CTN and CEA over multiple time points to determinedoubling time which play an important role in the followupand management of MTC Calcitonin doubling times of yearsseem to be associated with a better longterm prognosis thanthose of months ltration on MTC Dendritic cellThe role of immunotherapy in MTC is not fully studiedHowever previous studies have identified evidence of TcellDCbasedimmunotherapy was also given in patients with solid tumorsincluding MTC and it was reported that vaccination withautologous tumorpulsed DCs generated from peripheral bloodwas safe and can induce tumorspecific cellular cytotoxicity Schott reported that subcutaneous injectionof calcitonin and CEA loaded DC vaccine in patients withmetastatic medullary thyroid cancer showed clinical benefitCalcitonin and CEA decreased in of patients and one of thesepatients had complete regression of detectable liver metastasisand reduction of pulmonary lesions A phase I study using theheatkilled yeastCEA vaccine GI6207 was performed at theNational Cancer Institute NCI A total of patients wereenrolled in a classic phase I design at dose levels One patientwith MTC had a significant ‚ammatory response at the sitesof her tumors and a substantial and sustained antigenspecificimmune response Furthermore the relatively low toxicity profileof therapeutic cancer vaccines could be advantageous comparedto approved tyrosine kinase inhibitors TKIs for some patientswith indolent recurrent or metastatic MTC Here we presenta case of a patient with recurrent MTC who was enrolled ona clinical trial with yeastbased vaccine targeting CEA Uponsurgical resection after vaccine his tumor was found to expressprogrammed deathligand PDL1 which may explain thepatient™s subsequent reponse to a PDL1 inhibitorCASE PRESENTATIONWe report a 61yearold male who initially presented with anenlarging anterior neck mass that was biopsied and found tobe consistent with the diagnosis of MTC no known somatic ermline mutation of the RET protooncogene Subsequentlyhe underwent a total thyroidectomy with bilateral neck lymphnode dissection He then had multiple local recurrences resultingin a total of five neck surgeries the last one occurring years after diagnosis Based on the elevated CTN levels andpersistent local recurrence he then started systemic treatmentwith oï¬label sunitinib years after diagnosis Whileon sunitinib his CTN levels nadired to pgml reference pgml down from pgml months after startingtreatment He continued for years and then stopped due toside eï¬ects His CTN levels after discontinuing sunitinib roseto pgmlOn followup imaging studies there was no evidence ofdistant metastases and he presented to the NCI with diseaseinvolving his thyroid bed and cervical nodes most of which werenot amenable to resection He then enrolled on a clinical trialwith yeastbased therapeutic cancer vaccine targeting CEA aphase study of GI6207 in patients with recurrent medullarythyroid cancer NCT01856920 During a 6monthprotocol“mandated surveillance he had a CTN of pgmLand CEA of ngmL reference “ ngmL that increasedto pgmL and CEA of ngmL CTN doubling timeof days During the subsequent 3month vaccine periodhis doubling time improved to days nadir CTN was pgmL and CEA ngmL He then chose to have electivesurgery to remove a neck lymph node and per protocol thevaccine was discontinuedApproximately months after surgery his calcitonin hadrisen to pgml and CEA ngmL and the patient wasenrolled on a phase I trial of avelumab a PDL1 inhibitor phase I label multipleascending dose trial to investigate the safetytolerability pharmacokinetics biological and clinical activity ofavelumab MSB0010718C a monoclonal antiPDL1 antibodyin subjects with metastatic or locally advanced solid tumorsNCT01772004 He then had five consecutive declines inhis calcitonin to pgml and CEA levels remained overallstable at ngmL while on the immune checkpoint inhibitoravelumab a decline not previously seen in his NCIclinical course A Response assessment by RECIST v11reported stable disease Figure These findings coincided with an immunerelated adverseevent asymptomatic rise in grade lipase that led to protocolmandated treatment discontinuation A subsequent analysis ofthe patient™s lymph node resected postvaccination revealed thatthe tumor was PDL1 positive B No baseline samplewas available for evaluation given that the patient was diagnosedover years prior to the latest surgery ImmuneAnalysisSufficient cryopreserved peripheral blood mononuclear cellsPBMCs were available from this patient to analyze CEAspecificCD4 and CD8 T cell responses before vaccination and aftersix and seven vaccinations with yeastCEA corresponding to and months respectively PBMCs were also examined daysfollowing one cycle administered every weeks for days ofavelumab Figure 3A This assay involves intracellular cytokinestaining ICS following a period of in vitro stimulation IVSwith overlapping 15mer peptide pools encoding the tumorassociated antigen CEA or the negative control pool HLA aspreviously described The patient did not have preexisting CEAspecific T cells but displayed a notable increase inCEAspecific T cells months following yeastCEA vaccinationfollowing subtraction of background and any value obtainedprior to vaccination there were CD4 cells producingIFNγ and CD4 cells producing TNF per — cellsplated at the start of the stimulation assay As visualized in thedot plots of Figure 3B the CD4 CEAspecific cells includedmultifunctional cells or cells producing cytokine Theincrease in CEAspecific T cells was not seen at the two later timepoints evaluatedFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCFIGURE A Five consecutive declines in the patient™s calcitonin levels while on the immune checkpoint inhibitor a decline B Robustly positive PDL1staining after surgical resection of a neck lymph node after vaccine higher power on the rightFIGURE Cross sectional imaging studies with computed tomography of the neck A prior to PDL1 administration and B after a decrease in calcitoninshowing stable thyroid bed recurrenceFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCFIGURE Induction of CEAspecific T cells and changes in peripheral immune cell subsets A Schema showing the timing of sequential immunotherapies andimmune assays B CEAspecific T cells were identified in PBMCs by intracellular cytokine staining following a period of in vitro stimulation with overlapping 15merpeptide pools encoding for the tumorassociated antigen CEA or the negative control peptide pool HLA Dot plots of IFNγ and TNF production by CD4 T cellsshowing induction of multifunctional CEAspecific T cells producing cytokine at months C“G PBMCs were assessed for the frequency of immune cellsubsets over the course of immunotherapy The most notable fluctuations were observed after initiation of avelumab indicated by black arrow The frequency overtime of Tregs C cDC D pDC E MDSC F and B cells G indicated as a percentage of total PBMCsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCThe frequency of PBMC subsets was also evaluatedin this patient over his course of treatment at the NationalCancer Institute using 11color flow cytometry on cryopreservedPBMC as previously described Supplemental Table PBMCs were assayed prior to vaccination and monthsfollowing yeastCEA vaccine as well as at time points preand post and days avelumab Figure 3A Using change as a cutoï¬ the first fluctuation in immune cell subsetswas observed months following vaccination with yeastCEAand included an increase in regulatory Tcells Tregs an inhibitory immune subset compared to prevaccine levelsFigure 3C After the patient completed vaccine and underwentsurgery and prior to the initiation of avelumab the patienthad more Tregs Figure 3C and more conventionaldendritic cells cDC Figure 3D a subset that is involved inantigen presentation compared to prevaccine levels The mostdramatic fluctuations in immune subsets were noted at thetime point after weeks of avelumab and included decreasesin Tregs Figure 3C cDC Figure 3D and plasmacytoid DCpDC Figure 3E compared to preavelumab therapy levels pDCare tolerogenic DC exhibiting poor immunostimulatory abilityand their interaction with T cells often favors the generationof Tregs Increases in myeloid derived suppressor cellsMDSCs Figure 3F another immune suppressive subset andB cells Figure 3G were also noted after avelumab comparedto preavelumab levels There were no alterations in the CD4CD8 natural killer NK or NKT compartments noted at anytime point examinedDISCUSSIONFor many years doxorubicin was the only US Food and DrugAdministration FDA“approved treatment for patients withadvanced thyroid cancer however response rate in patients withMTC is up to with significant toxicity “ Recentlyin advanced MTC several TKIs such as axitinib cabozantinibgefitinib lenvatinib imatinib motesanib sorafenib pazopanibsunitinib and vandetanib have been studied in phase I IIand III clinical trials Vandetanib an oral inhibitor of VEGFRvascular endothelial growth factor receptor RET and EGFRepidermal growth factor receptor was approved by theFDA in April after a phase III trial demonstrated improvedmedian progressionfree survival PFS compared to placebohazard ratio CI “ and overall response rateof Cabozantinib an inhibitor of hepatocyte growthfactor receptor MET VEGFR2 and RET was approved bythe FDA in after a phase III trial demonstrated improvedmedian PFS of months relative to months in theplacebo group The impact of toxicity on patientswas clearly indicated and for cabozantinib of patientsrequired dose reductions and required dosing delays Therefore toxicity of FDAapproved TKI agents limits their usein patients with small volume asymptomatic or indolent disease Furthermore no clear data exist from these studies thateither agent impacts overall survival In addition RETspecificTKIs in development are Selpercatinib previously LOXO292and Blue667 with more specific RETtargeting activity Theseagents have demonstrated evidence of efficacy in early trialresults however further treatments are warranted withless toxicityEvidence for cellmediated immunity to tumorspecificantigens has been found in medullary thyroid cancer andearly studies suggested that MTCspecific T cells exist Emerging data suggest that the immune system may be relevantin the treatment of MTC “ Furthermoreimmunebased treatments have been studied Dentritic cell“basedimmunotherapy was given in patients with solid tumorsincluding MTC and it was reported that vaccination withautologous tumorpulsed DCs generated from peripheral bloodwas safe and can induce tumorspecific cellular cytotoxicity This case report may demonstrate the potential for therapeuticcancer vaccines to synergize with immune checkpoint inhibitionsequentially in MTC and that principle could be applied as well toother cancers that may have tumor microenvironments TMEsdevoid of baseline immune recognition The therapeutic cancervaccine in this trial was a heatkilled yeastbased vaccine designedto stimulate an immune response against CEA After a phase Itrial demonstrated safety transient injection site reaction wasthe most common adverse event and preliminary evidence ofimmunologic and clinical activity a phase II study was developedin MTC NCT01856920 The phase I study included apatient with MTC who had substantial ‚ammation at sitesof disease that followed months of the vaccine It isalso possible that the patient™s previous sunitinib is relevant inthis case report In a model using CEAtransgenic mice bearingCEA tumors continuous sunitinib followed by vaccine increasedintratumoralltration of antigenspecific T lymphocytesdecreased immunosuppressant Tregs and MDSCs reducedtumor volumes and increased survival The immunomodulatoryactivity of continuous sunitinib administration can create a moreimmunepermissive environment Despite the significant recent advances of antiPD1 andantiPDL1 therapy they still impact only a minority of patientswhose TMEs express those molecules at baseline One hypothesisis that sequential use of vaccine can drive immune cells to theTME resulting in an adaptive reaction by tumor cells potentiallyfrom the presence of cytokines produced by active immune cellsin the TME upregulating PDL1 and perhaps defining a rolefor antiPDL1PD1 therapies in patients who may not haveotherwise benefited from such immunotherapies Basedon this perspective combining or sequencing vaccines with antiPDL1PD1 therapies could broaden the clinical benefit for allpatients with immunologically œcold tumor microenvironmentsdevoid of reactive immune cells to enhance the clinical efficacyamong cancer patients with a variety of tumor types This casemay provide an example of how increasing peripheral Tcellactivation with vaccines can enable immune cells to then migrateto the TME and improve response rates to antiPDL1PD1therapies Indeed existing data with the FDAapprovedtherapeutic cancer vaccine for prostate cancer sipuleucelTindicate that vaccine did increase T cells in the TME aftertreatment Induction of CEAspecific T cells was noted in the peripheralblood of this patient following vaccination with yeastCEA butnot at later time points It is possible the CEAspecific cellshomed in on the TME inducing PDL1 expression subsequentlyFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCseen on the tumor In addition fluctuations in the peripheralimmunome were noted in this patient over the course of therapywith yeastCEA vaccine and subsequent avelumab therapythese changes included both immunepotentiating and immunesuppressive alterations with the most notable fluctuationsoccurring after several administrations of avelumab The increasein suppressive elements may be a compensatory mechanisminduced to tamper down the immune activation induced by thediï¬erent immunotherapy treatments However as this patienthad metastatic diseaseit is unknown whether the changesin the peripheralimmunome were directly induced by thesequential immunotherapy regimens or potentially related todisease progressionAs with all case reports these presentations have limitationsthe fact that the patient did not have a biopsy at baseline prior tostarting the vaccine limits understanding of the baseline TMEThus it is unclear if the vaccine drove PDL1 expression orif it was preexisting in this patient Little data exist for thepresence of PDL1 expression on MTC tumor cells To furthercomplicate this case™s assessment the patient was previouslytreated with sunitinib which has been able to deplete Tregswhich alone or in combination with vaccine could have impactedthe PDL1 status of this patient Nonetheless data gleanedfrom using immunotherapy in such a rare disease are worthgreater examinationAlthough a decline in calcitonin does not directly correlatewith clinical responses in this case or in MTC in general themagnitude and consistency of the decline are noteworthy amidstdata that suggest the predictive value of calcitonin doubling timeand disease progression Also many patients with MTChave disease recurrence solely defined by serum tumor markersIn these patients the opportunity to impact calcitonin kineticswith immunotherapy may decrease the pace of the diseaseand delay progression to overt metastasis requiring systemictherapies TKIs that are associated with toxicity or ultimatelymetastatic diseaserelated morbidity Despite the eï¬ectivenessof TKIs in MTC opportunities for immunotherapy clinicaldevelopment may provide patients with additional treatmentoptions that are less toxic and could thus be used earlier in thedisease processETHICS STATEMENTWritten informed consent for publication of clinical detailsandor clinical images was obtained from the patientAUTHOR CONTRIBUTIONSJD RD and RM were responsible for study concept and designJD and RD acquired the data from the study and prepared themanuscript RD was responsible for the immune analysis andinterpretation RM reviewed the manuscript JM AG MB MRLC MM WD JS and JG read and approved the final versionof the manuscript All authors contributed to the andapproved the submitted versionFUNDINGThis work was supported by National Cancer Institute NationalInstitutes of Health Intramural Research Program This researchwas financially supported by Merck KGaA Darmstadt Germanyas part of an alliance between Merck KGaA and Pfizer given thatJAVELIN Solid Tumor is an alliancesponsored trialACKNOWLEDGMENTSThis work was selected for poster presentation at The EndocrineSociety 99th Annual Meeting Orlando FL in We arethankful for the support of the National Institutes of HealthClinical Center staï¬ including nurses clinical and researchfellows Merck KGaA Darmstadt Germany and Pfizer reviewedthe manuscriptfor medical accuracy only before journalsubmission The authors are fully responsible for the content ofthis manuscript and the views and opinions described in thepublication reflect solely those of the authorsSUPPLEMENTARY MATERIALfor this can be foundat httpswwwfrontiersins103389fendoThe Supplementary Materialonline202000490fullsupplementarymaterialREFERENCES Saad MF Ordonez NG Rashid RK Guido JJ Hill CS Jr Hickey RC Medullary carcinoma ofthe clinicalfeatures and prognostic factors in patients Medicine “ the thyroid A study of Kouvaraki MA Shapiro SE Perrier ND Cote GJ Gagel RF Hoï¬ AO RETprotooncogene a review and update of genotypephenotype correlationsin hereditary medullary thyroid cancer and associated endocrine tumorsThyroid “ 101089thy200515531 Del Rivero J Edgerly M Ward J Madan RA Balasubramaniam S Fojo T Phase III trial of vandetanib and bortezomib in adults with locallyadvanced or metastatic medullary thyroid cancer Oncologist “e4 101634theoncologist20180452 Saad MF Fritsche HA Jr Samaan NA Diagnostic and prognostic valuesof carcinoembryonic antigen in medullary carcinoma of the thyroid J ClinEndocrinol Metab “ 101210jcem585889 Saad MF Ordonez NG Guido JJ Samaan NA The prognostic value ofcalcitonin immunostaining in medullary carcinoma of the thyroid J ClinEndocrinol Metab “ 101210jcem595850 Brandi ML Gagel RF Angeli A Bilezikian JP BeckPeccoz P Bordi C et alGuidelines for diagnosis and therapy of MEN type and type J ClinEndocrinol Metab “ 101210jcem86128070 French JD Bible K Spitzweg C Haugen BR Ryder M Leveraging the immunesystem to treat advanced thyroid cancers Lancet Diabetes Endocrinol “ 101016S2213858716302777 Eng C Mulligan LM Healey CS Houghton C Frilling A Raue F et alHeterogeneous mutation of the RET protooncogene in subpopulations ofmedullary thyroid carcinoma Cancer Res “ Stift A Friedl J Dubsky P BachleitnerHofmann T Schueller G Zontsich T Dendritic cellbased vaccination in solid cancer J Clin Oncol “ 101200JCO200302135Frontiers in Endocrinology 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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

"Sphingosine1phosphate receptor S1PR1 is involved in vascular development a key process intumorigenesis This study aimed to evaluate its roles in tumor development and prognosisMethods S1PR1 expression levels were analyzed using TIMER and Oncomine database and the prognosticsignificance of S1PR1 was assessed using PrognoScan and KaplanMeier plotter databases The relationship betweenS1PR1 and tumorinfiltrated immune cells was analyzed using TIMERResults S1PR1 expression was remarkably lower in breast and lung cancer tissues than in the correspondingnormal tissues Lower expression was related to poor overall survival and diseasefree survival in breast invasivecarcinoma BRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC A functional networkanalysis confirmed the function of S1PR1 in regulating vasculogenesis In addition S1PR1 levels were significantlynegative with regard to the tumor purity of BRCA r ˆ’ P 176e66 LUAD r ˆ’ P 605e16 andLUSC r ˆ’ P ˆ’ 520e20 Furthermore S1PR1 levels were significantly positive with regard to infiltratingCD8 r P 591e35 and CD4 T cells r P 103e26 macrophages r P 367e12neutrophils r P 203e7 and dendritic cells DCs r P 914e11 in BRCA S1PR1 levels weresignificantly positive with regard to CD8 T cells r P 361e12 macrophages r P 101e17neutrophils r P 415e8 and DCs r P 416e6 in LUAD and positive with regard to B cells r P 157e15 CD8 r P 383e26 and CD4 T cells r P 398e14 macrophages r P 261e45 neutrophils r P 179e25 and DCs r P 212e40 in LUSCConclusions S1PR1 levels are positively correlated with multiple immune markers in breast and lung cancer Theseobserved correlations between S1PR1 and the prognosis and immune cell infiltration provide a foundation forfurther research on its immunomodulatory role in cancerKeywords S1PR1 Breast cancer Lung cancer Tumorinfiltrating Prognosis biomarker Correspondence caodl126com wenrenlyf2008163com1The First Affiliated Hospital Guangzhou University of Chinese Medicine No Airport Road Baiyun District Guangzhou China2Department of Laboratory Medicine Guangdong Second Provincial GeneralHospital No Xingang Middle Road Haizhu District Guangzhou Guangdong Province ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhong BMC Cancer Page of BackgroundSphingosine1phosphate S1P produced by sphingosine kinase Sphkis a biologically active signalinglipid [] S1P regulates vascular development andfunction including vascular maturation [ ] S1P receptor S1PR1is a biologically active sphingolipidmetabolite that mediates S1P activity and promotescell proliferation and survival [ ] S1PR1 is widelyexpressed in vascular endothelial cells and is requiredfor embryonic vascular development and maturation[] Estrogen the growthstimulating hormone inbreast cancer cells was shown to stimulate endothelial cell growth via S1PR1 [ ] In the tumor microenvironment S1P exhibits multiple functionsitincreases the survival of macrophages b it serves asthe œcomeandgetme signal of dead cells attractingand enhancing macrophage migration by combiningwith S1PR1the polarization ofTAMM2 macrophages by activating S1PR124 [“] Accumulating evidence demonstrated that tumorprogression requires new blood vessel growth whichis achieved by producing angiogenic factors that canactivate vascular endothelial cells [] Tumor cellsreleasevascularendothelial growth factor VEGFa which leads toangiogenesis and tumor growth [] Studies haveshown that S1PR1 inhibits VEGF signaling by prointeraction between VEcadherin andmoting theVEGFR2thereby inhibiting VEGFinduced vascularsprouting [ ]angiogenicstimulatesstimulicitasuchasHowever the role of S1PR1 in tumorigenesis andits prognostic value are unclear A preclinical studyon human breast cancer cells found that S1PR1 antibody can enhance the cytotoxic and antiproliferativeeffect of carboplatin on MDAMB231 and SKBR3HER2 subtype cells respectively [] Lei et alfound that S1PR1 signaling has tumorsuppressiveeffects and survival benefits in breast cancer []Therefore it is necessary to clarify the role of S1PR1in tumor development and progression Transcriptome analysis can be used to predict important issues such as the intrinsic subtype of the primarytumor tumor grade drug reactivity and recurrencerisk [“]Herein we used Oncomine KaplanMeier plotterPrognoScan UALCAN and GEPIA datasetstoanalyze S1PR1 expression and its relationship withthe prognosis of cancer patients Furthermore westudied the correlation between S1PR1 and tumorinfiltrated immune cells in the tumor microenvironment using TIMER Our results shed light on theimportant role of S1PR1 in breast and lung cancerand determined that it is closely related to tumorimmunityMethodsOncomine database analysisThe Oncomine database wwwoncomineresourceloginhtml was used to evaluate the expressionlevel of S1PR1 in various types of cancers [] Thethresholds were a Pvalue of fold change of and data type was mRNAPrognoScan database analysisThe PrognoScan database wwwprognoscan wasused to test S1PR1 expression and survival in varioustypes of cancers [] The threshold was an adjustedCox Pvalue of CBioPortal database analysiscBioPortal httpcbioportal contains multidimensional cancer genomics data sets [] S1PR1 mutationsand copy number variation CNV in breast and lungcancers were analyzed using cBioPortal The OncoPrinttab was used to obtain an overview of the genetic alterations for each samplethe prognosticKaplanMeier plotterKaplanMeier Plotter kmplotcom was applied to assessvalue of S1PR1Grouped according to the median expression ofS1PR1 high vs low expression all patients were analyzed for overall survivalOS and progressionfreesurvival PFS and KaplanMeier was used to draw asurvival chart []Immune infiltrates analysis using the TIMERTIMER cistromeshinyappsiotimer wasused to analyze immune infiltrates across different typesof cancer [] Especially the expression of S1PR1 in different cancer types and the correlation between the expression of S1PR1 and the abundance ofimmuneinvasion was determined In addition the correlation between S1PR1 expression and tumor infiltrating immunecell gene markers was also explored through relatedmodulesGene correlation analysis using GEPIAGEPIA httpgepiacancerpkucnindexhtml wasused to confirm the genes with significantly correlatedexpression levelsin TIMER [] The Spearmanmethod was used to determine the correlation coefficients The tumortissue datasets were used foranalysisLinkedOmics database analysisThe LinkedOmics database httpwwwlinkedomicsloginphp was used to analyze S1PR1 coexpressioncorrelationPearson™sbasedon 0cZhong BMC Cancer Page of coefficients The results were visually evaluated usingvolcano plots and heat maps The function moduleof LinkedOmics was used to analyze gene ontologyGOKyotoEncyclopedia of Genes and Genomes KEGG pathways by a gene set enrichment analysis GSEA Therank criterion was FDR and simulationswere performed []biologicalprocessesBPandUALCAN database analysisUALCAN httpualcanpathuabeduincluded theCancer Genome Atlas TCGA level RNA sequencesClinical data from cancer types were used toanalyze the relative expression of genes in tumorand normalstagetumor grade or other clinicopathological characteristics []according to tumorsamplesS1PR1 mRNA expression level analysisGene expression data of breastinvasive carcinomaBRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloadedin UCSC Xena xenabrowsernet S1PR1 mRNAexpression level was compared between cancerous andnormal tissue using MannWhitney test with P setting as a cutoffStatistical analysisGene expression data in the Oncomine database wasanalyzed using pvalue fold change and mRNA datatype The survival curves were generated via KaplanMeier plots and PrognoScan database are displayedwith HR and P or Cox Pvalues from a logrank testSpearman correlation analysis was used to evaluatethe correlation of gene expression in TIMER and LinkedOmics databases P was considered statistically significantResultsS1PR1 mRNA expression levels in different types ofhuman cancersThe Oncomine database was used to analyze S1PR1mRNA levels in tumor tissues and normal tissues ofvarious cancer types S1PR1 expression was lower inmost tumor tissues including sarcoma bladder brainleukemiacentral nervous system breast colorectallung myeloma and ovarian cancer tissuesthan innormal tissues Fig 1a The mRNAseq data fromTCGA were analyzed using TIMER to verify thesefindings Data from TCGA shown that the differentialexpression of S1PR1 between the tumor and adjacentnormal tissues is shown in Fig 1b Compared withadjacent normal tissues S1PR1 expression was significantlycarcinomaBLCA BRCA cholangiocarcinoma CHOL colonadenocarcinomacarcinomaESCA head and neck squamous cell carcinomaHNSC kidney chromophobe KICH kidney renalpapillary cell carcinoma KIRPliver hepatocellularcarcinoma LIHC LUAD LUSC prostate adenocarREADcinomaskinstomachrectum adenocarcinomacutaneous melanomain bladderesophagealurothelialSKCMreducedCOADPRADFig S1PR1 expression levels in different types of human cancers a Differences in S1PR1 between cancer tissues and normal tissues based ondata in the Oncomine database P 1E04 Fold change Data type mRNA b Human S1PR1 expression levels in different tumor types fromTCGA database were determined using TIMER P P P 0cZhong BMC Cancer Page of adenocarcinoma STAD and uterine corpus endometrial carcinoma UCEC However S1PR1 expression was significantly higher in kidney renal clear cellcarcinoma KIRC and thyroid carcinoma THCAthan in adjacent normal tissues Fig 1b These datashowed that alterations in S1PR1 expression dependon the tumor type suggesting that this gene exertsdiverse functions in various tumorscancerand GSE12276in three breastPrognostic evaluation of S1PR1 in cancersWe investigated whether S1PR1 expression is relatedto prognosis The effect of S1PR1 expression on survival wasevaluated by PrognoScan Two probes204642_at and 239401_at matching S1PR1 were detected NotablycohortsGSE1456GPL96 GSE7378lowS1PR1 expression was significantly associated with apoorer prognosis breast cancer Fig 2a“f We usedthe KaplanMeier plotter database to further examinethe prognostic value of S1PR1 in breast cancer Poorprognosis based on recurrencefree survival RFS inbreast cancer was significantly correlated with lowS1PR1 expression HR P 71e13 but a significant correlation was not observed for overall survival OS HR P and postprogressionsurvival PPS HR P Fig 2g“i Its determined that the low expression of S1PR1 is an inderisk factorpendentfor poor prognosis of breastcancer In additionlow S1PR1 expression was alsorelated to poor prognosis in two cohorts of patientswith lung cancer GSE31210 and GSE8894 as determined using two probes 204642_at and 239401_atFig 2j“l KaplanMeier plotter database also showedthat low expression of S1PR1 was an independent riskfactor for poor prognosis of lung cancer overall survival HR P 69e08 recurrencefree survivalHR P but notrelated to postin lung cancer HR P progression survival Fig 2m“oFurthermore we found that low S1PR1 expressionwas associated with a poor prognosis in patientswith soft tissue blood and brain cancers Fig S1a“c In contrastlow S1PR1 expression was an independent risk factor for a good prognosis in gastricFig S1d“g These results confirmed thecancerprognostic value of S1PR1 in specific types of cancer both high and low S1PR1 expression was associated with prognosis depending on thetype ofcancer Based on the consistent results for the associations between S1PR1 expression and survivalinbreast and lung cancers we focused on the preciseeffects of S1PR1 in these two cancer types as wellas the underlying mechanismsP Correlations between clinical characteristics and S1PR1expression in breast cancer and lung cancerWe used the KaplanMeier plotter to study the relationship between S1PR1 expression and clinical characteristics in patients with breast cancer and lungcancer Low expression of S1PR1 was associated withworse overall survival OS in male and female patients with lung adenocarcinoma P Table In particular low S1PR1 mRNA expression was correlated with worse OS in stage P 920E13 andearlystage AJCC stage Mlung cancerTable Low S1PR1 mRNA expression was relatedto poor OS in patients with P or withoutP a smoking history Table In additionlow S1PR1 mRNA expression was related to worseOS in patients who did not receive chemotherapy orradiotherapy These findings strongly suggest that lowS1PR1 mRNA expression is correlated with poor OSin lung cancer Table In BRCA low S1PR1 mRNAexpression was related to poor OS in ERpositive orHER2negative patients and in the luminal androgenreceptor subtype Table Taken together high expression of S1PR1 could be considered a good prognostic indictor for breast and lung cancers dependingon the clinical characteristicsDecreased expression of S1PR1 in breast cancer and lungcancer patientsWe further analyzed the expression of S1PR1 inbreast and lung cancers Gene expression data ofbreast invasive carcinoma BRCAlung adenocarcinoma LUAD and lung squamous cell carcinomain TCGA were downloaded and S1PR1LUSCmRNA expression level was compared between tumorand normal tissue As shown in Fig 3a the expression of S1PR1 was significantly decreased in tumortissues of BRCA LUAD and LUSC Fig 3a In comparison with normal control tissues breast cancer andlung cancertissues presented lower expression ofS1PR1 which was also observed by GEPIA analysisFig 3b Furthermore we analyzed TCGA data usingthe UALCAN database Compared to normal tissuesS1PR1 mRNA expression was significantly decreasedin primary tumors and tumor stages stage stage stage and stage of BRCA LUAD and LUSCFig 3c“e Taken together these data confirmed thedownregulation of S1PR1 expression in breast cancerand lung cancer patientsRegulators of S1PR1 in breast cancer and lung cancerWe used the LinkedOmics function module to detectthe S1PR1 regulatory network to further understandthe biological role of S1PR1 in breast cancer and lung 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig Prognostic value of S1PR1 in cancers a“f KaplanMeier survival curves comparing high and low expression of S1PR1 in breast cancersusing PrognoScan Survival curves based on OS DSS and DFS in three breast cancer cohorts [GSE1456GPL96 n GSE7378 n andGSE12276 n ] g“i Survival curves for breast cancers based on mRNAseq data from TCGA of Kaplan“Meier plotter databases j“l Kaplan“Meier survival curves comparing high and low expression of S1PR1 in lung cancers using PrognoScan Survival curves based on RFS in two threelung cancer cohorts [GSE31210 n and GSE8894 n ] m“o Survival curves for lung cancers based on mRNAseq data from TCGA ofKaplan“Meier plotter databases OS Overall survival RFS RelapseFree Survival PPS Postprogression survival DSS Diseasespecific survivalDFS Diseasefree survivalcancer Figure 4a“c shows genes with significantlypositive dark red dots and negative dark greendots correlations with S1PR1 false discovery rateFDR The top positively and negatively related genes are shown in a heat map in Fig 4d“f AGene Ontology GObased gene set enrichment analysis GSEA showed that genes that are coexpressedwith S1PR1 are enriched for vasculogenesis and thepurinergic receptor signaling pathway while genes related to mitochondria and RNA transcript processingwere inhibited in breast cancer Fig 4g SimilarlyGO annotation resultscoexpressed with S1PR1 are primarily associated withvasculogenesis the purinergic receptor signaling pathway and the phospholipase Cactivating G proteincoupled receptortRNAmetabolic process RNA modification and RNA transcript processing werecancerFig 4h“i A KEGG pathway analysis showed enrichlineage Staphylococcusmentaureusinboth breast cancer and lung cancer SpliceosomeDNA replicationand proteasome pathways wereinhibited in both tumor types Fig 4jl These resultssuggest that S1PR1 contributes to various processesin tumor development at least partially through regulate vasculogenesisinfection and renin secretion pathwayssignaling pathway whilefor hematopoietic cellshowedthatgenesinhibited in lungamplifications and deletionsGenomic alterations in S1PR1 in breast cancer and lungcancercBioPortal database was used to determine the typesand frequencies of S1PR1 alterations in BRCA LUADand LUSC S1PR1 was altered in of patients withBRCA These alterations included mRNA missensemutationsFig 5aS1PR1 was altered in of patients with LUAD and of patients with LUSCincluding mRNA missense mutations truncating mutations amplificationsand deletions Fig 5a Moreover S1PR1 CNV wasassociated with OS in LUAD but not with OS or DFSin BRCA and LUSC Fig 5b“d These results suggestthat mutations in S1PR1 are associated with prognosisin LUADRelationship between immune and S1PR1 expression inbreast cancer and lung cancerTumor infiltrating lymphocytes TIL are lymphocytes that leave the blood circulation and migrate tothe vicinity of the tumor The amount of TIL in thetumor is an important indicator to predict the prognosis of cancer patients and the response to immunotherapy [ ] Tumor purity is a key factorin analyses ofimmune infiltration by genomic approaches [] Therefore we use TIMER to investigate whetherthe expression of S1PR1 in breastcancer and lung cancer is related to immune infiltration We found a significant negative correlation between the S1PR1 expression level and tumor purityin both breast cancer and lung cancer Fig 6a“fLeft S1PR1 was a determinant of immune infiltration in BRCA tumor purity r ˆ’ P 176e including subtypes of BRCA BRCABasal r ˆ’ P 128e06 BRCAHer2r ˆ’ P 444e06 and BRCALuminal r ˆ’ P 915e S1PR1 was related to immune infiltration inincluding LUAD tumor purity r ˆ’lung cancer P 605e16 and LUSC tumor purity r ˆ’ P 520e20Furthermore the relationship between S1PR1 andspecific immune infiltrates in breast cancer and lungcancer were analyzed The S1PR1 expression levelwas significantly positively correlated with levels ofr P 597e35infiltrating CD8 T cellsCD4 T cells r P 103e26 macrophagesr P 367e12 neutrophilsr P 203e07 and DCs r P 914e11 in BRCAFig 6a In BRCABasal there were slight positivecorrelations between S1PR1 expression levels andinfiltrating CD8 T cells r P levels of176e03 and CD4 T cells r P 852e03Similarly there were positive correlations with infiltrating levels of CD8 T cells r P 113e CD4 T cells r P 200e05 neutror philsP 135e04 in BRCAHer2 S1PR1 expression levelswere positively correlated with levels ofinfiltratingCD8 T cells r P 343e21 CD4 T cellsr P 626e14 macrophages r P r P 857e03 and DCs 0cZhong BMC Cancer Page of Table Correlation between S1PR1 mRNA expression andprognosis in lung cancer with respect to clinicopathologicalfactorsClinicopathologicalcharacteristicsOverall survivalNHazard ratioPvalueTable Correlations between S1PR1 mRNA expression andclinical prognosis in breast cancer with respect toclinicopathological factorsClinicopathologicalcharacteristicsOverallNHazard ratioPvalue “ “490E05ER statusER positiveER negativePR status “ “ “ “590E06PR positivePR negativeHER2 status920E13HER2 positive113E01920E01HER2 negativeIntrinsic subtypeSexFemaleMaleHistologyAdenocarcinomaSquamous cell carcinomaStageGradeIIIIIIAJCC stage TAJCC stage NAJCC stage MSmoking historyExclude those never smokedOnly those never smokedChemotherapyNoYesRadiotherapyNoYes “ “ “NA “ “ “ “ “ “ “ “ “ “NA “NANA “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “NA “NABasalLuminal ALuminal BHER2Lymph node statusLymph node positiveLymph node negativeGradeTP53 statusMutatedWild typePietenpol subtypeBasallike Basallike ImmunomodulatoryMesenchymalMesenchymal stem likeLuminal androgen receptorSystemically untreated patientsIncludeExclude “ “71E13Bold values indicate P NA noneBold values indicate P NA none414e04 neutrophils r P 667e04 andDCs r P 644e07tumors Fig 6a We also found that S1PR1 expressionlevels were positivelycorrelated with levels ofin BRCALuminalr P 361e12infiltrating CD8 T cellsr P 101e17 neutrophilsmacrophagesr P 415e08r P 416e06 in LUAD In addition there were positiveinfiltrating B cells r correlations with levels ofand DCs 0cZhong BMC Cancer Page of Fig Decreased expression of S1PR1 in breast and lung cancer patients a Gene expression data of breast invasive carcinoma BRCA lungadenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloaded in UCSC Xena S1PR1 mRNA expression level wascompared between cancerous and normal tissue using MannWhitney test with P setting as cutoff b The expression of S1PR1 in BRCALUAD and LUSC were analysis using GEPIA T tumor N normal tissue NUM number c“e S1PR1 mRNA expression level was expressed as boxplots using the UALCAN database mRNA expression of S1PR1 in normal control and BRCA LUAD and LUSC tumors Left primary tumors Rightindividual cancer stage P P P 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig S1PR1 coexpression genes in breast and lung cancer a“c The S1PR1 highly correlated genes identified by Pearson test in BRCA a LUADb and LUSC c d“f The heat map shows that in BRCA d LUAD e and LUSC f the first genes are positively red and negatively bluecorrelated with S1PR1 g“i Significantly enriched GO annotations of S1PR1 in BRCA g LUAD h and LUSC i j“l Significantly enriched KEGGpathways of S1PR1 in BRCA j LUAD k and LUSC lin LUSC These results strongly suggest P 127e15 CD8 T cells r P r P 398e14383e26 CD4 T cellsr P 261e45 neutrophilsmacrophagesr P 179e25 and DCs r P 212ethatS1PR1 plays a special role in the immune infiltrationof breast and lung cancers and has a particularlystrong effect on T cells macrophages neutrophilsand DCs These observed correlations betweenS1PR1 and various types of immune cells in breastand lung cancers indicated that S1PR1 may havehigh prognostic valuefurthercorrelationsimmune cellsevaluated theCorrelations between S1PR1 expression and immunemarkersbetweenWeS1PR1 and markers of variousinbreast cancer and lung cancer using TIMER Table and GEPIA databases Table S1 The correlationsbetween S1PR1 expression and immune markergenes for different immune cell populations including CD8 T cells T cells general B cells monocytes TAMs M1 and M2 macrophages neutrophilsNK cells DCs and various functional T cells suchas Th1 cells Th2 cells Tfh cells Th17 cells andTregs as well as exhausted T cells were analyzed byTIMER After adjusting for tumor purity S1PR1 expression levels were significantly positively correlatedwith marker sets for various immune cells exceptfor NK cells Th17 and T cell exhaustion in BRCATable and Fig However S1PR1 expressionlevels were highly positively correlated with most immune marker sets and both T cell populations andexhausted T cells in LUAD and LUSC Table andFig We further analyzed the correlation betweenS1PR1 expression and the markers using the GEPIAdatabaseincluding data for BRCA LUAD andLUSC The results for correlations between S1PR1and markers of immune infiltrating cells were similarto those of the TIMER analysis Table S1 This further confirms that S1PR1 is significantly related toimmune infiltrating cells in lung and breast cancersuggesting that high levels of S1PR1 could induceimmune activity in the lung and breast cancermicroenvironmentin normalDiscussionWe systematically analyzed the expression levels ofS1PR1 and the prognostic value in different types ofcancers Compared with levelstissuesS1PR1 expression was significantly lower in BLCABRCA CHOL COAD ESCA HNSC KICH KIRPLIHC LUAD LUSC PRAD READ SKCM STADand UCEC and was significantly higher in KIRC andTHCA Accordingly S1PR1 expression patterns depend on the type of cancer Prognostic data fromKaplanMeier plotterlow levels ofS1PR1 are significantly related to poor prognosis inbreast cancer and lung cancershowed thatsuch a complex biologicalThe downregulation of S1PR1 was associated withworse prognosis in breast cancer and lung cancer andwassignificantly related to clinical characteristicssuch as gender population smoking status and stageThese results suggested that S1PR1 is a prognosticbiomarker in breast cancer and lung cancer Howeversome literatures have reported the oncogenic role ofS1PR1 in breast cancer Lee H demonstrated thatStat3induced S1PR1 expression as well as S1PS1PR1 pathway is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression [] This maybe one of the molecular mechanisms by which S1PR1mediatesresponse Weconsidered that the main reason for this inconsistencyis that our study analyzed the expression of S1PR1 atthe overalllevel We further verified the significantreduction of S1PR1 expression in breast cancer andlung cancer patients through TCGA analysis Anotherstudy has also claimed a survival function benefit ofS1PS1PR signaling in BRCA patients which mightexplain the obstacle to relative antagonist therapy inclinics [] A recent study determined that attenuated endothelial S1PR1 function led to increasedtumor growth and metastasis whereas S1PR1 overexpression led to smaller tumors and strategies to enhance S1PR1 function in the tumor vasculature maypotentiate the efficacy of cytotoxic and targeted anticancer therapies [] These studies support our findings that high expression of S1PR1 is beneficialfortumor survivalThe tumor microenvironment refers to noncancerimmunecells in and around tumorsinfiltrated of 0cZhong BMC Cancer Page of Fig S1PR1 genomic alterations in breast and lung cancer a OncoPrint of S1PR1 alterations in BRCA LUAD and LUSC Different types of geneticalterations highlighted in different colors b“d The relationship between genetic alterations and S1PR1 OSDFS in BRCA b LUAD c and LUSCd Logrank test was used in analysis of OSDFScells in the tumor microenvironment plays a vitalfunction in the occurrence and development of tumors [ ] Lymphocyte infiltration in the tumormicroenvironment is an independent predictor of cancer patient survival and lymph node metastasis [] Studies have shown that S1PR1 can affect the 0cZhong BMC Cancer Page of Fig Correlations between S1PR1 expression and immune infiltration levels in breast and lung cancer a S1PR1 expression was significantlynegatively related to tumor purity and significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in BRCA n b S1PR1 expression was significantly negatively related to tumor purity and was significantpositively correlated with infiltrating levels of CD8 T cells CD4 T cells and dendritic cells in BRCABasal n c S1PR1 expression wassignificantly negatively related to tumor purity and was significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cellsneutrophils and dendritic cells in BRCAHer2 n d S1PR1 expression was significantly negatively related to tumor purity and wassignificantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophages neutrophils and dendritic cells in BRCALuminal n e S1PR1 expression was significantly negatively related to tumor purity and was significantly positively correlated withinfiltrating levels of CD8 T cells macrophages neutrophils and dendritic cells in LUAD n f S1PR1 expression was significantly negativelyrelated to tumor purity and was significant positively correlated with infiltrating levels of B cells CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in LUSC n Spearman™s correlation coefficients were used for analyses P P P proliferation and differentiation of lymphocytes in thetumor microenvironment [] The evaluation of immune cell infiltration in breast and lung cancers usingthe TIMER database revealed strong negative correlations between S1PR1 and tumor purity in BRCALUAD and LUSC Furthermore the S1PR1 expression level was positively correlated with levels ofCD8 T CD4 T neutrophils macrophages and DCsin BRCA The correlation between S1PR1 expressionand immune cell marker genes suggests that S1PR1regulates lung cancer tumor immunity through multiple immune cell populations These results indicatethat high levels of S1PR1 could increase the cytotoxicity of the immune system and immune activation in 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMERLUSCPurityLUADPurityBRCAPurityDescriptionGene markersCD8 T cellT cell generalB cellMonocyteTAMvarXCD8ACD8BCD3DCD3ECD2CD19CD79ACD86CD115 CSF1RCCL2CD68IL10M1 MacrophageINOS NOS2M2 MacrophageIRF5COX2 PTGS2CD163VSIG4MS4A4ANeutrophilsCD66b CEACAM8CD11b ITGAMNatural killer cellDendritic cellTh1Th2CCR7KIR2DL1KIR2DL3KIR2DL4KIR3DL1KIR3DL2KIR3DL3KIR2DS4HLADPB1HLADQB1HLADRAHLADPA1BDCA1CD1CBDCA4NRP1CD11c ITGAXTbet TBX21STAT4STAT1IFNg IFNGTNFa TNFGATA3STAT6corˆ’ˆ’p126E17242E08471E12715E19320E10738E07198E08128E01129E10468E04463E01835E02176E16618E01490E28772E02114E02196E13203E01824E01155E24727E01110E01395E01263E03319E02875E01268E01389E14211E02717E07226E11176E53158E59621E03472E13592E19261E04784E01808E 143E02669E13corˆ’ˆ’ˆ’ˆ’ˆ’p219E04166E02128E02885E07400E04537E05121E04297E07310E08386E02586E11110E09793E18355E01358E02436E14975E10539E17195E08816E06257E13130E06996E04506E01104E04879E02581E01834E03413E09479E02869E07453E07100E09107E04269E03481E05366E03310E01913E02930E02301E01220E03corˆ’p651E21122E17760E21282E26799E24378E13329E13127E45267E56589E24118E30149E36864E02431E01237E06152E57477E53928E54299E06266E30170E33136E03263E07845E04485E11168E06343E01105E06386E52884E20129E48187E52869E24669E28158E24517E20473E32103E04185E02134E01300E07625E01 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMER ContinuedTfhTh17TregSTAT5AIL13BCL6IL21STAT3IL17AFOXP3CCR8STAT5BTGFb TGFB1T cell exhaustionPD1 PDCD1CTLA4LAG3TIM3 HAVCR2GZMBBRCAˆ’ˆ’181E07127E01352E08977E01175E01929E02394E01671E01858E20321E25412E04575E01600E04219E01782E02LUADˆ’227E08115E01801E03234E01265E05462E01198E01461E04467E12943E06256E01727E02439E01178E06599E01LUSCˆ’422E21120E05924E01492E06609E04409E01415E19727E27247E03164E01380E16388E20311E06844E46333E09shown in recently reports endothelialBRCA LUAD and LUSC by increasing the infiltrationof CTLs CD4 T cells and DCs On the contrarylow expression of S1PR1 could lead to reduced infiltrated effector cells in the tumor microenvironmentAsloss ofS1PR1 led to a reduction in CD45 cells macrophages and DCs which influences tumor growth andmetastasis [] In addition S1P is involved in enhancing endocytosis and migration of mature dendriticcells through S1PR3 an event that may increase theimmune response to cancer cells Our findings areconsistent with such reports and these discoveriesimply that S1PR1 plays an important role in recruiting and governing immune infiltration in BRCALUAD and LUSCthe purinergicTo further elucidate the molecular mechanismsunderlying the role of S1PR1 in b

Thyroid_Cancer

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

"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adults™population of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [“] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [“] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients™ intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at ˆ’ °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients™ intakeUsual Macronutrients™ intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants™ physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrients™intake and physical activity between different FTO genotypes The post hoc Tukey™s test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrients™protein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [“] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ‰¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableˆ’Calorieˆ’ˆ’ˆ’ˆ’ATˆ’ˆ’ˆ’ˆ’P valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele ß ˆ’ kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrients™intakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [“] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients™ intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3“ and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors™ contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet “Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States “ JAMA “Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol “Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab “Fredriksson R Hagglund M Olszewski PK Stephansson O Jacobsson JAOlszewska AM Levine AS Lindblom J Schiöth HB The obesity gene FTO isof ancient origin upregulated during food deprivation and expressed inneurons of feedingrelated nuclei of the brain Endocrinology May “Doaei S Hajiesmaeil M Aminifard A MosaviJarrahi SA Akbari MEGholamalizadeh M Effects of gene polymorphisms of metabolic enzymeson the association between red and processed meat consumption and thedevelopment of colon cancer a literature review J Nutritional Sci Doaei S Kalantari N Mohammadi NK Izadi P Gholamalizadeh M EiniZinabH Salonurmi T Jarrahi AM Rafieifar S Janipoor R Sadeghypor M Upregulation of FTO gene expression was associated with increase in skeletalmuscle mass in overweight male adolescents Arch Med Sci Sep155Hakanen M Raitakari OT Lehtimäki T Peltonen N Pahkala K Sillanmäki LLagstrom H Viikari J Simell O Ronnemaa T FTO genotype is associatedwith body mass index after the age of seven years but not with energyintake or leisuretime physical activity J Clin Endocrinol Metab Apr “Thorisson GA Smith AV Krishnan L Stein LD The international HapMapproject web site Genome Res Nov “Smemo S Tena JJ Kim KH Gamazon ER Sakabe NJ GómezMarín C AneasI Credidio FL Sobreira DR Wasserman NF Lee JH Obesityassociatedvariants within FTO form longrange functional connections with IRX3Nature Mar5077492371“ Antonio J Knafo S Kenyon M Ali A Carson C Ellerbroek A Weaver CRoberts J Peacock CA Tartar JL Assessment of the FTO gene 0cMehrdad Lipids in Health and Disease Page of Ronkainen J Huusko TJ Soininen R Mondini E Cinti F Mäkelä KAKovalainen M Herzig KH Järvelin MR Sebert S Savolainen MJ Fat massandobesityassociated gene Fto affects the dietary response in mouse whiteadipose tissue Sci Rep Mar Poritsanos NJ Lew PS Fischer J Mobbs CV Nagy JI Wong D Rüther UMizuno TM Impaired hypothalamic Fto expression in response to fastingand glucose in obese mice Nutr Diab 2011110e19 Doaei S Kalantari N Izadi P Salonurmi T Jarrahi AM Rafieifar S Azizi Tabesh GRahimzadeh G Gholamalizadeh M Goodarzi MO Interactions between macronutrients™ intake FTO and IRX3 gene expression and FTO genotype in obeseand overweight male adolescents Adipocyte Jan “ Olszewski PK Fredriksson R Olszewska AM Stephansson O Alsiö JRadomska KJ Levine AS Schiöth HB Hypothalamic FTO is associated withthe regulation of energy intake not feeding reward BMC Neurosci Dec1011“ Razquin C Martinez JA MartinezGonzalez MA A 3year intervention with aMediterranean diet modified the association between the rs9939609 genevariant in FTO and body weight changes Int J of Obesity “ McTaggart JS Lee S Iberl M Church C Cox RD Ashcroft FM FTO isexpressed in neurones throughout the brain and its expression is unalteredby fasting PLoS One 2011611e27968 httpsdoi101371journalpone0027968Stratigopoulos G Padilla SL LeDuc C A Watson E Hattersley AT McCarthyMI Zeltser LM Chung WK Leibel RL Regulation of FtoFtm gene expressionin mice and humans Am J Physiol Integr Comp Physiol 2008294R1185“ Wang P Yang FJ Du H Guan YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May ““ Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3“ N Engl J Med Sep “ Wardle J Carnell S Haworth CM Farooqi IS O™Rahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab “ httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E O™Daly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspolymorphisms rs1421085 rs17817449 and rs9939609 in exercisetrainedmen and women the effects of a 4week hypocaloric diet J Int Soc SportsNutr Dec Blundell JE Lawton CL Cotton JR Macdiarmid JI Control of humanappetite implications for the intake of dietary fat Annu Rev Nutr “Ludwig DS The glycemic index physiological mechanisms relating toobesity diabetes and cardiovascular disease Jama “Sonestedt E Roos C Gullberg B Ericson U Wirfält E OrhoMelander M Fatand carbohydrate intake modify the association between genetic variationin the FTO genotype and obesity Am J Clin Nutr Sep “Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relativevalidity of food group intake in a food frequency questionnaire developedfor the Tehran lipid and glucose study J Epidemiol “ Azar M Sarkisian E Food composition table of Iran National Nutrition andfood research institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes “ Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455“ Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May “ Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov “ Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul “ Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov “Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript levels but does not altertheir DNA methylation profiles in rat white adipose tissues Gene “ 0c"

Thyroid_Cancer

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

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

Thyroid_Cancer

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

Thyroid_Cancer

" 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

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

Thyroid_Cancer

"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

"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves™ disease induced by ICIs We report a case of newonsetGraves™ disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves™ disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves™ disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves™ diseaseis developed with negative and positive TRAb titresKeywords Graves™ disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction [“]The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves™ disease induced by ICI treatment has not beenextensively explored Here we present a case of Graves™disease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves™ orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves™ diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH “ μIUmL FT3 “ pgmL FT4 “ ngdL TRAb IUL TSAb ‰ and Tg ‰ ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves™ disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves™ disease during nivolumabtherapy other than the present case have been reported[“] Table Iadarola [] reported a case ofGraves™ diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Graves™diseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves™ disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves™ disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves™ diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves™ diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves™ disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves™ diseaseGraves™ disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves™ disease Gan et al[] reported a case oftremelimumabinduced Graves™ hyperthyroidism Yajima [] reported a case of Graves™ disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Graves™disease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves™ disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves™ diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves™ diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves™ diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves™ disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves™ disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors™ contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patient™s next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist “ Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer “Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary “Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med “Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J Med“Orlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab “Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci “Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J “Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig“ Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves™ disease Hum Immunol“ Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab “ Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves™ disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J “ Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject –º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add –º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice –º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access“ months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles “ weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigator™s discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA “ measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA “ level by ‰¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ‰¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ‰¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA “ PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics “ years “ years ‰¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA “ decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of CA “ patients or normalisation PET SUVmax in In Arm B had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an œexceptional CA “ response and had an ˜exceptional™ PET response as defined by either ‰¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection status“““““““““AAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA “ decrease FDG PET SUVmax decrease ‰¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA “ PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA “ decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA “ response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA “ and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options

Thyroid_Cancer

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

Thyroid_Cancer

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

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'

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

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

Thyroid_Cancer

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

peripheral serum metabolomic profiles inform central cognitive impairmentJingye Wang1 Runmin Wei12 Guoxiang Xie1 Matthias Arnold Alexandra Kueider‘Paisley Gregory Louie Siamak Mahmoudian Dehkordi3 colette Blach5 Rebecca Baillie Xianlin Han7 Philip L De Jager David A Bennett9 Rima Kaddurah‘Daouk Wei Jia The incidence of Alzheimer™s disease AD increases with age and is becoming a significant cause of worldwide morbidity and mortality However the metabolic perturbation behind the onset of AD remains unclear In this study we performed metabolite profiling in both brain n and matching serum samples n to identify differentially expressed metabolites and metabolic pathways associated with neuropathology and cognitive performance and to identify individuals at high risk of developing cognitive impairment The abundances of metabolites glycolithocholate GLCA petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and the deoxycholatecholate DCACA ratio along with the dysregulation scores of metabolic pathways primary bile acid biosynthesis fatty acid biosynthesis and biosynthesis of unsaturated fatty acids showed significant differences across both brain and serum diagnostic groups P‘value Significant associations were observed between the levels of differential metabolitespathways and cognitive performance neurofibrillary tangles and neuritic plaque burden Metabolites abundances and personalized metabolic pathways scores were used to derive machine learning models respectively that could be used to differentiate cognitively impaired persons from those without cognitive impairment median area under the receiver operating characteristic curve AUC for the metabolite level model median AUC for the pathway level model Utilizing these two models on the entire baseline control group we identified those who experienced cognitive decline in the later years AUC sensitivity specificity for the metabolite level model AUC sensitivity specificity for the pathway level model and demonstrated their pre‘AD onset prediction potentials Our study provides a proof‘of‘concept that it is possible to discriminate antecedent cognitive impairment in older adults before the onset of overt clinical symptoms using metabolomics Our findings if validated in future studies could enable the earlier detection and intervention of cognitive impairment that may halt its progressionAlzheimer™s disease AD one of the top leading causes of death in the United States is an increasing challenge for health care systems and will result in increased economic burden as increasing numbers of new cases are diagnosed annually12 Currently there is no therapy to prevent or slow AD progression which may be due to the inability to detect AD before its progression into evident cognitive decline Identification of early 1University of Hawaii Cancer Center Ilalo Street Honolulu HI USA 2Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI USA 3Department of Psychiatry and Behavioral Sciences Duke University Durham NC USA 4Institute of Computational Biology Helmholtz Zentrum M¼nchen German Research Center for Environmental Health Neuherberg Germany 5Duke Molecular Physiology Institute Duke University Durham NC USA 6Rosa Co LLC San Carlos CA USA 7University of Texas Health Science Center at San Antonio San Antonio TX USA 8Center for Translational Computational Neuroimmunology Columbia University College of Physicians and Surgeons Department of Neurology New York NY USA 9Rush Alzheimer™s Disease Center Rush University Medical Center Chicago IL USA 10Institute of Brain Sciences Duke University Durham NC USA 11Department of Medicine Duke University Durham NC USA email kaddu001mcdukeedu wjiacchawaiieduScientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cbiomarkers associated with preclinical symptoms would allow early intervention or preventive strategies to be developed3 Research has identified multiple neurochemical perturbations in AD including amyloid precursor protein metabolism phosphorylation of tau protein and a wide range of metabolic perturbations4 Unfortunately current biomarkers for early disease including cerebrospinal fluid CSF betaamyloid and tau levels5 structural and functional magnetic resonance imaging6 the recent use of brain amyloid imaging7 or inflammaging8 and CSF markers to track brain atrophy and deposition of cortical betaamyloid and neurofibrillary tangles are limited because they are either invasive timeconsuming or expensiveRecent studies have focused on obtaining biomarkers to identify features that differentiate persons a0with cognitive impairment from persons without cognitive impairment Molecular markers sensitive to the underlying pathogenic factors would be highly relevant to early disease detection and facilitation of disease monitoring and treatment responses Metabolomics is an unbiased approach to study smallmolecule metabolites that offers hope for the discovery of more biomarkers for AD This profiling technology has already been used to identify differential metabolites that can distinguish mild cognitive impairment MCI subjects who will develop AD from stable MCI9 Mounting evidence suggests that AD is closely accompanied with the abnormal bile acid BA metabolism10“ free fatty acid FFA metabolism14“ lipid metabolism1718 and neurotransmitter metabolism19 BAs have become increasingly recognized as important metabolic signaling molecules that modulate lipid glucose and energy metabolism20 More importantly BAs in brain act as neuroactive steroids21 Different classes of BAs can either inhibit or potentiate GABAα a0and inhibit NMDA receptors while also exerting neuroprotective effects Recent crosssectional studies have shown differences in blood BAs in AD compared with noncognitively impaired individuals2425 Additionally researchers found an accumulation of FFAs in the hippocampus and cortex of AD mice compared to control mice2627 Another animal study examined the role of elevated FFA in the pathogenesis of AD and established a potential mechanism of FFA causing hyperphosphorylation of tau through astrogliamediated oxidative stress28 Alterations of FFAs have also been detected in postmortem AD brains tissues14 and serum samples16 which may indicate an alternative fuel source before the onset of clinical symptoms29 These observations have given rise to the possibility that metabolic perturbations could presage the onset of cognitive impairment and therefore aid in the identification of individuals with higher risks by providing additional information to use with standard clinical markersIn this study we performed metabolomic profiling in participants from a large longitudinal cohort with the goal of identifying metabolic changes as well as key metabolic pathways that might serve as new predictors of future cognitive impairment in older adultsMaterials and methodsParticipants The Religious Orders Study ROS which began in is a longitudinal clinicalpathologic cohort study of risk factors of cognitive decline and incident dementia run from the Rush Alzheimer™s Disease Center that is comprised of individuals from religious communities eg Catholic brothers nuns and priests across the USA3031 The Rush Memory and Aging Project MAP which began in includes participants from northeastern Illinois USA with a broader range of socioeconomic status and life experiences31 Participants in both studies enroll without known dementia agree to annual clinical evaluation and an donation Both studies were approved by an Intuitional Review Board of Rush University Medical Center All subjects signed an informed consent an Anatomic Gift Act and a repository consent to allow their biospecimens and data to be used for ancillary studies All research was performed in accordance with relevant guidelinesregulations set forth by the Rush University Medical Center Both studies are conducted by the same team of examiners and share a large common core of data collection at the item level to allow for efficient merging of dataCognitive performance tests Cognitive performance was measured using a battery of cognitive performance tests of which could be summarized in five cognitive domains ie episodic memory working memory semantic memory perceptual orientationvisuospatial ability and perceptual speed Table a0S1 Domains are created by averaging the zscores based on mean and standard deviation from all baseline data for tests in each domain The global cognitive function score is calculated by averaging zscores for all tests to yield a global measure of cognitive function Additionally the MiniMental State Examination was also administered to characterize the cohortClinical diagnoses Medical conditions were documented via selfreport and clinical evaluation Clinical diagnoses each year were determined blinded to previously collected data A threestep process starts with an actuarial decision tree based on the history of cognitive decline and impairment ratings in five cognitive domains based on cutoffs for cognitive tests32 a0followed by clinical judgment by a neuropsychologist for cognitive impairment and determination of dementia and its causes by a clinician ie neurologist geriatrician second neuropsychologist geriatric nurse practitioner33 The diagnosis of AD follows the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer™s Disease and Related Disorders Association NINCDSADRDA34 Participants were categorized as a AD b MCI if diagnosed cognitive impairment by the neuropsychologist but not diagnosed dementia by the clinician32 and c no cognitive impairment NCI if diagnosed without AD or MCI35 At the time of death brain autopsies and histopathological exams were performed by clinicians to confirm the diagnosis After an autopsy was completed a spectrum of neuropathologic diagnoses was obtained such as a pathologic diagnosis of AD as defined using the modified NIA Reagan criteria However many other pathologies were present in the brains of older individuals the mean age of death is a0years old in ROSMAP and they were catalogued for each participantScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cNCI converters were those participants who were cognitively normal at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followupNeuropathology Upon death a postmortem neuropathological evaluation was implemented and the procedures follow those outlined by the pathologic dataset recommended by the National Alzheimer™s Disease Coordinating Center Brains of deceased subjects were removed weighed cut into one cmthick coronal slabs and stored Each brain was examined for the neuropathological indices of common pathologies that contribute to cognitive impairment The location age and volume of all macroscopic infarcts were recorded and tissue was obtained for histological confirmation in addition to the identification of microscopic infarctions as previously described3637 AD pathology was identified using the modified Bielschowsky silver stain technique and by use of the Consortium to Establish a Registry for Alzheimer™s Disease CERAD criteria38 and NIAReagan criteria39 while the assessment of neurofibrillary tangles was based on Braak criteria40 as described previously41 The CERAD score a semiquantitative measure of neuritic plaque burden is made of levels no AD possible AD probable AD and definite AD As recommended CERAD scores were reclassified to a binary level score score Seven categories of Braak stages were based on the region and severity of neurofibrillary tangles pathologyMetabolites quantification Using targeted metabolomics protocols42 and profiling protocols43 established in previous studies BAs were quantified by ultraperformance liquid chromatography triple quadrupole mass spectrometry UPLCTQMS Waters XEVO TQS Milford USA and other metabolites were quantified by gas chromatography timeofflight mass spectrometry GCTOFMS Leco Corporation St Joseph USA Details are described in the Supporting InformationStatistical analysis Stratifying by clinical diagnosis continuous demographic variables were expressed as mean [standard deviation SD] and tested by Wilcoxon ranksum test while categorical demographic variables were expressed as n percentage and tested by Chisquare test Missing values in quantitative metabolites due to limits of quantification were regarded as leftcensored missing and imputed by GSimp4445 Individual BA concentrations were normalized to the total BAs concentration ie the proportion of total BAs Metabolites were reported as median quantile quantile and tested by univariate analysis Wilcoxon ranksum test Due to the limited sample size of the AD group participants in serum samples we combined MCI and AD participants into an aggregate group MCIAD for the following data analysis Logtransformed abundances were used in the following data analysis We additionally generated BA ratios based on the BA metabolic pathway topologyTo identify metabolites differentially expressed in participants with cognitive decline we used ordinal logistic regression to compare metabolites across three groups NCI MCI AD for brain samples and logistic regression across two groups NCI MCIAD for serum samples To control the positive false discovery rate Qvalues were calculated based on Pvalues Additionally for serum samples we adjusted for potential confounders eg fasting status supplements diabetic and lipid lowering medications using logistic regressions The relationships between logtransformed brain metabolites levels with neurofibrillary tangle burden and neuritic plaque burden were expressed as boxplots across Braak scores Kruskal“Wallis test and CERAD scores Wilcoxon ranksum test respectively Using Spearman™s rank correlation test we further evaluated the associations between the abundances of each identified metabolite and the global cognitive function score in both brain and serum samples Linear regression models with each individual metabolite used as the predictor and each cognitive test as the response variable adjusted for age gender years of education and presence of APOE ε4 were used to test the associations between metabolite and cognitive function Similar analyses with an additional adjustment of BMI were conducted for serum samples The Wilcoxon ranksum test was carried out to explore whether identified variables were differentially expressed between NCI converters vs NCI nonconverters and between NCI converters vs MCIAD in sera Then we built a random forest RF predictive model to differentiate NCI nonconverters vs MCIAD using glycolithocholate GLCA deoxycholatecholate DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and age as the predictorsTo differentiate MCIAD vs NCI nonconverters we randomly split the data into training set and testing set times Each time we trained an RF model on the training set to differentiate the MCIAD from NCI nonconverters and evaluated it on the testing set using the area under the receiver operating characteristic curve AUROC sensitivity SE and specificity SP A final model was built on the whole NCI nonconverters and MCIAD dataTo investigate the preclinical predictive potentials as well as to validate the classification performance of our model we utilized this model on the entire baseline NCI group to identify those NCI converters from NCI nonconverters The differences of RF scores between NCI nonconverters vs NCI converters and NCI nonconverters vs MCIAD groups were tested by the Wilcoxon ranksum test To determine whether RF scores could independently differentiate NCI converters from NCI nonconverters in the presence of potential confounders we used the logistic regression method with RF scores as the predictor adjusting for gender years of education APOE ε4 and BMI Additionally we fit linear mixed effects models to evaluate correlations between RF scores with global cognitive function and each of the five cognitive domains separately with a random effects term for education and BMI and fixed effects terms for RF score gender and APOE ε4For the personalized pathway level analyses we extracted metabolite information from the Human Metabolome Database HMDB46 and metabolic pathway information from the Kyoto Encyclopedia of Genes and Genomes KEGG database47 to map affiliated metabolites to metabolic pathways We used the pathifier Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cOverallNCIMCIADBrain samplesNAge mean SDMale n Education mean SDAPOE ε4carrier n Serum samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Serum NCI samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Overall Overall NCI MCIAD NCI nonconverters NCI converters Table Detailed demographic characteristics of study samples Chisquare test Pvalue comparing AD vs NCI Wilcoxon rank sum test Pvalue comparing MCIAD vs NCI Wilcoxon rank sum test Pvalue comparing NCI converters vs NCI nonconverters Chisquare test Pvalue comparing NCI converter vs NCI nonconverter NCI cognitively normal MCI mild cognitive impairment AD Alzheimer™s disease APOE ε4 apolipoprotein E epsilon allele SD standard deviation algorithm48 to transfer metabolic level information of each sample to pathway level by generating a pathway dysregulation score PDS For each pathway each sample was projected onto a directed principal curve49 which was yielded depending on leading principal components of the pathway to optimally pass through the cloud of samples PDS was the distance along the curve between the projection of each sample and that of NCI Thus PDS could capture the pathwaylevel extent of abnormality increments or decrements for each participant relative to those with NCI We performed similar data analysis on pathway level data to what we did on metabolomics level data We tried to identify differential pathways using ordinal logistic regression across NCI MCI and a0AD groups in brain samples and logistic regression for NCI and MCIAD in serum samples Next we explored the associations between identified pathways with neuropathology Kruskal“Wallis test for Braak scores Wilcoxon ranksum test for CERAD scores and cognitive performance Spearman™s rank correlation test for the global cognitive function linear regression with adjustments for each cognitive test Then we examined the predictive potential of identified pathways in serum samples using univariate analysis Wilcoxon ranksum test for NCI converters vs NCI nonconverters NCI converters vs MCIAD Finally we built RF models on training sets and tested them on testing sets according to times random splitting on the model construction data and applied the final model on the validation data using ROC SE SP as evaluation methods The overall workflow chart of the data and the analysis are shown in Fig a0S9Data were analyzed using R version with packages including pROC pathifier randomForest ggplot2 ggsignif and MASS The statistically significance was determined by a threshold of unadjusted Pvalues and Qvalues ResultsParticipants and characteristics For the joint analyses of the ROSMAP study we measured metabolomics of serum samples NCI MCI and AD at the blood draw and postmortem brain tissues from dorsolateral prefrontal cortex NCI MCI and AD at the time of death Among brain samples and serum samples a total of participants had both brain and blood metabolomics data NCI participants n were further categorized into œNCI converters and œNCI nonconverters NCI converters were those participants who were cognitively normal NCI at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followup Among NCI participants the time between the blood draw and conversion ranged from to a0years with a median of a0years Fig a0S10 Detailed demographic characteristics of the serum samples and postmortem brain samples are included in Table a0 Among participants with postmortem brain samples AD patients tended to have at least one APOE ε4 allele compared to the NCI group as expected The mean age of NCI and MCIAD group at the time of blood draw among serum samples was a0years SD and SD respectively Similarly the age and the percentage of APOE ε4 Scientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cFigure a0 Brain metabolome and serum metabolome composition and alterations a Left panel the brain metabolome composition Right panel “ a0log10 Pvalue across clinical groups of brain tissues NCI MCI AD b Left panel the serum metabolome composition Right panel “ a0log10 Pvalue across clinical groups of serum tissues NCI MCIADcarriers were higher in the NCI converters group than the NCI nonconverters group We did not observe other significant demographic characteristics differences across clinical groups Table a0Identifying metabolites differentially expressed in participants with cognitive impairment In this study metabolites and metabolites overlapping metabolites were detected in brain tissues and serum samples respectively Tables a0S7 S8 Amino acids BAs carbohydrates anic acids and fatty acids were the predominant types of annotated metabolites accounting for of all the metabolites in brain tissues and in serum samples Fig a01ab left panel A total of seven a0metabolites BA BA ratio anic acid known as a longchain fatty acid fatty acids showed significant differences across clinical groups in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a01ab right panel Tables a0S14 S15 After adjusting for confounders ie fasting status supplement use diabetic and lipid lowering medications most of serum metabolites remained statistically significant Table a0S18 In brain tissues increments of the levels of GLCA DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid followed the pattern NCI MCI AD We observed increments of GLCA DCACA ratio and decrements of petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid in sera of MCIAD compared to controls Table a0 The trend of increments of identified metabolites in brain samples increments of BAs and decrements of FFAs in serum samples were further validated within individuals with both brain and serum samples From NCI to MCI and AD groups increments of identified metabolites were observed in brain samples Table a0S11 The increasing trend of GLCA DCACA ratio and decreasing trend of FFAs among MCIAD group relative to NCI group were detected in sera Table a0S11The seven brain metabolites were all negatively correlated with global cognitive function where higher scores indicate better cognitive performance ρ ˆ’ a0 for GLCA ρ ˆ’ a0 for DCACA ratio ρ ˆ’ a0 for petroselinic acid ρ ˆ’ a0 for linoleic acid ρ ˆ’ a0 for myristic acid ρ ˆ’ a0 for palmitic acid and ρ ˆ’ a0 for palmitoleic acid using Spearman™s rank correlation analysis Fig a02a Similarly after adjusting for age gender years of education and APOE ε4 all identified metabolites remained negatively correlated with tests in five Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cBrain samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]Serum samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]NCIMCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ]NCI [ ] [ ] [ ] [ ] [ ] [ ] [ ]MCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Table Levels of metabolites differentially expressed in participants a0by diagnostic group Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing AD vs NCI Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing MCIAD vs NCI NCI cognitively normal MCI mild cognitive impairment AD Alzheimer™s disease IQR interquartile rangeFigure a0 Associations between metabolites level and global cognitive function a Boxplots showing group differences and P values for identified metabolites across Braak groups for brain tissue abundances b Boxplots showing group differences and significances for identified metabolites across CERAD groups for brain tissue abundances ρ correlation coefficient of Spearman™s rank correlation testScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0ccognitive domains and the MiniMental State Exam see Table a0S2 for significant correlation pairs The serum concentration of two BAs showed negative correlations with global cognitive function ρ ˆ’ a0 for GLCA ρ ˆ’ a0 for DCACA ratio conversely fatty acids demonstrated positive correlations ρ for petroselinic acid ρ for linoleic acid ρ for myristic acid ρ for palmitic acid and ρ for palmitoleic acid Fig a02b Linear regression revealed similar consistent results in serum samples with adjustment for age gender years of education APOE ε4 and BMI see Table a0S2 Results of associations between identified metabolitesratio and each cognitive performance domains are shown in Table a0S13 Correlations with global cognitive function were further validated in individuals with both brain and serum samples and the directions were consistent with our previous findings among the a0entire cohort Seven identified metabolites were all negatively correlated with global cognitive function in brain samples while two BAs showed negative correlations and five FFAs showed positive correlations in serum samples Fig a0S6 Additionally the serumbrain ratio of identified FFAs were positively correlated with global cognitive function ie lower levels of identified FFAs in serum and higher levels of identified FFAs in brain were associated with worse cognition Fig a0S7Identified metabolites predicted antecedent cognitive impairment before the manifestation of clinical symptoms The concentrations of GLCA and DCACA were significantly lower in the NCI nonconverters group than in the NCI converters group By contrast the abundances of petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid were higher in the NCI nonconverters group than in the NCI converters group Fig a03a There were no significant differences in these metabolites between participants in NCI converters group vs MCIAD group Fig a03a Using the seven metabolites and age we built RF models on the training set according to 100times randomly splitting approach to differentiate MCIAD patients from NCI nonconverters group The median of times AUC on testing set was CIs “ with SE CIs “ and SP CIs “ using Youden™s index to maximize the sum of SE and SP Fig a03b RF models showed decent classification performances in differentiating MCIAD group from NCI nonconvertersNext we were interested in studying the model™s early diagnostic capability for predicting NCI converters before clinical diagnosis The model was thus applied on the entire NCI group at baseline to differentiate NCI converters from NCI nonconverters We achieved an AUC of CIs “ SE SP at the cutoff value of Fig a03c with significant differences in RF scores between NCI converters vs NCI nonconverters between NCI nonconverters vs MCIAD group using the Wilcoxon ranksum test Pvalue Fig a03d After additional adjustment for gender years of education APOE ε4 and BMI fasting status and medications supplements diabetes lipid lowing the RF scores remained significant as an independent predictor with a coefficient of Pvalue Table a0S3 Additionally the RF scores showed significant negative correlations with global cognitive function and the five cognitive domains with the same adjustment in mixed effects models Table a0S12Personalized metabolic pathway‘based study for the association and prediction of cognitive impairment Considering altered metabolite levels were significantly associated with cognitive impairment and showed early predictive value of clinical symptoms onset we then employed the pathifier algorithm to summarize metabolite information to pathways level for further examinations All PDS scores ranged from to where larger scores represent the higher extent of the abnormality in the corresponding metabolic pathway out of metabolites detected in brain tissues and out of metabolites detected in sera were successfully mapped to the KEGG metabolic pathways This method identified metabolic pathways in brain tissues and metabolic pathways in serum samples overlapping pathways Figs a0S1ab left panel Table a0S9 Table a0S10 three of which ie primary BAs biosynthesis FFAs biosynthesis and biosynthesis of unsaturated FFAs were significantly shifted in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a0S1ab right panel Table a0S16 Table a0S17 We noted increased PDS for all three identified pathways from NCI to MCIAD that suggested dysregulation of these metabolic pathways in MCIAD patients compare to NCI Detailed PDS of these pathways stratified by diagnostic groups are described in Table a0S4 Results also indicated that higher PDS were significantly associated with lower global cognitive function ie worse cognitive performance in both brain ρ ˆ’ a0 for primary BAs biosynthesis pathway ρ ˆ’ a0 for FFAs biosynthesis pathway ρ ˆ’ a0 for biosynthesis of unsaturated FFAs pathway Fig a0S4 and serum samples ρ ˆ’ a0 for primary BAs biosynthesis pathway ρ ˆ’ a0 for FFAs biosynthesis pathway ρ ˆ’ a0 for biosynthesis of unsaturated FFAs pathway Fig a0S5 respectively In Table a0S5 we show the significant negative associations between each cognitive test and PDS of three pathways after adjusting for age gender years of education and APOE ε4 additional adjustment for BMI in serum samples Two fatty acid pathways showed significantly different PDS between the NCI nonconverters group and the NCI converters group Pvalue and Pvalue respectively A gradually increasing trend was noted for the BAs pathway across groups ie NCI nonconverters NCI converters and MCIAD Fig a04aWe then constructed a discriminant RF model in training data and tested on testing data based on three identified metabolic pathways along with age to differentiate MCIAD from NCI nonconverters in model construction data using 100times randomly splitting approach The median AUC on the a0testing set was CIs “ with SE CIs “ and SP CIs “ Fig a04b Applying the RF model to the whole NCI data at baseline could successfully discriminate NCI converters from NCI nonconverters with an AUC of CIs “ SE SP cutoff value Fig a04c Similarly predictive RF scores were significantly different between NCI converters vs NCI nonconverters and NCI nonconverters vs MCIAD group Pvalue Fig a04d After adjusting for gender Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cFigure a0 The identified panel of metabolites and its predictive performance a Boxplots showing group differences and P values for identified metabolites across NCI nonconverters NCI converters and MCIAD for serum abundances b ROC curves of metabolite models trained on the training data and tested on the testing data according to 100times randomly training“testing splitting c The ROC curve of the final metabolite model on the validation data d RF scores of the final metabolite model across NCI nonconverters NCI converters and MCIAD Pvalue Pvalue Pvalue Wilcoxon rank sum test The optimal cutoff was determined by the Youden index AD Alzheimer™s disease AUC area under

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"Sleep disturbance is an issue reported by caregivers Waking at night is a feature of dementia and byproxy sleep disturbance among caregivers is reported to be high Little is known about the characteristics ofdementia caregivers™ sleep and the factors that may influence sleep disruptionThe purpose of this study was to investigate the sleep characteristics and disturbances of Australian caregivers of aperson living with dementia In addition it evaluated the psychological wellbeing of caregivers by evaluatingassociations between mood and sleep in this populationMethods This study used a crosssectional descriptive correlation design Participants were recruited with theassistance of Alzheimer™s Australia Dementia Australia and targeted social media advertising In total adultprimary informal caregivers of people with dementia participated completing a questionnaire on demographiccharacteristics the Depression Anxiety and Stress Scale DASS21 and the Pittsburgh Sleep Quality Index PSQIResults In this study of caregivers were female who had been caring for someone living with dementia onaverage for years of participants had two or more comorbidities namely cardiovascular disease osteoarthritisand diabetes of participants were poor sleepers with with difficulty initiating sleep and reporting havingdifficulty maintaining sleep Overall psychological distress was common with high levels of moderate to severedepression anxiety and stress Global PSQI scores were significantly positively associated with depression and anxietywith the strongest correlation seen with stress scores Depression scores were also moderately associated with daytimedysfunction Stress was identified as a significant predictor of overall sleep qualityConclusions Sleep problems are common within the population of dementia caregivers Due to the nature andduration of caregiving and the progression of dementia of the care recipient there is the potential for a decline in thecaregivers™ mental and physical health Caregivers of those living with dementia are more likely to have comorbiditiesdepression anxiety and stress Sleep quality is correlated with emotional distress in dementia caregivers although thedirection of this association is unclear Therefore sleep and psychological wellbeing may be intertwined withimprovements in one aspect resulting in a positive impact in the otherKeywords Carers Caregivers Sleep Mood Psychological wellbeing Dementia Correspondence aislingsmythecueduau1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA AustraliaFull 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 0cSmyth BMC Geriatrics Page of BackgroundDementia is an inclusive term used to describe a numberof neurological conditions resulting in cognitive impairment and can include Alzheimer™s Disease senile dementia frontotemporal dementia vascular dementia LewyBody dementia Korsakoff syndrome alcohol related braininjury and younger onset dementia [“] Globally thenumber of individuals with a formal dementia diagnosisrose from million in to million in []A further million new cases of dementia worldwideare predicted each year [] as populations around theglobe continue to live longer [] These figures could bevery much under estimated as it is widely accepted thatbetween and of people affected have no formaldementia diagnosis [] This growing epidemic effects notonly the individuals living with dementia but also theirfamilies caring for them the communities they live in andthe health care systems they rely uponFamily or friends are often key informal caregivers forpeople living with dementia [ ] Globally over billion hours ofinformal care is provided to peopleliving with dementia [ ] Whilst providing this carecan be highly rewarding it has also been described as achronic stressor with caregivers reporting low qualityand quantity of sleep [] and high levels of stress anddepression [] In fact sleep disruption is prevalentamongst dementia caregivers with over of caregiversexperiencing sleep disturbances []The National Sleep Foundation recommend that olderadults ‰¥ years require seven to h sleep for optimalphysical and psychological wellbeing [] Yet numerousstudies have illustrated that dementia caregivers sleepsignificantly less than that with estimates reporting mostcaregivers sleep less than h per night [] Not only arecaregivers sleeping less than they should the sleep theydo get is of significantly lower quality than their noncaregiver counterparts []Poor sleep is associated with a myriad of negative physical and psychological outcomes including hypertensionobesity mood disorders and dementia [] A disruptedsleep pattern is also recognised as a significant factor inpredicting caregiver strain [ ] and perhaps more importantly in predicting placing an individual into long termcare [] Enabling people living with dementia to stay athome rather than transfer to longterm care is the optimaloutcome for many families However this cannot be to thedetriment of the caregiver™s own physical andor psychological wellbeing Therefore in order to support the personliving with dementia PLWD to remain in the communitymaintaining caregiver health is vital Given the pivotal rolesleep has in a myriad of physiological processes it is essential to optimise and preserve caregivers sleepDespite the important role sleep plays in dementiacaregiver health it remains an understudied populationparticularly within the Australian context In fact arecent report by Carers Australia identified noAustralian studies on sleep disruption in dementia caregivers Only sixteen international studies were identifiedwhich subjectively measured sleep in the dementiacaregivers population with the majority of studies notreporting on the causes or consequences of disturbedsleep []In order to address this paucity of Australian data thepurpose of this study was to elucidate the sleep characteristics and disturbances of Australian caregivers ofPLWD Furthermore this study will determine whetherthere is a relationship between sleep and psychologicalwellbeing among caregivers of communitydwellingpeople living with dementia Lastly we will aim to identify significant predictors of poor sleep which in turncould offer a therapeutic target of poor sleep in dementia caregiversMethodsRecruitmentThis study used a crosssectional descriptive correlationdesign One hundred and four informal caregiversof people with dementia living in the community wereenrolled in the study Participants were invited via anumber of anisations including Alzheimer™s AustraliaDementia Australia and targeted social media advertising Mail outs were conducted and online questionnairesdistributed The inclusion criterion required the participant be an adult years or older primary informalcaregiver of a communitydwelling person living withdementia Power analysis was undertaken to computeminimum number of sample size required A samplesize of at least participants is necessary to detect amedium to large effect with power assuming atwotailed ttestEthicsThis study was granted ethical approval by Edith CowanUniversity Human Research Ethics Committee No Informed written consent was obtained from allparticipants prior to participation Participants receivedno incentive for taking partMaterialsThe survey collected demographic characteristics including gender age body mass index BMI as well as information about preexisting medical history caregivinghistory and use of respite The survey also included the questions from the Depression Anxiety and StressScale DASS21 [] and the Pittsburgh Sleep QualityIndex PSQI []The DASS21 is a selfreport questionnaire assessinglevels of caregiver stress anxiety and depression over the 0cSmyth BMC Geriatrics Page of previous seven [] day period The results provide anindication of the individual™s perception of their experience of depression anxiety and stress The DASS21 isoften used as a screen for high levels of distress whenthe depression andor anxiety scores are high and as ascreen for the presence of a significant life event orproblem if the stress score is elevated DASS scorescorrespond to ranges of severity normal mild moderate severe extremely severe for depression anxiety andstress individually []The PSQI [] is a selfreport questionnaire assessinglevels of perceived quality and patterns of sleep over theprevious month across seven components subjectivesleep quality sleep latency sleep duration habitual sleepefficiency sleep disturbances use of sleep medicationand daytime dysfunction Each component has a potential score of three with a higher score indicating poorersleep related performance The total score for the sevencomponents creates a global score range to wherea score greater or equal to five ‰¥ indicates that theperson is a œpoor sleeper with severe difficulties in atleast two of the seven components or moderate difficulties in three or more components []Presence of comorbidities was determined by participantshaving one or more chronic conditions As per WorldHealth anisation WHO chronic conditions wereidentified as those requiring ˜ongoing management over aperiod of years or decades™ covering a wide range of healthproblems such as heart disease diabetes asthma immunodeficiency disorder depression and schizophreniaStatistical analysesData analysis was undertaken using IBM SPSS Version and GraphPad Prism Descriptive statistics weregenerated for demographic characteristics DASS21 andPSQI scales Spearmans rho r correlation analysis wasused to assess the relationship between categorical components of DASS21 depression anxiety and stress andboth PSQI global scores and individual componentscores subjective sleep quality sleep latency sleep duration sleep efficiency sleep disturbances use of sleepingmedication and daytime dysfunction For logistic regression all variables were categorical age in years genderBMI length of care in months comorbidities DASS21component scores An r of was considered a mediumcorrelation and an r of was considered a large correlation [] Nonpaired ttests were used to determine ifthese values were statistically significant Results wereconsidered statistically significant if p Multivariatestepwise regression analysis was undertaken to determinethe predictive factors of global PSQI scores and identify variables significantly associated with sleep quality Independentvariables included in multivariate stepwise regression analyses were age gender BMI length of care comorbiditiesand DASS21 subscale scores Subjects with missing data oneither PSQI or DASS21 were excluded from inclusion incorrelation and regression analysesResultsCharacteristics of the caregiversOne hundred and four n surveys were completedin either hardcopy n or online n Table As the surveys were widely distributed by service providers and links made available online the response ratecannot be determinedParticipating caregivers were predominantly femalen with a mean age of years Range “years The average BMI for respondents was kgm2range “ kgm2 Table which lies within theoverweight category While half of the participants n had a BMI within the healthy weight range“ twenty two participants were classified as underweight BMI less than and participants were classified as overweight The averagelength of time in the caregiving role in this study was months years ranging from months to yearsOnly n of participants reported using formalrespite services and all were female n of participants reported no comorbidities of participants n reported one comorbidity and n reported two or more comorbidities Themost commonly reported comorbidities were cardiovascular disorders n mainly hypertension and hypercholesterolemia bone and joint disorders n mainlyTable Demographic of participantsCharacteristicGendern or meanˆ’ SDMaleFemaleAllAgeBMI kgm2Caregiver role MonthsComorbiditiesNoneOneTwo TotalUse of RespiteYesNoTotal99a94b96c ± ± ± a n missingb n missingc n missing 0cSmyth BMC Geriatrics Page of osteoporosis and arthritis and endocrine disorders n mainly prediabetes diabetes and thyroid dysfunctionTable Characteristics of Caregivers™ sleepMeasuren Subjective Sleep qualityThe majority of participants n reported aglobal sleep score equal or greater than which indicated they had clinically significant sleep issues in thepreceding month with a mean global score of ± Table The highest scoring individual subcomponentsincluded sleep latency ± subjective sleep quality ± and sleep disturbances ± n of participants had a sleep latency period greater than min indicative of issues initiating sleep Table ofparticipants had sleep efficiencies less than suggestingissues with maintaining sleep while in bed of participants had taken sleep medication at least once a weekover the previous month Participants were invited to addany additional relevant comments to their PSQI sleep assessment Forty participants provided additional information regarding their sleeping issues and identified issueswhich fell broadly into three themes Sleep disruption dueto caregivers physical needs such as pain and restless legs caregivers emotional distress such as stress anxietyand worrying and responding to care recipientneeds Depression anxiety and stress in caregivers of participants reported mild depression scores of participants reported mild anxiety scores and of participants reported mild stress scores Over athird of respondents reported moderate to extremelysevere stress levels n and depression n and more than a quarter n of respondents reported moderate to extremely severe anxietylevels Table In the bivariate analyses numerous subcomponents ofthe PSQI scale were significantly associated with measures of depression anxiety and stress Table Theglobal PSQI score is significantly positively associatedwith depression r anxiety r and stressscores r Stress scores also significantly correlatedwith other PSQI subcomponents including subjectivesleep quality r sleep latency r and daytimedysfunction r Depression scores were moderatelyassociated with daytime dysfunction r Global ScoreSubjective Sleep QualityVery GoodFairly GoodFairly BadVery BadSleep Latency‰¤ mins“ min“ min minsSleep duration h“ h“ h hSleep Efficiency‰¥ ““ Sleep DisturbancesDaytime DysfunctionFrequency of SleepingMedicationNeverOnce per weekTwice per weekThree per week PSQI ComponentMean ˆ’ SD ± ± ± ± ± ± ± ± Predictors of Sleep quality in caregiversTo understand the relationship between the predictorsand global PSQI scores multivariate stepwise regressionanalyses were conducted to assess variables significantlyassociated with sleep quality The dependent variablewas the global PSQI score and the independent variableswere age gender BMI use of respite length of carecomorbidities and scores on the subscales of DASS21scale Depression Anxiety and Stress Stress was theonly significant covariate of global PSQI scores Stressscores could statistically significantly predict PSQIGlobal scores accounting for of variance Thestandardised coefficient was which was statisticallysignificant p as was the overall model F p All other variables were excluded from themodel due to nonsignificance Forward stepwise regression 0cSmyth BMC Geriatrics Page of Table Depression anxiety and stress in caregivers DASS21Clinical classificationTotalDepression n Anxiety n Stress n NormalMildModerateSevereExtremely Severe analyses were then undertaken with global PSQI scores asthe dependent variable and stress scores as a predictorwhile adjusting for the following confounders gender agecomorbidities of the caregiver and length of care Whilethese confounders were not statistically significant theywere retained within the model to adjust the effects ofstress on PSQI After adjusting for these factors that modelremains statistically significant F p andaccounts for of the adjusted variance in sleep scoresFor every one unit increase in stress scores PSQI Globalscores increases by a score of t p Thisregression model confirms that stress is a key significantcovariate of selfreported sleep issuesDiscussionA recent Australian report highlighted a significant gapin the literature around Australian caregivers of a PLWD[] Our study provides a comprehensive overview ofAustralian dementia caregiverssleep characteristicsassociations between psychological wellbeing and sleepand highlights a predictive role for stress in sleep qualityAll previous international studies identified an averagePSQI in dementia caregiver studies [] highlightingthe prevalence of the issue As expected and consistentwith previous studies [] Australian caregivers ofPLWD have a high prevalence of poor sleep with ofparticipants classified as poor sleepers The PSQI globalscore ± was higher than those found in somedementia caregiver studies [ ] but comparable withTable Correlation between PSQI subcomponent scores andDASS21 subcomponent score in dementia caregiversPSQI componentDASS21DepressionDASS21AnxietyDASS21StressPSQI Global ScoreSubjective sleep qualitySleep latencySleep durationSleep efficiencySleep disturbancesUse of sleeping medicationDaytime dysfunctionp p ˆ’ˆ’ˆ’others [] The PSQI subcomponents which greatestcontributed to the overall score were sleep latency timetaken to fall asleep sleep quality overall subjectivequality and sleep disturbances Sleep latency and sleepdisturbances have previously been identified as the mostcommon contributors to sleep quality in caregivers ofPLWD in a recent systematic review [] Given thedearth of Australian specific data this study identifiedimportant depth of detail around caregiver™ssleepcharacteristics of caregivers took longer than therecommended min to fall asleep only of caregivers slept more than h of caregivers had sleepefficiency lower that the recommended and ofcaregivers used sleep aiding medication in the previousmonth Taken togetherfindingspresent a novel and ominous overview of the poor sleephealth conditions that Australian dementia caregiversexperiencethese descriptiveCaregivers of community dwelling people living withdementia reported poor sleep quality and high levels ofdepression anxiety and stress This study found that poorsleep was correlated with subjective feelings of depressionanxiety and stress which were in keeping with previousliterature around caregiving and psychological distressMore than half of the caregivers surveyed reported symptoms of depression and stress and respectivelyand reported anxiety These findings are in keepingwith that of a recent study of dementia caregivers in ruralVictoria where of caregivers reported depression orstress and reported anxiety []Previous work has identified an association betweendepression and poor sleep among dementia caregivers[ ] Reduced quality of sleep and depression arehigher among caregivers of people living with dementia[] Furthermore caregivers of people living withdementia who were also depressed experience a greatervariation in sleep patterns []This study reveals sleep scores were significantly associated with measures of depression anxiety and stressThese findings illustrate the interplay between sleepquality and quantity and psychological wellbeing incaregivers providing care for an individual living withdementia Further exploration of predictive factors incaregiverssleep quality identified stress as a keypredictor of poor overall sleep quality whilst adjustingfor age gender comorbidities and length of care This isa novel finding and represents a potential therapeutictarget to improve sleep quality in dementia caregiversDespite the high prevalence of selfidentified poorsleep overnight respite was only used by of participants which is in keeping with previous research thatcites of Australian caregivers have never usedrespite services [] Access to respite care remains oneof the major means of easing caregiver burden and is 0cSmyth BMC Geriatrics Page of frequently identified as needed by caregivers yet remainsunderutilised Whilst a high proportion of dementiacaregivers report a need for respite services there isinsufficient awareness of and access to respite servicesfor caregivers [] Respite service availability can beinvaluable with caregivers reporting lower stress levelsand improved health after use [] Sleep disturbanceshave also been shown to be partially reversed for caregiversduring periods of respite care [] perhaps due to reversalof hyper stressed state Respite services have been cited asan important measure to allow caregivers time to attend totheir own health imperative to supporting the caregiver tocontinue in providing care for their loved one []A recent Australian parliamentary inquiry into sleephealth identified sleep as a foundation of positive healthand wellbeing alongside diet and exercise [] Furthermore the report urged government to prioritise sleephealth as a national priority given its pivotal role inmaintaining health It is clear that caregivers of PLWDhave suboptimal sleep which is associated with poorerpsychological wellbeing and potential increased risk ofdeveloping chronic health conditions such as diabetesand cardiovascular disease [] Interestingly cardiovascular disease and endocrine disease such as diabeteswere the most prevalent comorbidities in our studypopulation Previous studies have also identified highblood pressure diabetes and arthritis as the most prevalent chronic disease in the dementia caregiver groupwhich is in keeping with our findings []Managing sleep and its associated mediators will havedirect impact on both the caregiver™s own health as wellas the caregiver care recipient relationship It is imperative to provide educational support around sleep considerpractical interventions such as overnight respite and toaddress stress management interventions for caregivers inturn reinstating and preserving sleep of this critical population of informal caregiversLimitationsA limitation of this study was the small sample size despitenumerous attempts to recruit participants and involvementof national anisations Although over participantsprovided demographic details only participants completed all questionnaires and were included in statisticalanalyses Challenges related to recruiting caregivers andparticularly caregivers of people living with dementia hasbeen described in numerous publications [ ]Caregivers of people with dementia living in the community are more likely to be female and this was representedin our population Furthermore caregivers who are underthe greatest stress may also be those least likely to participate However this remains one of the larger studies ofAustralian dementia caregivers sleep Another limitationwithin this study was that no data was collected regardingthe severity of dementia or behavioural disturbances whichmay impact sleep of the caregiverConclusionSleep problems are widespread within the population ofdementia caregivers Given that the majority of peopleliving with dementia are reliant on family caregiversminimising health and psychologicalimpact on caregivers should be of major concern Furthermore sleepquality is correlated with emotional distress in dementiacaregivers so improving the sleep of caregivers may inturn improve their psychological wellbeing Converselystress is a significant predictor of poor sleep so managing stress may have a positive impact on sleepDementia caregivers are often older with coexistingmorbidities and high levels of psychological distress Withprolonged caregiving and the progression of dementia ofthe care recipient there is the potential for a concurrent decline in the caregivers mental and physical health In orderto support the caregiver in their role it is of the upmost importance that we promote and maximises their health andwellbeing By managing and minimising the negative factorsassociated with caregiving we can enhance caregiving satisfaction and gratification In turn this can ensure optimalcaregivercare recipient relationship supporting the personliving with dementia to remain at home as long as possibleAbbreviationsDASS21 Depression Anxiety and Stress Scale21 PLWD PersonPeopleliving with dementia PSQI Pittsburgh Sleep Quality Index WHO WorldHealth anisationAcknowledgementsThe authors acknowledge the statistical advice of Dr Mark Jenkins and DrPeter PalamaraAuthors™ contributionsAS was involved in study design data collection data interpretation andmanuscript preparation LW supported study design data interpretation andediting of the manuscript CV supported study design and manuscriptpreparation EQ advised on use of psychological tools interpretation of theirdata and manuscript preparation LE was involved in data collection andmanuscript preparation All authors have read and approved the manuscriptFundingThis research was supported by an Edith Cowan University ECU Early CareerResearcher Grant awarded to Dr Aisling Smyth ECU had no role in thedesign data collection and data interpretation of the studyAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was granted ethical approval by Edith Cowan University HumanResearch Ethics Committee No Informed written consent wasobtained and participants received no incentive for taking partConsent for publicationNot applicableCompeting interestsThe authors state that they have no competing interests 0cSmyth BMC Geriatrics Page of Cohen J Statistical power analysis for the behavioral sciences 2nd edHillsdale NJ Erlbaum Wilcox S King AC Sleep complaints in older women who are familycaregivers J Gerontol B Psychol Sci Soc Sci 1999543P189“Simpson C Carter P Dementia behavioural and psychiatric symptomseffect on caregiver's sleep J Clin Nurs ““Kochar J Fredman L Stone KL Cauley JA Sleep problems in elderlywomen caregivers depend on the level of depressive symptoms resultsof the caregiver“study of osteoporotic fractures J Am Geriatr Soc “Lee D Heo S Yoon S Chang D Lee S Rhee H Sleep disturbances andpredictive factors in caregivers of patients with mild cognitive impairmentand dementia J Clin Neurol “Johnson E Respite reconsidered a discussion of key issues and futuredirections for carer respite Sydney Carers Australia Phillipson L Johnson K Cridland E Hall D Neville C Fielding E et alKnowledge helpseeking and efficacy to find respite services an exploratorystudy in helpseeking carers of people with dementia in the context ofaged care reforms BMC Geriatr “ O™connell B Hawkins M Ostaszkiewicz J Millar L Carers™ perspectives ofrespite care in Australia an evaluative study Contemp Nurse “Lee D Man K Lindesay J Effect of institutional respite care on the Sleepof people with dementia and their primary caregivers J Am Geriatr Soc“ Parliament of the Commonwealth of Australia Bedtime Reading inquiry intoSleep health awareness in Australia Canberra Commonwealth of Australia [Available from httpsparlinfoaphgovauparlInfodownloadcommitteesreportrep024220toc_pdfBedtimeReadingpdffileTypeapplication2Fpdf] Accessed Mar Moon H DilworthAnderson P Baby boomer caregiver and dementiacaregiving findings from the National Study of caregiving Age Ageing“ Bristow M Cook R Erzinclioglu S Hodges J Stress distress and mucosalimmunity in carers of a partner with frontotemporal dementia Aging MentHealth “ McConaghy R Caltabiano ML Caring for a person with dementia exploringrelationships between perceived burden depression coping and wellbeing Nurs Health Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsAuthor details1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA Australia 2School of Arts and HumanitiesPsychological Services Centre Edith Cowan University Joondalup AustraliaReceived April Accepted August ReferencesAlzheimer's 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recommendations finalreport Sleep Health “Jay S Vincent G Kovac K Dorrian J Thomas M Reynolds A Ferguson SReport Reducing Sleep Disruption in Carers Australia Carers Australia httpswwwsleephealthfoundationaufilesResearch_GrantsSpecial_reportsReport_Carers_Australia_FINALv2pdf Accessed Mar2020 Worley SL The extraordinary importance of Sleep the detrimental effects ofinadequate Sleep on health and public safety drive an explosion of Sleepresearch P T “ Rowe MA McCrae CS Campbell JM Benito AP Cheng J Sleep patterndifferences between older adult dementia caregivers and older adultnoncaregivers using objective and subjective measures J Clin Sleep Med“Schulz R Belle SH Czaja SJ McGinnis KA Stevens A Zhang S Longtermcare placement of dementia patients and caregiver health and wellbeingJAMA “ Gaugler JE Yu F Krichbaum K Wyman JF Predictors of nursing homeadmission for persons with dementia Med Care “Lovibond SH Lovibond PF Manual for the Depression Anxiety Stress ScalesSydney Smyth C The Pittsburgh Sleep quality index PSQI Insight “ Buysee DJ Reynolds CF Monk TH Berman SR Kupfer DJ The PittsburghSleep Qulaity index a new instrument for psychiatric practice and researchPsychiatry Res

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"Vitamin D is a fatsoluble vitamin vitamin D is essential to sustain health and it protects againstosteoporosis It is crucial to the human body™s physiology in terms of muscular movement and neurological signaltransmission and to the immune system in defense against invading pathogensCase presentation This was a case of a 26yearold Sudanese woman who presented with a 2year history ofanosmia recurrent nasal polyps back pain and chronic fatigue She was diagnosed as having a case of vitamin Ddeficiency and responded well to treatmentConclusion There is an association between vitamin D deficiency and recurrent allergic nasal conditionsKeywords Vitamin D deficiency Allergy Nasal polyps Backache Chronic fatigabilityBackgroundVitamin D is a fatsoluble vitamin it is naturally presentin some foods and as dietary supplements It is also produced endogenously through exposure to ultraviolet raysfrom sunlight Vitamin D obtained from sun exposurefood and supplements is biologically inert and mustundergo two hydroxylations in the body for activationTheand produces hydroxyvitamin D 25OHD also known as calcidiolThe second occurs in the kidney and forms the physiologically active 125dihydroxy vitamin D 125OH2D alsoknown as calcitriol []first occursin theliverVitamin D is found in cells throughout the body vitamin D is essential to sustain health and it protectsagainst osteoporosis It is crucial to the human body™sphysiology in terms of muscular movement and neurological signal transmission and to the immune system indefense against invading pathogens []Although there are different methods and criteria fordefining vitamin D levels the criteria Holick proposed Correspondence drmuhanadkamalhotmailcom1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum SudanFull list of author information is available at the end of the have been widely accepted In this proposal vitamin Ddeficiency is defined as blood level of less than ngmlinsufficiency of vitamin D is defined as blood levels ranging between and ngml and sufficiency if greaterthan or equal to ngml [] About one billion peopleglobally have vitamin D deficiency and of the population has vitamin D insufficiency The majority ofaffected people with vitamin D deficiency are the elderlyobese patients nursing home residents and hospitalizedpatients Vitamin D deficiency arises from multiplecauses including inadequate dietary intake and inadequate exposure to sunlight Certain malabsorption syndromes such as celiac disease short bowel syndromegastric bypass some medications and cystic fibrosis mayalso lead to vitamin D deficiency []Vitamin D deficiency is now more prevalent than everand should be screened in highrisk populations Manyconflicting studies now show an association betweenvitamin D deficiency and cancer cardiovascular diseasediabetes autoimmune diseases and neuropsychiatricdisorders [ ] The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of two functional endoscopic sinusCase presentationThis was a case of a 26yearold Sudanese woman married who has a 3yearold boy This woman presentedto our ear nose and throat ENT department complaining of anosmia for the past years She had a history ofsurgeriesFESSs for nasal polyps the first one was years agoand the second one was years prior to presentationShe complained of being highly sensitive to different irritants including dust weather change perfumes andpetsShe also stated that she attended more than three different physicians due to generalized fatigue and gettingtired easily after simple daily activity in addition to sleeping for more than hours a dayShe attended an orthopedic clinic for unspecified lower back pain that was notrelated to any type of trauma or physical activity a lumbosacral magnetic resonance imaging MRI was done andrevealed no abnormal findingsShe mentioned that she isknown to be anxious most of the time and aggressive toward simple reactions from her family members She hadno psychiatric history and was not using any medicationsShe was not known to be diabetic or hypertensive orto have any chronic illnesses she was not on any regularmedication She is a housewife of high socioeconomicstatus she is well educated graduated from dentalschool with a bachelor™s degree but currently notemployed She has never consumed tobacco or alcoholshe practiced regular cardio exercisesOn examinationshe looked healthy well not pale or jaundiced Herpulse rate was 74minute and her blood pressure was Her body mass index BMI was All systems examinations were normal except for bilateral nasalpolyps Complete blood count CBC renal function testREF electrolyte liver function test LFT thyroid function test TFT urine analysis general urine test antinuclear antibody ANA and rheumatoid factor RFwere all normal An imaging profile included lumbosacral MRI a computed tomography CT scan of her sinuses and electrocardiogram ECG which were normalexcept for bilateral nasal polyps and severe sinusitis thatlooked allergic to fungi in natureShe underwent FESSsurgery to remove the polyps and clean out her sinusesup to weeks after surgery she used nasal steroidsmometasone furoate two times a day but hersymptoms regarding anosmia were not improved MRIof her brain and a CT scan of her sinuses were done andboth revealed normal features A vitamin D deficiencywas suggested and the laboratory results revealed a lowvitamin D level of ngml Treatment with vitamin Dsupplement was prescribed at international unitsIU weekly for weeks and then IU maintenancedose daily she was advised to take food rich in vitaminD and get exposed to sunlight for minutes three timesa week after the loading dose of supplement She was atregular followup for months at rates of weekly for thefirst month every weeks for the second month andmonthly for the rest of the followup period At eachvisit she was assessed with clinical history and examination It was noticed that the symptoms of tirednesssleeping anosmia and back pain were dramatically improving during that period At the months followupher blood level of vitamin D was normal she describedher condition as free from all symptoms and shereturned back to normal physical activityDiscussion and sThis was a nonclassical case of vitamin D deficiency ofa 26yearold woman who presented with chronic anosmia and recurrent nasal polyps She was diagnosed ashaving a case of vitamin D deficiency and respondedwell to vitamin D replacement therapy This case correlated an association between decreased levels of vitaminD and recurrent nasal polyps that led in time to chronicanosmia as a result of chronic high sensitivity reactionstriggered by our patient™s autoimmune system The literature links chronic rhinosinusitis with nasal polypsCRSwNP with asthma and allergic rhinitis but the cellular and molecular mechanisms that contribute to theclinical symptoms are not fully understood Sinonasalepithelial cell barrier defectsincreased exposure topathogenic and colonized bacteria and dysregulation ofthe host immune system are all thought to play prominent roles in disease pathogenesis []Despite all the previous surgical and medical interventions over the past years our patient™s condition did notimprove and she still complained of anosmia A study revealed that this patient was experiencing excessive allergicreactions that led to recurrent nasal polyps It is wellknown that classical clinical effects of vitamin D deficiencyare bones and musculoskeletalrelated disorders severallines of evidence demonstrate the effects of vitamin D onproinflammatory cytokines regulatory T cells and immune responses with a conflicting interpretation of theeffects of vitamin D on allergic diseases []The working diagnosis was suggested in relation tosome musculoskeletal symptoms and chronic fatigue especially when the imaging profile for her lower back andall routine investigations were normal It has been suggested that clinicians should routinely test for hypovitaminosis D in patients with musculoskeletal symptomssuch as bone pain myalgias and generalized weaknesswhich might be misdiagnosed asfibromyalgia andchronic fatigue [] The most common causes of anosmia were assessed as well and they were negative theseincluded sinonasal diseases post infectious disorder andposttraumatic disorder and congenital defects and disorders caused by neurodegenerative disease [] 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of Authors™ contributionsMI analyzed and interpreted the findings of the case report and was themajor contributor in writing the manuscript ME reviewed the report andadded valuable comments All authors read and approved the finalmanuscriptFundingNoneAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from Albasar Institutional Review BoardConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum Sudan 2Preventive Medicine and EpidemiologyAlneelain University Khartoum SudanReceived March Accepted July Thus blood level for vitamin D was requested and theresults were of low D levelIn the past history of the previous nasal polyps surgeries our patient noted that there was no anosmia and hermain complaints were classic complaints of sinusitis including sneezing nasal blockage and headache Soonafter surgery her symptoms improved except for theallergyrelated symptoms despite usage of inhaled steroids spray She stated that at the last time the presentation was different since it was only anosmia indicatingthat there was significant inflammation that affected thesmell receptors around the olfactory epithelium Afterthe last nasal polyps and sinuses drainage surgery thesymptoms related to allergic reactions including chronicsneezing did not improve for up to weeks and she wasstill suffering from hyposmia although that was a fairpostoperative period for recoveryThe symptoms of anosmia and sneezing and othersystematic symptoms gradually started to improve aftervitamin D supplements indicating that the main reasonbehind her symptoms was vitamin D deficiency She wasfollowed up for up to months after establishment ofvitamin D supplements and at the last followup she hada normal sense of smell and she was free from backpain fatigue and allergyrelated symptomsThis was a nonclassical presentation as our patientwas young and she did not have alkaline phosphatasecalcium and phosphorus abnormalities [] that are expected in cases of vitamin D deficiencyThis case revealed an association between decreasedlevels of vitamin D and recurrent nasal polyps that ledto anosmia as a result of hypersensitive reactions produced by the body™s systemsAlthough vitamin D deficiency is prevalent measurement of serum 25OHD level is expensive and universalscreening is not supported However vitamin D testingmay benefit those at risk for severe deficiencyIt is highly recommended to consider vitamin D deficiency among all patients with unspecified symptoms orin cases of nondiagnosed disorder regardless of the presenting complaintIn there is an association between vitaminD deficiency and recurrent allergic nasal conditionsAbbreviationsCRSwNP Chronic rhinosinusitis with nasal polyps CT Computedtomography ECG Electrocardiogram ENT Ear nose and throatFESS Functional endoscopic sinus surgery BMI Body mass indexCBC Complete blood count RFT Renal function test LFT Liver function testTFT Thyroid function test ANA Antinuclear antibody RF Rheumatoid factorIU International unitReferencesNational Institutes of Health Vitamin D fact sheet for health professionals httpsodsodnihgovfactsheetsVitaminDHealthProfessionalen2Accessed Apr National Institutes for Health NIH Vitamin D fact sheet for consumers httpsodsodnihgovfactsheetsVitaminDConsumer Accessed Dec Kuriacose R Olive KE Vitamin D insufficiencydeficiency managementSouth Med J “ httpsdoi101097SMJSizar O Khare S Givler A Vitamin D deficiency Treasure Island Stat PearlsPublishing httpswwwncbinlmnihgovbooksNBK532266 PMID Accessed Dec Wlliam B Fatme A Meis M Targeted 25hydroxyvitamin D concentrationmeasurements and vitamin D3 supplementation can have important patientand public health benefits Eur J Clin Nutr “ httpsdoi101038s4143002005640Hanmin W Weiwen C Dongqing L Xiaoe Y Xiaode Z Nancy O et alVitamin D and chronic diseases Aging Dis “ httpsdoi1014336AD20161021 Whitney W Ropert P Robert C Chronic rhinosinusitis with nasal polyps JAllergy Clin Immunol Pract “ httpsdoi101016jjaipThacher TD Clarke BL Vitamin D insufficiency Mayo Clin Proc “ httpsdoi104065mcp20100567Kennel KA Drake MT Hurley DL Vitamin D deficiency in adults when totest and how to treat Mayo Clin Proc “ httpsdoi104065mcp20100138Sanne B Elbrich M Duncan B Antje W Veronika S Joel D et al Anosmia aclinical review Chem Senses “ httpsdoi101093chemsebjx025Shikino K Ikusaka M Yamashita T Vitamin Ddeficient osteomalacia due toexcessive selfrestrictions for atopic dermatitis BMJ Case Rep httpsdoi101136bcr2014204558AcknowledgementsNot applicablePublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Understanding of the RelevantRole of LINE1 Retrotransposition inHuman Disease and ImmuneModulationXiao Zhang12 Rui Zhang12 and Jinpu Yu12 Cancer Molecular Diagnostics Core Tianjin Medical University Cancer Institute Hospital National Clinical ResearchCenter of Caner Key Laboratory of Cancer Prevention and Therapy Key Laboratory of Cancer Immunology and BiotherapyTianjin China Tianjin™s Clinical Research Center for Cancer Tianjin ChinaLong interspersed nuclear element1 LINE1 retrotransposition is a major hallmark ofcancer accompanied by global chromosomal instability genomic instability and geneticheterogeneity and has become one indicator for the occurrence development andpoor prognosis of many diseases LINE1 also modulates the immune system andaffects the immune microenvironment in a variety of ways Aberrant expression of LINE retrotransposon can provide strong stimuli for an innate immune response activatethe immune system and induce autoimmunity and ‚ammation Therefore inhibitionthe activity of LINE1 has become a potential treatment strategy for various diseasesIn this review we discussed the components and regulatory mechanisms involved withLINE1 its correlations with disease and immunity and multiple inhibitors of LINE1providing a new understanding of LINE1Keywords retrotransposons LINE1 regulatory mechanisms cancer immune inhibitorINTRODUCTIONLong interspersed nuclear elements LINEs are the only autonomous and active retrotransposonswhich include LINE1 LINE2 and LINE3 Cordaux and Batzer de Koning Also “ of LINE2 and LINE3 sequences in the human genome are as a truncated molecularfossil Doxiadis Ardeljan LINE1 retrotransposons are one of the mostabundant and eï¬ective classes of mobile DNAs that account for of the human genomeLander Hancks and Kazazian Fulllength LINE1 is “ kb and containsa 5cid48untranslated region 5cid48UTR Swergold two reading frames ORF1 and ORF2and a 3cid48UTR punctuated with a polyA tract Babushok and Kazazian Beck Denli revealed a new reading frame ORF0 It is located in the 5cid48UTR of theLINE1 transcript and on the strand opposite of the ORF1 and ORF2 structural genes Antisensepromotor ASP can initiate fusion transcripts and regulate ORF0 to enhance LINE1 mobilityRomanGomez Weber Criscione Both ORFs are required for LINE1 retrotransposition process ORF1 encodes an RNAbindingprotein named ORF1P that has nucleic acid chaperone activity and ORF2 encodes a proteinnamed ORF2P that has endonuclease and reversetranscriptase activities Mathias Feng The first step occurs when RNA polymerase II binds to the 5cid48UTR promoterregion of LINE1 and mediates the transcription of fulllength mRNA of LINE1 Lavie The LINE1 mRNA is exported to the cytoplasm where ORF1 and ORF2 are translatedand combined to form a ribonucleoprotein RNP p The RNP is then incorporated intoEdited byTrygve TollefsbolThe University of Alabamaat Birmingham United StatesReviewed bySandra Rose RichardsonThe University of QueenslandAustraliaApiwat MutiranguraChulalongkorn University ThailandJohn LaCavaThe Rockefeller UniversityUnited StatesCorrespondenceJinpu YuyujinputjmuchcomSpecialty sectionThis was submitted toEpigenomics and Epigeneticsa section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted July Published August CitationZhang X Zhang R and Yu J New Understanding of theRelevant Role of LINE1Retrotransposition in Human Diseaseand Immune ModulationFront Cell Dev Biol 103389fcell202000657Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe nucleus and the ORF2P endonuclease in the RNP identifiesand cuts specific sequences on the bottom DNA strand atthe consensus site 3cid48ˆ’AATTTTˆ’5cid48 Subsequently the free 3cid48hydroxyl generated at the fracture is utilized by the ORF2P andLINE1 mRNA in the RNP is used as the template for reversetranscription to produce the complementary DNA of the LINE gene Wei Hancks and Kazazian Wang andJordan The distribution of LINE1 in the human genomeis selective LINE1 endonuclease activity and DNA replicationdetermine LINE1 insertion preference Flasch Forexample LINE1 preferentially inserts into nucleosomedepletedDNA primarily as a result of its ATrich sequences Sultana The direction of the DNA replication fork aï¬ects LINE insertion preference because the cleaved strand is usually thelagging strand templateLINE1 elements play a crucial role in the course of speciesformation and evolution On one hand derepressed LINE1functions as a driver of many diseases and even a diagnosticmarker for some diseases Pedersen and Zisoulis On theother it can aï¬ect the developmental processes and ‚uencethe behavior by generating multiple gene products and causingvariable deleterious eï¬ects on the structure of the host genomethrough new insertions deletions and recombinations GarciaPerez LINE1 RNA and protein overexpression isrelated to apoptosis DNA damage and repair cellular plasticityand stress responses and can even promote tumor progressionMorrish Belgnaoui SinibaldiVallebona DNA damage caused by genomewide or intersperse repetitive sequences hypomethylation caninduce ‚ammatory microenvironment Lindqvist Teerawattanapong Here we reviewed the correlationbetween LINE1 and disease as well as immune systemmeanwhile conducted a new exploration in LINE1 inhibitors bycombining its regulation mechanismsFigure shows the relative positions of the 5cid48 untranslatedregion 5cid48UTR the reading frames ORF0 ORF1 andORF2 the 3cid48 untranslated region 3cid48UTR and the Poly A tailORF2 encodes endonuclease EN reverse transcriptase RTand cysteinerich domain C Fulllength LINE1 mRNA wasgenerated using the sense promoter at 5cid48UTR The LINE1mRNA is exported to the cytoplasm where ORF1 and ORF2are translated and combined to form a ribonucleoprotein RNPp The RNP is then incorporated into the nucleus andthe ORF2P endonuclease in the RNP identifies and cuts specificsequences on the bottom DNA strand at the consensus site3cid48ˆ’AATTTTˆ’5cid48 Subsequently the free 3cid48 hydroxyl generatedat the fracture is utilized by the ORF2P and LINE1 mRNA in theRNP is used as the template for reverse transcription to producethe complementary DNA of the LINE1 gene Richardson Kazazian and Moran LINE1 AND DISEASELINE1 and CancerWhen LINE1 retrotransposition is out of control it can lead todiseases More than s focusing on LINE1 and cancerare available in the PubMed archive Rodic theglobal hypomethylation ofLINE1 Hypomethylation and Cancergenome promotesThechromosomalinstability genomic instability and geneticheterogeneity because specific changes in DNA methylation canaï¬ect a variety of genome sequences especially the intergenicand intronic regions of the DNA resulting in chromosomeinstability and mutations Wilson LINE1 promoterhypomethylation is a biomarkerfor genomewide DNAhypomethylation which is itself a major hallmark of cancerThayer first demonstrated the methylation statusof LINE1 in cancer cells Since then LINE1 hypomethylationof tumors has attracted widespread attention Thayer LINE1 hypomethylation was reported to be associatedwith poor survival in more than cases of gastric cancersuggesting its potential as a prognostic biomarker Shigaki This phenomenon was also subsequently foundin lung cancer liver cancer esophageal cancer prostate cancerand endometrial cancer Iwagami Kawano Lavasanifar Ogino analyzed colon cancer samples from two independent prospectivecohorts demonstrating a linear correlation between LINE hypomethylation and aggressive tumor behaviorIt hasbeen reported that global DNA hypomethylation promotesaggressive tumor behavior by amplifying oncogenes or throughabnormal expression of microRNAs Baba In esophageal cancer with high mortality and poor endoscopicscreening sensitivity LINE1 hypomethylation can serve as agood diagnostic biomarker thereby improving 5year survivalShah LINE1 hypomethylation can also be seenin some precancerous lesions For examplein colorectalcancer LINE1 hypomethylation had no significant diï¬erencebetween adenomas and cancerous tissues but it was significantlylower in adenomas than in normal tissues Dawwas Therefore LINE1 hypomethylation also can be used as an earlybiomarker for cancerHoweverthere was no significant diï¬erencein thehypomethylation of LINE1 between the blood samplesof patients with leukemia and those of normalsubjectsBarchitta LINE1 Integrations and CancerMany tumor tissues have been found to present a high levelof LINE1 activity that can rapidly increase their copy numberthrough the œcopyandpaste mechanism Dunaeva LINE1 can be used as cisregulatory elements to regulatethe expression of host genes Wanichnopparat Pancancer Analysis of Whole Genomes analysis of cancer genomes from histological subtypes revealed thataberrant LINE1 integrations could lead to gene rearrangementRodriguezMartin LINE1mediated rearrangementcan trigger oncogene amplification In breast cancer Morse andcolleagues first proposed that hypomethylation activates LINE which can utilize the target primed reverse transcriptionpathway to insert into the oncogene MYC causing tumorspecificrearrangement and amplification Morse LINE was found to induce the amplification of CCND1 oncogenein esophageal tumor by inducing the breakage“fusion“bridgeFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseaseFIGURE Structure of LINE1 and LINE1 retrotransposition cyclecycles RodriguezMartin LINE1 can mediatethe deletion of tumor suppressor genes It may be through Xinactivation mechanism that LINE1 mRNA forms facultativeheterochromatin in the inactivated region or LINE1 mRNAand premRNA form RISC complex to degrade complementarymRNA Allen Aporntewan In coloncancer Miki reported that LINE1 insertion disrupts thetumor suppressor gene APC which can lead to gene inactivationMiki In lung squamous cell carcinoma wefound that LINE1 insertion into tumor suppressor gene FGGYpromotes cell proliferation and invasion in vitro and facilitatestumorigenesis in vivo Zhang High Expression of ORF1 and ORF2 ofLINE1 and CancerThe activation of LINE1 increases the translation of ORF1 andORF2 which are not expressed in normal somatic tissues ORF1encodes an RNAbinding protein and high expression level ofORF1 was proved to be more common in most of the cancersand therefore as a diagnostic marker In breast cancer highexpression of nuclear ORF1 is associated with distant metastasisand poor prognosis Harris In highgrade ovariancarcinoma the ORF1 level was high and correlated to the lossof TP53 Rodic The expression of both the LINE1ORF1 and cMet protein was significantly increased and peakedin early stage in ovarian cancer suggesting that LINE1 ORF1significantly activates cMet Ko In tumor cellexperiments increased mRNA and protein expression of LINE1ORF1 can result in significant enhancement in cell proliferationand colony formation Tang It is worth noting thatthe expression of ORF1 was heterogeneous and had histologicalspecificity Cancers originating in the endometrium such asbiliary tract esophagus bladder head and neck lung and colonexhibit ORF1 overexpression whereas other cancers such asrenal liver and cervical cancer show little expression of ORF1Ardeljan Recent studies have shown that an ELISAmethod to measure ORF1 in serum can be better in prostatecancer detection Hosseinnejad ORF2 encodes a protein with reverse transcriptase andendonuclease activities High expression of endonuclease inducesdoublestrand DNA breakage that can aggravate DNA damagerepair and increase genomic instability Kines Reverse transcriptase activation can promote cell proliferationthe noncoding RNAand diï¬erentiation and also altertranscription spectrum and otherepigenetic phenotypesresulting in alterations in cell regulatory networkstumordevelopment and other important pathological processes Rodicand Burns Burns Christian ORF2 canexpress early in the tumorigenesis process as it can be detectedby a highly specific monoclonal antibody mAb chA1L1 in bothtransitional colon mucosa and prostate intraepithelial neoplasiasDe Luca However studies have shown that chA1L1 recognizes both ORF2p and the transcriptional regulatorBCLAF1 so it is not specific Briggs But recentlytumor proteome profiling studies based on mass spectrometryhave shown that ORF2p was difficult to be detected and afteraffinity capture of ORF1p ORF2p has not been detected in stemcell LINE1 proteome analysis Vuong Ardeljan Therefore the detection and application of ORF2 intumors are still worth exploringLINE1 and Metabolic DisordersNew research has shown that LINE1 is also associated with bloodsugar and lipid levels Turcot LINE1 methylation isassociated with type diabetes mellitus T2DM Studies showedthat compared with hypermethylation LINE1 hypomethylationwas associated with a higher risk of worsening metabolic statusindependent of other classic risk factors MartinNunez This discovery highlights the potential role for LINE1DNA methylation as a predictor of the risk of T2DM orFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseaseother related metabolic disorders LINE1 DNA methylation isassociated with increased LDL cholesterol and decreased HDLcholesterol levels and these metabolic changes increase the riskof cardiovascular disease Pearce LINE1 DNAmethylation is also associated with many bloodbased metabolicbiomarkers In fetal neural tissue with neural tube defects it wasfound that the low methylation level of LINE1 was associatedwith the significant reduction of vitamin B12 in maternal plasmaas well as lower folate levels and increased concentrations ofhomocysteine Wang Folic acid and other B vitaminsplay an important role in the biosynthesis of new purines andpyrimidines Therefore the methylation status of LINE1 can be apredictor of some metabolic diseases Current studies have shownthat LINE1 can also regulate metabolism by inserting metabolicgenes It was reported that LINE1 insertions in the FGGY genecan upregulate cytochrome P450 arachidonic acid metabolismand glycerolipid metabolism These metabolic disorders can leadto the occurrence of a variety of diseases and poor prognosisZhang LINE1 and Neurological DisordersLINE1 can aï¬ect the developing brain at diï¬erent stages ofhealth and disease Suarez Ataxia telangiectasiaAT is a progressive neurodegenerative disease caused byataxia telangiectasia mutated ATM gene mutation In researchers found that in nasopharyngeal carcinomas with ATMdeficiency LINE1 retrotransposition increased and ORF2 copynumber increased in AT neurons thus verifying the correlationbetween LINE1 retrotransposition and ATM deficiency Coufal High expression of LINE1 was found in Rettsyndrome caused by mutation of methyl CpG binding protein MeCP2 in the Xlinked gene which was caused by theinclusion of LINE1 5cid48UTR sequence in the MeCP2 targetleading to methylationdependent repression Muotri LINE1 is involved in the aging process In patientswith frontotemporal lobe degeneration LINE1 transcripts werefound to be elevated Li LINE1 hypomethylationhas been observed in most psychiatric studies Increased copynumbers of LINE1 as a result of LINE1 hypomethylation werealso found in patients with schizophrenia bipolar disorder andmajor depressive disorder Liu Li Thelink between LINE1 methylation levels and Alzheimer™s diseaseis still being studiedLINE1 and Genetic DisordersLINE1 is reported to be related to chromosome disordersThe first observation of LINE1 insertion was in whenKazazian observed a new exon of F8 LINE1 insertion inthe Xlinked gene which is a gene encoding coagulation factorVIII in a patient with hemophilia A Kazazian Then a LINE1 insertion was found in the CHM gene of apatient diagnosed with choroideremia The reverse integrationof a LINE1 element into exon resulted in aberrant splicingof the CHM mRNA van den Hurk FurthermoreLINE1 can also promote mobilization of other RNAs intrans Alu and SVA which can be transmobilized leading togene insertions Kemp and Longworth Retrotransposoninsertions were found to account for up to of all NF1mutations Wimmer Neurofibromatosis type I isan autosomal dominant disorder caused by NF1 gene mutationsMessiaen Alu insertion is located bp upstreamof NF1 exon causing the exon to skip and change the reading frame Payer and Burns Only two cases werethought to be a result of independent SVA insertion in SUZ12Paccompanied by 867kb and 1Mb deletions that encompassedthe NF1 gene Vogt In autosomal recessive geneticdisease such as Fanconi anemia caused by SLX4FANCP deficiencyand Aicardi“Goutieres syndrome AGS of threeprime repairexonuclease mutations LINE1 expression was upregulated andpro‚ammatory cytokines were produced through the cGAS“STING pathway Brégnard Suarez LINE1 AND IMMUNE REGULATION OFDISEASELINE1 and Autoimmune DiseaseHypomethylated and highly expressed LINE1 has been foundin autoimmune diseases such as systemic lupus erythematosusSLE Sjögren™s syndrome SS and psoriasis Schulz Yooyongsatit Mavragani LINE1 RNAis characterized by viral RNA and exists as RNP ps whichcan be recognized by RNA sensors and activate innate immuneresponses Mavragani Cell studies demonstrated thatLINE1 activates the production of IFNβ by RNA pathway Zhao When LINE1 retrotransposition was inhibited byRT inhibitors significant reductions were observed in IFNαIFNβ and IFNγ mRNA levels Brégnard LINE1transcripts and p40 protein a ˆ’kDa RNA binding proteinthat LINE1 encodes have been detected in SLE and SS patientsIt has been demonstrated that LINE1 can induce the productionof IFNI in vitro by TLRdependent and TLRindependentpathways Mavragani In MRL autoimmunelymphoproliferative syndrome LINE1 ORF2 encoding an RTand its products are associated with an MHC class I molecule onthe cell membrane Benihoud In Fanconi anemia andAGS LINE1 was found to be associated with the activation of theautoimmune system LINE1 also regulates immunity by actingas a cisregulatory element through the mechanism of LINE1mRNA and premRNA forming RISC complex to degrade thecomplementary mRNA Wanichnopparat LINE1 and Tumor ImmunityIn TCGA cancer samples the scientists measured thetranscriptional activity of pathways and found that of immune pathways were significantly negatively correlatedwith LINE1 Jung LINE1 is inversely correlatedwith the expression of immunologic response genes Less LINE activity was found in tumors with high immune activityIn esophageal cancer tissues scientists found that the LINE1methylation level in tumors was significantly positively associatedwith the peritumoral lymphocytic reaction Kosumi The activities of regulatory T cells and PD1 signaling as reportedin cancer immune evasion and chronic ‚ammatory conditionsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasealso have negative correlations with LINE1 It is reported that thenegative correlation between LINE1 and immune activity maybe caused by the destruction of LINE1 inhibition but the specificmechanism is still unclear LINE1 may also mediate immunetolerance which may change from immune stimulation mode toimmunosuppression mode through continuous IFN signaling ordirectly aï¬ect lymphocyte signalingtumorLINE1 and MetabolismInducedImmunityLINE1 is also associated with blood sugar and lipid levelsAbnormal glucose and lipid metabolism can lead to metabolicreprogramming in tumor cells The most classic metabolismofis Warburg eï¬ect where a large amount ofglucose is absorbed to fulfillthe need for proliferationand produce lactic acid Lunt and Vander Heiden The acidic microenvironment caused by lactic acid leads toimpaired Tcell activation and proliferation prevents NK cellactivation stabilizes HIF1α to stimulate the polarization of anti‚ammatory M2 macrophages and inhibits the production ofIFNγ in tumorltrating T cells Husain Colegio Brand Abnormal lipid metabolism intumor cells also can lead to local immunosuppression in themicroenvironment Hao LINE1 can aï¬ect localimmune homeostasis by inserting elements into metabolismrelated genes FGGY is known to encode a protein thatphosphorylates carbohydrates and is associated with obesity andsporadic amyotrophic lateral sclerosis Zhang LINE retrotransposons suppress FGGY leading to lipid metabolismdisturbance and dietinduced obesity in mice Taylor Lung squamous cell carcinoma patients with L1FGGYtissue have a poor prognosis have low levels of CD3 Tcells and have high levels of CD68 macrophages and CD33myeloidderived cells Zhang L1FGGY alsoregulates the abnormal transcription of cytokines related to theimmunosuppressive micromilieuLINE1 INHIBITIONThe correlation between LINE1 and disease as well as immunitywas analyzed Figure The life cycle of LINE1 providesa plethora of ways to target and inhibit LINE1 expressionBanuelosSanchez The inhibition of LINE1 hasbecome a treatment strategy for some diseasesTargeting LINE1 Methylationa CpGFulllength LINE1 transcription is driven bydinucleotiderich internal promoter Hypomethylation of LINE causes the activation of LINE1 which causes retroelementtransposition and chromosomal alteration Saito The hypomethylation of LINE1 has become an important factorin the occurrence and development of diseases so maintainingthe state of LINE1 methylation has become a key methodfor the treatment of diseases Soy isoflavone supplementationcan regulate the level of LINE1 methylation in head and necksquamous cell carcinoma HNSCC In a clinical trial of patients with HNSCC who took a soy isoflavone supplement mgday orally for weeks before surgery a positivecorrelation was found between LINE1 methylation level anddaily isoflavone intake Rozek Some cellbasedstudies and clinical data have shown that LINE1 dysregulationis associated with tumor drug resistance Zhu Lavasanifar It was found in breast cancer cellstreated with paclitaxel that DNMT3a a member of the DNAmethyltransferase family could enhance the methylation level inthe gene by binding to the inner region of the LINE1 gene andthen upregulate the expression level of LINE1 Downregulatingthe expression of DNMT3a can eï¬ectively inhibit the expressionof LINE1 Wang LINE1 retrotransposon silencedalso through histone modifications Histone demethylaseKDM4B may enhance the LINE1 retrotransposition efficacywhereas depletion of KDM4B reduced itin breast cancerXiang Elevated LINE1 expression was found inPC9 drugtolerant persister DTP cancer cells treated withthe EGFR inhibitor erlotinib HDAC inhibitors can suppressLINE1 in DTP cancer cells Guler Currently DNAmethyltransferase inhibitors and histone deacetylase inhibitorshave entered clinical trials Gaillard Targeting RT ActivityLINE1 elements harbor ORF1 and ORF2 which has reversetranscriptase RT activity and RT inhibition may be anovel noncytotoxic anticancer therapy strategy Sciamanna RT is a key player in retrotransposition andfunctions by transcribing LINE1 mRNA or other RNAs toFIGURE The relationship between LINE1 and diseases and their regulatory mechanismsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseasecDNA at the integration sites Khalid Specificreverse transcription inhibitorsincluding nevirapine NVRand efavirenz EFV which target the HIV1encoded RT andare currently used in AIDS therapy reduce cell proliferationand promotes diï¬erentiation of a variety of cancer cell linesof unrelated histological origin Mangiacasale Landriscina Sciamanna In vivoassays using murine models inoculated with various humancancer cell lines revealed that daily treatment of animals withEFV significantly delayed the progression of tumors Oricchio NVR and EFV dramatically countered L1FGGYabundanceinhibited tumor growth attenuated metabolismdysfunction and improved the local immune evasion in lungsquamous cell carcinomas Zhang EFV hasrecently undergone a phase II clinical trial in patients withmetastatic prostate cancer Houédé Another RTinhibitor F2DABOs has shown antiproliferative activity innude mice helping to promote cell diï¬erentiation and inhibittumor growth Sbardella Later the nucleosidereverse transcription inhibitor abacavir was also shown to inhibitcell growth migration and invasion Carlini Capsaicin is the main chemical component of Asiasari radixand Capsicum annuum as well as the major component of aChinese traditional herbal medicine Shoseiryuto Friedman Capsaicin suppresses LINE1 by inhibiting theRT activity of LINE1 ORF2P which is the LINE1encodedRT responsible for LINE1 activity Nishikawa A recent study revealed that azidothymidine AZT inhibitsthe RT activity of ORF2P in a fetal oocyte attrition modelExperiments showed that AZTtreated oocytes have a reductionof LINE1 ORF1 ssDNA compared with untreated oocytesTharp It is important to note that RT inhibitorsdo not eliminate the tumor but only control its progressionTherefore in addition to the antiAIDS drugs approved by theFDA the combination of Chinese and western medicine can beregarded as an emerging treatmentCombined ImmunotherapyRecent studies suggest that LINE1 hypomethylation may be apositive indicator of immunotherapy DNA methyltransferaseDNMT is an important epigenetic molecule that catalyzedDNA methylation and can induce the development of varioustumors Downregulating the expression of DNMT can eï¬ectivelyinhibitthe expression of LINE1 Wang SoDNA methyltransferase inhibitors DNMTis play an importantrole in the antitumor process DNMTI can improve tumorimmunogenicity promote NK cells and CD8 T cells toplay a cellmediated cytotoxic role and promote immuneresponse to participate in antigen commission by regulatingimmunosuppressive cells Chiappinelli DNMTican enhance the expression of cancertestis CT antigenmaking the tumor more susceptible to CT antigen vaccine Thecombination of decitabine a DNA methyltransferase inhibitorand cancertestiscancergermline antigen NYESO1 vaccinehas a good therapeutic eï¬ect in the primary treatment ofhuman recurrent epithelial ovarian cancer Odunsi A clinical trial has shown that combination therapywith carboplatin and antiprogrammed death1 has a goodtherapeutic eï¬ectin lung cancer because carboplatin caninduce LINE1 expression Langer ThereforeLINE1 can be used as a target of combined immunotherapyin tumor therapyOther InhibitorsRecently a number of other regulatory approaches have beenreported In somatic cells microRNAs miRNAs or miRs alsoregulate the activity of LINE1 Idica MiR128regulates LINE1 activity in somatic cells by targeting the nuclearimport factor transportin1 TNPO1 3cid48UTR which mediatesnuclear import and requires RanGTP for cargo delivery into thenucleus Twyï¬els MiR128 inhibits the expression ofTNPO1 mRNA and protein and TNPO1 deficiency suppressesLINE1 mobilization by inhibiting nuclear import of LINE“RNP Idica MiR128 also guides the miRNAinduced silencing complex to bind directly to a target siteresiding in the ORF2 RNA of LINE1 Hamdorf At present a novel target of miR128 has been identified asheterogeneous nuclear ribonucleoprotein A1 hnRNPA1 whichis required for LINE1 retrotransposition Goodier Fung MiR128 represses hnRNPA1 mRNA andprotein by targeting the CDS of hnRNPA1 which interactswith LINE1 ORF1p via RNA bridge to promote LINE1mobilization Goodier This interaction results intranslational repression of the LINE1 retrotransposition therebyreducing the risk of LINE1mediated mutagenesis Pedersenand Zisoulis Therefore microRNAs can be a target forLINE1 inhibitionAryl hydrocarbon receptor AHR is a ligandactivatedtranscription factor that activates LINE1 expression Teneng AHR is overexpressed in breast and thyroid cancerssuggesting that these tumors also overexpress LINE1 Powell Lai found that biseugenol a novel AHRinhibitor impeded cancer growth and inhibited EMT in gastriccancer cells Lai These findings suggest that targetingAHR with small molecule inhibitors may be a novel therapeuticapproach ORF1P phosphorylation by protein kinase A is alsorequired for LINE1 Kinase inhibitors specifically designed totarget LINE1 ORF1P phosphorylation may be associated withinhibition of LINE1 Bojang Therefore there isroom for drug development research focusing on targeting andinhibiting LINE1 ORF1P phosphorylationCONCLUSIONThe activation of LINE1 retrotransposon is associated with avariety of human diseases and is involved in the occurrenceand progression of disease through retrotranspositiondependentand retrotranspositionindependent mechanisms Currently ithas even become a marker of tumorigenesis and prognosis andis related to immune regulation The eï¬ective inhibition ofLINE1 activation has become a treatment for some diseasesThe inhibition of LINE1 in animal experiments can inhibitFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe occurrence and development of tumors so the clinicalapplication of LINE1 inhibitors is imminent In addition toexploring some known inhibitors other mechanisms of LINE inhibition should also be explored We summarized therelationship between LINE1 and diseaserelated immunity andproposed that LINE1 may aï¬ect the immune status of thebody by regulating metabolismleading to poor prognosisMetabolic substances can aï¬ect the immune microenvironmentfor examplelactic acid can lead to immunosuppressivemicroenvironment leading to poor prognosis of tumors Thedysregulation of LINE1 can lead to the disorder of glucoseand lipid metabolism and the inhibition of glucose and lipidmetabolism may reverse the disease progression caused byLINE1 Now the antitumor eï¬ect of regulating the body™smetabolism has entered clinical trials such as the significanteï¬ect of metformin in the treatment of tumors Therefore themetabolic status of diseases caused by LINE1 can be checkedMetabolic therapy combined with LINE1 inhibitors may inhibitthe progression of LINE1 and may improve the immunemicroenvironment to achieve the optimal therapeutic eï¬ectAUTHOR CONTRIBUTIONSXZ wrote the RZ and JY reviewed and revised the All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by the National Natural ScienceFoundation of China Grant Nos and National Science and Technology Support Program of ChinaGrant No 2018ZX09201015 and Project of Science andTechnology of Tianjin Grant No 18JCQNJC82700REFERENCESAllen E Horvath S Tong F Kraft P Spiteri E Riggs A D High concentrations of long interspersed nuclear element sequence distinguishmonoallelically expressed genes Proc Natl Acad Sci USA “ 101073pnas1737401100Aporntewan C Phokaew C Piriyapongsa J Ngamphiw C Ittiwut CTongsima S Hypomethylation of intragenic LINE1 repressestranscription in cancer cells through AGO2 PLoS One 6e17934 journalpone0017934Ardeljan D Taylor M S Ting D T and Burns K H The human longinterspersed element1 retrotransposon an emerging biomarker of neoplasiaClin Chem “ 101373clinchem2016257444Ardeljan D Wang X Oghbaie M Taylor M S Husband D DeshpandeV LINE1 ORF2p expression is nearly imperceptible in humancancers Mob DNA 101186s1310001901912Baba Y Watanabe M Murata A Shigaki H Miyake K Ishimoto T LINE1 hypomethylation DNA copy number alterations and CDK6amplification in esophageal squamous cell carcinoma Clin Cancer Res “ 10115810780432CCR131645Baba Y Yagi T Sawayama H Hiyoshi Y Ishimoto T Iwatsuki M Long interspersed element1 methylation level as a prognosticbiomarker in gastrointestinal cancers Digestion “ Babushok D V and Kazazian H H Jr Progress

Thyroid_Cancer

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

Thyroid_Cancer

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

structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These findings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to find newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodified leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant anti‚ammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] anti‚ammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was first measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their specified regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the specific enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose fifty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the first group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationfive mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a significant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was confirmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a flavoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosignificant change in MDA level as compared to normal nonirradiated mice Fig A significant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modifies ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were significantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a significant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisfinding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were significantly lower ascompared to vehicletreated irradiated ones but still significantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different from control group significantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB file 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have five subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferroni€™s post test significantly different from control group significantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test There were no significant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferroni€™s multiple comparison test significantly different fromcontrol group significantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 confirmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientific GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSO€‘d6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was refluxed for h The solid product formed wascollected by filtration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr ʋ cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSO€‘d6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h filtered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr ʋ cmarom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSO€‘d6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSO€‘d6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this figure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr ʋ cm arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr ʋ cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSO€‘d6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr ʋ cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSO€‘d6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSO€‘d6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr ʋ cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSO€‘d6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSO€‘d6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidifiedatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent flatbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The final DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespecific activity of NQO1 was determined using menadione as asubstrate as described [] Briefly the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t

Thyroid_Cancer

Computed Tomography Features andClinicopathological Characteristicsof Gastric Sarcomatoid CarcinomaYiyang Liu1 Pan Liang1 Kaixiang Feng2 Kuisheng Chen3 Songwei Yue1 Jiang Ji4Weiwei Li1 Xitong Zhao1 and Jianbo Gao1 Department of Radiology The First Affiliated Hospital of Zhengzhou University Zhengzhou China Departmentof Thyroid Surgery The First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of PathologyThe First Affiliated Hospital of Zhengzhou University Zhengzhou China Department of Radiology General Hospital NingxiaMedical University Yinchuan ChinaPurpose Gastric sarcomatoid carcinoma GSC is a very rare malignant tumor Thepurpose of this study is to describe the clinical computed tomography CT andpathologic features of GSC to increase awareness of this entityEdited byZiwen LiuPeking Union Medical CollegeHospital CAMS ChinaMethods The CT features and clinical data of five patients with pathologicallydocumented GSC were retrospectively analyzed and compared with the correspondingdata of gastric adenocarcinoma and lymphomaReviewed bySavio Gee BarretoMedanta The Medicity IndiaHaruhiko SugimuraHamamatsu University Schoolof Medicine JapanXiuying XiaoShanghai Jiao Tong University ChinaCorrespondenceJianbo GaofccyisunshinegszzueducnSpecialty sectionThis was submitted toGastrointestinal Cancersa section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu Y Liang P Feng K Chen KYue S Ji J Li W Zhao X and Gao J Computed TomographyFeatures and ClinicopathologicalCharacteristics of GastricSarcomatoid CarcinomaFront Oncol 103389fonc202001611Results Among the patients were male and was female The median agewas years Of the cases of GSC were in the gastric fundus and cardia was in the gastric body and was in the gastric fundus The gastric wall had localthickening in cases and mass formation in case with stenosis and deformationof the adjacent gastric cavity The longaxis diameter of the lesions ranged from to cm mean cm and was cm in cases and cm in case The tumorshowed predominantly inhomogeneous density with radiodensity values ranging from to HU In addition ulcers with an irregular base and slightly raised borders wereobserved in of cases After an injection of contrast material heterogeneous n or homogeneous n enhancement was observed After contrast medium injectionobvious enhancement was seen in cases and moderate enhancement was seen in cases the peak tumor signal was observed in the portal phase Two of the patientsdemonstrated evidence of lymph node involvement and in one patient the boundarybetween the lesion and the left lobe of the liver was unclear with low attenuation in theright lobe of the liver with circular enhancement The remaining two patients showed noevidence of metastasisConclusion Although GSC is extremely rare it should be considered in the differentialdiagnosis of gastric adenocarcinoma and lymphoma CT findings combined withpatient age and sex can provide support for the diagnosis of GSC However the finaldiagnosis must be confirmed with histopathologyKeywords sarcomatoid carcinoma stomach gastric cancer tomography Xray computed diagnosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alINTRODUCTIONSarcomatoid carcinomas SCs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology The features of this tumor were first described in by Snover SCs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands However these tumors are rarein the digestive tract especially in the stomach As of April there are only six cases of gastric sarcomatoid carcinomaGSC reported in the English medical literature These previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor Due to the more invasive nature and poorerprognosis of GSC than pure gastric adenocarcinoma GACand gastric lymphoma GL it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography CT characteristics of GSC The present studyanalyzed our experience in diagnosing five patients with GSCin terms of the imaging findings and clinical features To thebest of our knowledge our study represents the largest seriesof GSCs to dateIn addition due to the rarity of GSC the diï¬erential diagnosisbetween GSC and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of GSC from GAC and GLMATERIALS AND METHODSThe protocol was approved by the Medical Ethics Committeeof Zhengzhou UniversityInformed consent was obtainedfrom all patientsPatient SelectionFrom August to January we searched the pathologyrecords and the Picture Archiving and Communication SystemsPACS of our hospital The search terms included stomachand sarcomatoid carcinomas A total of five patients wereidentified as having SC and were enrolled in the present study Weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensCT EvaluationFive GSC patients underwent CT examinations The CTscans were acquired with a 64row multidetector deviceDiscoveryCT750HD GE Healthcare Waukesha WIUnited States Conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgmL GE Medical Systems mLkgand mLs through a dualhead pump injector MedradWarrendale PA United States The imaging parameters wereas follows tube voltage kV tube current mA fieldof view FOV mm matrix — mm and sectionthickness mm Finally a 20mL saline flush was performedat a rate of mLsGastric Sarcomatoid CarcinomaContrastenhanced CT scans were acquired with scanningdelays of s arterial phase AP and s portal venous phasePP after the iv injection of the contrast agent started The CTdose index volume for the three phases was mSvImage AnalysisTwo experienced radiologists and years of abdominal CTexperience performed a retrospective analysis of the CT imagesAll analyses were performed with an AW47 workstation GEHealthcare and the radiologists were blinded to the clinicalinformation of the patients The evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement The enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the APimage The degree of enhancement of the tumor was based ondynamic CT imaging using HU attenuation where œobviousenhancement was defined as HU œmoderate enhancementas HU and œmildly enhancement as HUThe GSCs were staged with the Union for InternationalCancer Control UICC TNM staging standard All imagingfindings were compared with the postoperative pathologicalfindings The accuracy rate the number of CTs coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — Pathological EvaluationThree patients underwent gastrectomy and two underwentendoscopic biopsy The three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm The remaining specimen was a soft massmeasuring cm — cm — cm For biopsy multiple sampleswere acquired and the diameter of each sample was cmAccording to the relevant literature the diagnostic criteria fSC were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue In addition if biopsywas performed the sarcomatoid component can be seen in everysample Furthermore sarcomatoid regions express epithelialmarkers such as CK or EMAThe specimens were fully stretched fixed and soaked in formaldehyde solution for h All biopsy specimenswere analyzed The specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin HE staining Immunohistochemicalstaining was performed using a Roche BenchMark XT automaticimmunohistochemical detector The antibodies used in thisstudy included AE1AE3 CKL CK818 epithelial membraneantigen EMA vimentin P40 P63 and antigen KI67 Ki67All antibodies listed above were purchased from DAKO DakoGlostrup DenmarkFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Comparison between GSC and GAC GLAge median age rangeMain symptomsEpigastric discomfortpainIntermittent vomitingAcute hematemesisBloody stoolDysphagiaLocationCardia and FundusBodyAntrumThe longaxis diameter median size rangeShapeFocal thickeningDiffuse thickeningMassSerosal surfacebare areaClearUnclearUlcersYesNoDensity characteristicsHeterogeneousHomogeneousEnhancement patterHeterogeneousHomogeneousLymph node involvementYesNoLiver involvementYesNoTherapyResectionChemotherapyResection and ChemotherapyNeoadjuvant chemotherapyRadiation therapyGSCGACGL “ years “ years “ years “ cm “ cm “ cm Comprehensive Comparative AnalysisEach patient with GSC was matched by age ± years year ofdiagnosis and sex to four patients with GAC GL patients witheach disease were retrieved from PACS Patients with GSC werecompared with those with GAC GL in terms of demographicclinical and CT characteristics Table RESULTSPatient CharacteristicsThe patients included four men and one woman ranging inage from to years with a median age of years Theclinical and CT features of these patients are summarized inTables All patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingThe other presenting symptoms included hematemesis or weightloss Three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery And two patients chose to deny treatment Inaddition we also reviewed the upper gastrointestinal radiographyresults Figure The laboratory findings revealed that patient was positivefor tumor abnormal protein TAP and patient was positivefor carbohydrate antigen CA125 Before treatmenthemoglobin and erythrocyte count decreased in three patientsFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and pathological factors of the five GSC patientsCaseSexAge yearsComplaintLocationMaximumdiameterTumor markercmAnemiaTherapyMetastasisMMFMMSuddenhematemesisEpigastricdiscomfortIntermittentvomitingEpigastric painEpigastric painEpigastric painLesser curvatureRemnant stomachCardia andFundusCardia and FundusFundusCardia and FundusNormalTAP CA125 NormalNA““RRnNoneNoneRC“““œ yespresentpositive œ“ noabsentnegative F female M male age in years R Radical gastrectomy Rn Remnant gastrectomy C chemotherapyNA not availableTABLE Computed tomography features of the five GSC patientsCaseGross features of the tumorUlcersGrowth modeDensity characteristicsEnhancement patterFocal thickeningFocal thickeningMassFocal thickeningFocal thickening“IntracavityIntracavityIntracavityIntracavityIntracavityHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousHeterogeneousHeterogeneousHomogeneousHeterogeneousHeterogeneousMarginUnclearUnclearUnclearClearUnclearœ yespresentpositive œ“ noabsentnegativeFIGURE Characteristics of Xray examinations of a 65yearold male patient with GSC AB Reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines The surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and Pathological FeaturesMicropathologically the gastric tumor cells showed infiltrativegrowth The cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics Microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged Mitotic figures were visibleFigures 2AB On immunohistochemical examination thetumor cells showed positive staining for AE1AE3 CKLCK818 EMA P40 vimentin The Ki67 index was higher than Figures 2C“I All five tumors were diagnosed as GSCIn addition the sarcomatoid component showed spindle cellsarcomatoid morphologyCT FindingsOf the cases of GSC were in the gastric fundus and cardiaFigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach The CT manifestations of this tumor included localthickening n mass formation n The longaxisFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Histological and immunohistochemical features of GSC AB Hematoxylineosin HE staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei Mitotic figures visible Tumor cells showed infiltrative growth Cells were stained withhematoxylin and eosin stain magnification A — B — By immunohistochemistry the tumor cells were positive for AE1AE3 C CKL D CK818 E EMAF P40 G and vimentin H Moreover of them were positive for Ki67 I The final diagnosis was SC [magnification C“I —]diameter of the lesions ranged from to cm mean size cm In addition ulcers with an irregular base and slightlyraised borders were observed in of cases Among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaAmong the two patients with biopsyproven GSC one patientexhibited tumor invasion of the gastric bare area The majorchanges in the CT imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat Initially the CT findings were interpreted as GAC in fourcases and GL in The tumor showed predominantly inhomogeneous densityand the radiodensity values were “ HU in the noncontrastphase Heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement The radiodensity values on the APimages ranged from to HU and to HU in thevenous phase After contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase One of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement The remaining patientshowed noevidence of metastasis Among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on CTlobe ofCT Staging Versus Pathological Stagingof GSCNone of the GSCs were staged as T1T2 by CT or pathologyThe accuracy of CT staging T3 and T4 GSC was and respectively The overall diagnostic accuracy of CTfor determining the T stage of GSC was None of the GSCs were staged as N2N3 by CT or pathologyThe accuracy of CT in staging N3 and N4 GSC was and respectively The overall diagnostic accuracy of CT fordetermining the N stage of GSC was The comparison of TN staging based on CT and pathology isshown in Table Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaFIGURE Sarcomatoid carcinoma of the stomach in 62yearold women A Unenhanced CT image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region B“D Contrastenhanced CT image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase In perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenB Arterial phase of contrast enhancement image C Portal phase of contrast enhancement image D Portal phase of contrast enhancement coronal imageDISCUSSIONTABLE CT and pathological TN staging for comparisonSarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells However the spindle cells of SCsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation Moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved In the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofSarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur In the digestive system the incidencesof SCs in the esophagus and liver are relatively high but SCsare exceedingly rare in the stomach we could find only sixprevious reports in the English literature Table Between and patients with SC confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach The average age of the reported patientswas years range “ and that in our series was years range “ A previous study reported that the sexCaseNO NO NO NO NO CTT4aN0T3N0T3N1T3N0T4aN1NA not available T tumor N nodePathological stageT4aN1T3N0NANAT4aN0distribution of male to female GSC patients was and thecorresponding proportion in our patients was “ Ithas been noticed that SCs are more common in male patientsand sex is a probable risk factor GSC patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation Due to thickening of the gastricwall pain or discomfort in the upper abdomen is common Thesymptoms can last from a few days to several years withoutobvious specificityIn the present study of the cases of GSC were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach Four cases of GSC in the present study hadFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid CarcinomaTABLE Clinical and imaging features of six previously reported cases of GCSCaseGenderAgeyearsLocationSize cmShapeUlcersEnhanceappearanceRecurrenceMetastasisTherapyPrognosis MMFFMMRemnant stomachGreater curvatureLesser curvatureGastroesophageal junctionRemnant stomachDistal stomachPolypoidPolypoidPolypoidUlceratedPolypoidMass“““NENENENENEHyperNE“““““NoneSurgerySurgerySurgeryEndoscopySurgeryNA Mo D Mo D Mo D Mo D Mo DThe patient succumbed to heart failure before the surgical treatment An autopsy was performed œ yespresentpositive œ“ noabsentnegative Hyper hyperdenseNE no evaluate Mo Month D Dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmThe location distribution and longaxis diameters of the GSCs inour patients were similar to those in previous reports “in the actual diagnosis processThe diagnosis of SC has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma Carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsThe sarcoma components show typical specialized diï¬erentiation Howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases Therefore the understanding ofthese tumors has been hampered Nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective The CT finding SC in the stomach have notbeen previously scientific reported or even detailed descriptionThere are only four simple descriptions ChunChao reported that a patient with a giant SC presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa The hepatic flexure of the colon andgallbladder were also involved on CT Contrastenhanced CTimages showed obvious enhancement of the two lesions Sato reported a patient with SC of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter The other two reports only describeda soft tissue mass or a large tumor in the dilated stomach On the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than SC Gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ Tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion Enhanced abdominalCT showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes Yoshiyuki reported a 70yearoldJapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andCT revealed an ulcer on the lesion The contrastenhanced CTimages showed heterogeneous enhancement of the mass Thefinal pathological diagnosis was gastric carcinosarcoma Inthe present study we found that GSC showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers The site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach The signal of the tumor was homogeneousor heterogeneous on plain CT scans After contrast mediuminjection of tumors demonstrated heterogeneousenhancement on AP images due to cystic areas or necrosis inthe lesions In this study the enhancement degree of all tumorsreached a peak in the PP after contrast enhancement For thesetumors the enhancement degree in the delayed phase wasnot significantly reduced The overall enhancement mode wasdelayed enhancement In addition CT showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue These findingsreflect the metastatic and highly invasive characteristics of GSCOverall CT and contrastenhanced CT can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of GSCTomographic diagnosis of GSC has not been attemptedbecause of the rarity of this entity According to the findingsof our study GSC needs to be diï¬erentiated from GAC andGL on CT Adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach The incidenceof GAC is high in men and the median patient age is years The most common CT signs of GAC are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer Tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent The peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer Primary GL accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus The incidenceof GL is high among males with a median patient age of yearsThe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue The most common CT manifestations ofGL are diï¬use thickening of the gastric wall or a homogeneousFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall For GL becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on CT GL originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage After contrast medium injection most GL showedhomogeneous and slight enhancement in the delayed phase Lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis The CT findings may only reflect features of GSC but cannotaccurately diagnose GSC let alone explore the origin of thesarcomatous portion Immunohistochemistry IHC also failedto conclusively establish the origin of GSC Rodrigues usedfluorescence in situ hybridization FISH to confirm that SC andadenocarcinoma have a common origin that is the epithelium while primary GL originated from gastric submucosallymphoid tissueThe main treatment for localized lymphomas is eradicationof Helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone Surgery is the only treatment option for patients with GACAdjuvant chemotherapy and chemoradiotherapy are also oftenused Targeted therapy isin the exploration stage However there are currently no specific National ComprehensiveCancer Network guidelines for the treatment of only GSCbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method For examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for GSC and the efficacy ofchemotherapy remain controversial Domblides firstevaluated the efficacy of immune checkpoint inhibitors ICIs forSC and found that lung SC patients exhibited high response ratesand prolonged overall survival OS with ICIs This studyprovides a new idea for the treatment of GSCBecause GL tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of GAC Previous literature has found that SCin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ Similarly GSCpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis All these clinical manifestations suggest that GSChas a poorer prognosis than GAC and GL In additionGSC can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver This conclusion is consistent with ourresearch resultsCONCLUSIONThe incidence rate of GSC is extremely low so clinicians andradiologists are not familiar with the features of this tumorBased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that GSC is morecommon in men who are approximately years old and isoften accompanied by ulcers The disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced CTimages These characteristics can provide a reference for furtherresearch on GSCs in the future However an accurate diagnosisof GSC depends on the combination of clinical imaging andhistopathological features Due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with CT are essentialDATA AVAILABILITY STATEMENTThe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Medical Ethical Committee of the ZhengzhouUniversity The patientsparticipants provided their writteninformed consent to participate in this study Written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this AUTHOR CONTRIBUTIONSYL manuscript preparationliterature research and dataanalysis PL literature research and data analysis KF manuscriptreview and data collection KC guidance of pathologicalknowledge SY guidance of imaging knowledge JJ imaging datacollection and analysis WL and XZ manuscript editing JGstudy conception and design manuscript review and guarantor ofintegrity of the entire study All authors have read and approvedthe final manuscriptFUNDINGThis work was supported by the National Natural and ScienceFund of China No REFERENCES Zhu CC Li MR Lin TL Zhao G Sarcomatoid carcinoma of the stomach acase report and literature review Oncol Lett “ ol20153460 Snover DC Levine GD RosaiJ Thymic carcinoma Five distinctivehistological variants Am J Surg Pathol “ Zhou DK Gao BQ Zhang W Qian XH Ying LX Wang WL Sarcomatoidcarcinoma of the pancreas a case report World J Clin Cases “ 1012998wjccv7i2236 Xie Y Xiang Y Zhang D Yao X Sheng J Yang Y Sarcomatoidthe “ 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature Mol Med RepaFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alGastric Sarcomatoid Carcinoma Sato A Oki E Kohso H Endo Y Uchida H Hiroshige S Sarcomatoidcarcinoma of the remnant stomach report of a case Surg Today “ 101007s0059501204027 Nakayama Y Murayama H Iwasaki H Iwanaga S Kikuchi M Ikeda S et alGastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation Pathol Int “ 101111j144018271997tb04540x RobeyCaï¬erty SS Grignon DJ Ro JY Cleary KR Ayala AG OrdonezNG Sarcomatoid carcinoma of the stomach A report of three caseswith immunohistochemical and ultrastructural observations Cancer “ 101002109701421990040165730co2n Ruess DA Kayser C Neubauer J FichtnerFeigl S Hopt UT Wittel UACarcinosarcoma of the pancreas case report with comprehensive literaturereview Pancreas “ 101097mpa0000000000000904 Fujiie M Yamamoto M Taguchi K Iwanaga A Ohgaki K Egashira A et alGastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review Surg Case Rep 101186s407920160176z Tanimura H Furuta M Carcinosarcoma of the stomach Am J Surg “ 101016000296106790325x Kitamura S Study on carcinosarcoma of stomach Gan “ Kumagai K Kawai K Kusano H Matsuo K Irie J Tsuchiyama H A caseof socalled carcinosarcoma of the stomach Gan No Rinsho “ Bekki T Fujikuni N Tanabe K Yonehara S Amano H Noriyuki T Thegastric carcinosarcoma with severe venous invasion a case report Surg CaseRep 101186s4079201804218 Ikeda Y Kosugi S Nishikura K Ohashi M Kanda T Kobayashi T Gastriccarcinosarcoma presenting as a huge epigastric mass Gastric Cancer “ 101007s1012000604054 Cid³n EU Cuenca IJ Gastric adenocarcinoma is computed tomography CTuseful in preoperative staging Clin Med Oncol “ cmos2641 Hallinan JT Venkatesh SK Gastric carcinoma imaging diagnosis stagingand assessment of treatment response Cancer Imaging “ Gossios K Katsimbri P Tsianos E CT features of gastric lymphoma EurRadiol “ 101007s003300050069 Rodrigues DN Hazell S Miranda S Crespo M Fisher C de Bono JS Sarcomatoid carcinoma of the prostate ERG fluorescence insituhybridization confirms epithelial origin Histopathology “ 101111his12493 Levine MS Rubesin SE PantongragBrown L Buck JL Herlinger H NonHodgkin™s lymphoma of the gastrointestinal tract radiographic findings AJRAm J Roentgenol “ 102214ajr16818976941 Russo AE Strong VE Gastric cancer etiology and management in Asia and theWest Annu Rev Med “ 101146annurevmed081117 Domblides C Leroy K Monnet I Mazi¨res J Barlesi F Gounant V et alEfficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma JThor Oncol “ 101016jjtho202001014 Niu X Sarcomatoid carcinoma in the parotid gland a review of years ofexperience Laryngoscope “ 101002lary27474 Li S Jiang L He Q Wei W Wang Y Zhang X The prognostic significa

Thyroid_Cancer

Human transcription factor and protein kinase gene fusions in human cancerKari Salokas Rigbe G Weldatsadik Markku VarjosaloOncogenic gene fusions are estimated to account for upto of cancer morbidity Recently sequencelevel studies have established oncofusions throughout all tissue types However the functional implications of the identified oncofusions have often not been investigated In this study identified oncofusions from a fusion detection approach DEEPEST were analyzed in detail Of the oncofusions we found almost are expected to produce functional proteins with features from both parent genes Kinases and transcription factors were the main gene families of the protein producing fusions considering their role as initiators actors and termination points of cellular signaling pathways we focused our indepth analyses on them Domain architecture of the fusions and their wildtype interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins To understand overall oncofusion effects we performed differential expression analysis using TCGA cancer project samples Results indicated oncofusionspecific alterations in gene expression levels and lower expression levels of components of key cellular pathways in particular signal transduction and transcription regulation the sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling possibly via acquiring novel functionsAt any given moment multitudes of molecular networks are activated in cells throughout the body An important feature of these networks is highly concerted regulation of key signaling and deviation from homeostasis can result in diseases such as cancer Cancer is a complex progressive multistep disorder which stems from mutations caused by genomic instability1 The accumulation of genetic and epigenetic abnormalities ultimately leads to the transformation of normal cells into malignant derivatives Two highly enriched gene groups being mutated in the majority of cancer types are protein kinases PKs and transcription factors TFs23 PKs mediate most signal transduction events in cells by phosphorylation of specific substrates thus modifying their activity cellular localization andor association with other proteins TFs are the œtransistors of the cellular signaling circuits controlling the transcriptional outcome of activated signaling by binding to regulative elements of their corresponding target genes and driving or suppressing their expression Therefore it is easy to understand why mutational deregulation of these two gene groups can have such an impact on tumorigenesisIn addition to harboring activating or inactivating somatic point mutations PKs and TFs account for a large fraction of all human fusion genes involved in cancer COSMIC Catalogue of Somatic Mutations in Cancer cancersangeruk4 and dbCRID Database of Chromosome Rearrangements in Disease5 Chromosomal translocations creating fusion genes are among the most common mutation class of known cancer genes and they have long been identified as driver mutations in certain types of cancer6 Recently oncogenic fusion genes hereafter oncofusions OFs have been found in many hematological and solid tumors demonstrating that translocations are a common cause of malignancy78 Fusion mutations occur when two different gene regions fuse together via translocation Examples of consequences of chromosomal fusion to protein structure range from missense mutations to expressionchange inducing promotergene “combinations to fully functional fusion proteins with neomorphic properties A classic example of gained functions is the breakpoint cluster regionAbelson tyrosineprotein kinase BCRABL1 translocation in chronic myeloid leukemia9 Alternatively a protooncogene is fused to a strong promoter and thereby the oncogenic function is upregulated due to the strong promoter of the upstream fusion partner This is common in lymphomas where oncogenes are juxtaposed to the promoters of the immunoglobulin genes10 and also in prostate cancer where ETS TF ERG is fused with TMPRSS2 regulatory Systems PathologyBiology Research Group Institute of Biotechnology HiLIFE University of Helsinki Helsinki Finland email markkuvarjosalohelsinkifiScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0csequences thus obtaining androgen receptor ARresponsive expression11 The current understanding favors the aberrant gene function model rather than promoterinduced overexpressionThe frequency of recurrent OFs varies depending on the specific type of cancer12“ but identified translocations are estimated to account for up to of cancer morbidity8 Recent fusion prioritization study found that inframe transcripts were the most powerful predictor of driver fusions16 confirming the intuition that inframe transcripts are crucial to function Notably breakpoints were also observed to preferentially avoid splitting of domains Together with frameshift conservation such trends could reflect a selection on fusion proteins to maintain protein stability and evade degradation pathways17Nextgeneration sequencing NGS of genomes and transcriptomes from primary human cancer cells is constantly revealing new gene fusions that are involved in driving tumorigenesis including examples found in colorectal carcinoma bladder carcinoma breast cancer and acute lymphoblastic leukemia ALL1518“ Furthermore NGS has provided enough detailed sequence information of the fusion breakpoints allowing a0us to initiate systemslevel research on human oncofusions As a result various algorithms have been developed to mine OFs from large cancer datasets such as TCGA However the concordance among the different algorithms is very low that metacaller approaches utilizing consensus calls have been employed21 which limit novel OF discoveries Recently a new statistical method DEEPEST22 was developed to overcome these limitations In this study oncofusions that involve PKs and TFs were selected from the data produced by DEEPEST applied to the TCGA datasetIn most cases it is not possible to draw definite s about the mechanisms or extent by which individual translocations contribute to cancer Predicting protein function from a sequence has proven an extremely difficult task With gene fusions the task is even more daunting However an unexpectedly large number of PKs and TFs have been found to be mutationally activated or have increased expression due to gene amplification or translocation in cancer6 The high number of PKs and TFs with relatively low individual mutational frequency suggests either that a large number of signaling pathways can contribute to cancer or that many PKs and TFs can regulate the same pathways when activated unphysiologically Some additional support for this hypothesis comes from the interconnectivity of the PKTFoncofusionsIn this study fusions predicted to produce inframe proteins were analyzed to understand the proteinlevel implications of fusion events The fusions were analyzed from the perspective of their domain architecture to understand likely modes of action of the novel proteins Furthermore known interactomes of the participating wild type proteins were used to determine possible mechanisms of action pathways of interest and possible treatment vectors for affecting as many different fusions as possible As a result multiple cellular signaling pathways were found to intersect with major subsets of these fusions and multiple individual key interactors such as NTRK1 with over and EGFR with over interacting fusions were identified as potential targets of interestMaterials and methodsfusion selection and annotation Fusions that involve protein kinase genes23 and transcription factors24 were selected from the fusions that were identified by applying DEEPEST to the whole TCGA dataset22 Of these were determined to be unique by considering Ensembl gene IDs biotypes chromosomal breakpoints AGFusion assigned fusion effects and resulting protein sequences AGFusion was used to annotate these gene fusions to the human genome assembly GRCh38 v89 from Ensembl For analysis involving gene pairs the pair entry was used in alphabetical order eg ERGTMPRSS2 instead of TMPRSS2ERG in all cases Fusions were considered protein coding if both genes contributed over amino acids to the productclinical data Clinical data for TCGA samples was obtained from the GDC data repository The data was matched to AGFusion output data based on TCGA barcode eg TCGAWBA80K using a custom inhouse python script Stage information from the clinical data was simplified where possible eg Stage IIA was changed to Stage II Entries such as Stage Stage X and III NOS were ignored Tissue entries were simplified from detailed ICDO topographical codes to more general eg C569 †’ C56 and mapped to names accordingly Chromosomal sequence information from GRCh38 v89 was used to categorize breakpoints into chromosomal interval groupsInteractor analysis Interactors for wild type proteins of all fusion partners were obtained from IMEx consortium25 and any interactions that were not confirmed to be physical by experimental methods were discarded Interactors were added to the interactor set from each fusion while leaving out the fusion pair genes themselves Annotations for interactors were obtained using Uniprot and Reactome From Reactome mappings to all levels of pathway hierarchy were used Dijkstra™s algorithm26 implemented with a custom python script was then used to establish shortest paths to Reactome root nodes for each network node A weight of was used for all network edgesDomain annotation For the protein producing fusions sequences of the protein products were produced using the AGFusion tools Duplicate fusions based on fusion genes and protein sequence were discarded Domains were taken from AGFusion output and mapped to protein sequence in the wild type protein The intactness of domains was then determined by matching the WT domain sequence to the predicted fusion protein sequence and only full length intact domains were picked for further study A domain was classified as PK or TFspecific if ‰¥ of all its occurrences were in PK or TF proteins respectivelyData visualization Data illustrations were made with CorelDRAW Excel and inhouse python scripts using Matplotlib and Seaborn Cytoscape27 was used for creating network figuresScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cDifferential expression analysis Gene expression quantification HTSeqcounts “files were downloaded from GDC data portal Samples where OFs with intact fulllength PK or TF domains were detected were grouped together based on fusion gene pairs The groups were then analyzed with DESeq228 using other samples with protein producing nonPKTF fusions as controls For each pair group differential expression analysis against an equal number of control samples picked from samples in which other proteinproducing fusions were found Analysis was repeated times for each fusion pair For the resulting significantly differentially expressed genes qvalue basemean and expected values were averaged across all runs and a fold change value calculated based on these GO annotations were then added from ensembl annotations and Reactome pathways from first mapping ensembl gene IDs to Uniprot via Ensembl BioMart and then to Reactome lowest level pathway terms via Reactome Zscore value for pathway level overunderexpression was calculated by a method used in GOplot29 ieby deducting the number of underexpressed genes from the number of overexpressed genes and dividing the result by the square root of the number of significantly changed genes FDR corrected p ‰¤ ResultsDetection of oncofusions from TCGA dataset reveals enrichment of PK and TF fusions In this study we focused on protein producing OF genes Translocation of chromosomal regions can result in either inframe or outofframe OFs Fig a01A To characterize the proteins produced by known OFs in the TCGA dataset which currently contains data from different cancer projects we launched an analysis to understand the potential functional space of the protein producing fusions Fig a01B and especially those that involve either a PK or a TF or both PKTF fusionsThe DEEPEST dataset included fusions detected from cancer samples Of these were unique fusions Fig a01C upper panel Among the unique OFs were predicted to retain frame and also produce potentially functional proteins where both genes contributed over inframe amino acids Fig a01B Supplementary table a0S1 The limit of amino acids was the length of the shortest nonrepeat domain present in the fused proteins Examining the resulting protein producing OF set we noticed an abundance of those involving PK or TF Indeed these fusions constituted protein producing OFs Fig a01C lower panel Generally the proportion of PKTF fusions a0was under except in the PKTF”fusion prone cancers acute myeloid leukemia cholangiocarcinoma thyroid carcinoma and thymoma The number of OFs per sample varied across cancer types The types most prone to protein producing fusions were sarcoma SARC with an average of protein producing fusions per sample esophageal carcinoma ESCA fusions uterine corpus endometrial carcinoma UCEC stomach adenocarcinoma STAD breast invasive carcinoma BRCA uterine carcinosarcoma UCS and ovarian serous cystadenocarcinoma OV Due to the prevalence of PK and TF genes in the fusions we next investigated if they are enriched in particular cancers While in most cancers PKTF fusions made up around “ of all protein producing OFs the percentage reached in acute myeloid leukemia LAML samples in thymoma THYM in thyroid carcinoma THCA and and in kidney renal papillary cell carcinoma KIRP and cholangiocarcinoma CHOL respectively Fig a01C Acute myeloid leukemia is well known as an OFprone cancer30 However aside from the four fusions detected between ABL1 and BCR the high percentage was mostly TFdriven with KMT2A RUNX1 and RARA being found in and fusions respectively This is in contrast to the peak in THCA which is driven by BRAF fusions fusions of RET of NTRK1 and of NTRK3 among other protein kinasesReading frame retention is common in pK and tf oncofusions The fusions consisted of unique gene pairs and individual genes of the pairs did not have any protein producing fusions The top protein producing fusion was RPS6KB1VMP1 with unique protein producing fusions in the dataset all the others having less than Fig a02A There were fusion gene pairs that were predicted to produce protein in at least fusions in fusions in and in fusion Out of the fusion gene pairs that produced or more unique proteins were PKTF fusionsTo better understand the behavior of prolific gene pairs we next mapped tissue annotations from TCGA to fusions of each gene pair based on barcodes from samples where a fusion of the gene pair was present In contrast to RPS6KB1VMP1 and ITGB6RBMS1 which were seen in samples from different cancers pairs were seen in samples of only one cancer type Out of these cancerspecific fusions were predicted to produce or more unique proteins with ERGTMPRSS2 predicted to produce different unique proteins supplementary table a0S2 PKTF fusions featured different PK or TF genes ERG being the most common TF and ERRB2 the most common PK supplementary table a0S3 Between and percent of oncofusions in each cancer project were unique highest being sarcoma with unique gene pair combinations and thyroid carcinoma the lowest with supplementary table a0S4 Protein producing fusions followed a similar theme unique protein producing fusions making up between and of all oncofusions in each given cancer project supplementary table a0S4We next looked in more detail what cancer stages PK and TF fusions were detected in The most prominent group was stage II breast invasive carcinoma which also had the most samples in the data set Fig a02B In total of the PK and TF fusions were found in stage I samples in stage II in stage III and in stage IV On average samples had PKTF fusions per sample However in some cancers PK or TF fusions are enriched towards the more severe stages Discounting stage groups with less than samples groups had more than fusions per sample ESCA stage III samples in particular had PKTF fusions per sample while STAD and BRCA stage IV samples had and respectively and STAD stage I had Supplementary table a0S5 The distribution of protein producing OFs mirrored that of PKTF fusions quite closely supplementary figure S1A In terms of chromosomal breakpoint locations those in the PKTF fusions varied compared Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cFusion gene Fusion gene PromoterProtein coding regionetis noisuFTranslocationChr Chr Chr Chr Inframe fusionOutofframe fusionTCGA DEEPEST dataset gene fusionsAGFusionEnsemblGRCh38TCGAclinical dataUniprotKinases Manning et al TFs Lambert et al et al Gene expressionquantification dataprotein producing uniqueoncofusions Protein codingProtein kinase or Transcription factoroncofusionsPfamdomainsIMEx consortium interactor databaseCOSMICcancer genecensusReactomepathwaysClinical overviewDomain analysisInteraction analysis Pathway analysisDifferential expressionanalysisSamplesOncofusions per sampleABCselpmas fo rebmuNCCAACLBACRBCSECLOHCDAOCCBLDACSEMBGCSNHHCKICRKIPRKILMALGGLCHILDAULCSULOSEMVODAAPGPCPDARPDAERCRASMCKSDATSTCGTACHTMYHTCECUSCUMVUnietorp ni FT ro KP fo noitroporPsnoisufocno gnicudorpelpmas rep snoisufocno fo rebmuNACCBLCABRCACESCCHOLCOADDLBCESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diffuselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinoma Uterine carcinosarcomaUCSUVM Uveal melanomaScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0c—‚Figure a0 Schematic illustration of the gene fusions workflow and the number of gene fusions in human cancer A Schematic description of gene fusions formation Fusions are formed mainly via balanced and unbalanced chromosomal rearrangements such as translocations deletions inversions and insertions This usually leads to formation of a fusion gene with the ² end of Gene and ² end of Gene If the fusion occurs between two protein coding genes depending on whether the reading frame is violated and where exactly the fusion occurs a fusion protein may be transcribed with features and domains from both partners Other possible outcomes include full or truncated ² gene under the control of the promoter of the ² gene B Workflow used in this study Analysis progressed from the total set of fusions discovered by the DEEPEST method22 and moved towards more specific kinase TF containing protein producing oncofusions We started with TCGA databased fusion set from Dehghannasiri et a0al for which we generated protein sequences with AGFusion Domains were added by matching sequence to Uniprot proteins annotated with Pfam domains after which nonunique entries were dropped Fusions were classified as protein producing if both gene fragments were predicted to produce AA of protein sequence From this set the two most prominent protein groups were protein kinases and transcription factors and thus we focused further analysis on the unique protein kinase or transcription factor containing fusions using the full protein producing fusion set for comparison Known interactions for wild type fusion proteins were obtained from IMEx consortium and used for estimating maximal foreseeable effect on signaling pathways from Reactome Finally TCGA gene expression quantification data was used to probe observable effects of kinaseTF fusions using other protein producing fusions as background C Top Breakdown of samples and fusion mutations by TCGA project Largest single contributor of samples with fusions was TCGA breast invasive carcinoma project BRCA which had the highest number of samples and identified fusion mutations Bottom Proportion of protein producing fusions that include PK or TF genesto all protein producing fusion mutations but the prominent role of PKTF fusions is illustrated by overlapping hotspots supplementary figure S2Intact inframe domains are commonly retained in OFs To understand the contribution of each OF to the overall development or survival of cancerous cells the functional consequences of any given mutation and its impact on the pathways the proteins are involved in must be understood To this end we analyzed all identified unique protein producing fusions and the fulllength inframe domains of the fusion proteinsWhile AGFusion does predict protein sequence for each fusion partner and corresponding conserved or lost domains a domain is counted as conserved already if only amino acids are included in the sequence To adapt this to the study of fulllength domains we first mapped the Pfam identifiers of the domains to sequences in the wild type proteins from Uniprot The domains were then defined as conserved only if the full sequence was present in the fusion protein This resulted in conserved domains in all protein producing fusions Over of these domains are in PKTF fusions which account for of all protein producing fusions supplementary tables S1 S6 suggesting overall domain count strongly favors PK and TF genes perhaps indicating that these fusions produce more functional proteins in comparison to all protein producing fusionsThe most conserved domain was the protein tyrosine kinase domain Fig a03A supplementary table a0S6 which was conserved in fusions This was followed by the PH domain a common domain in intracellular signaling proteins and proteins of the cytoskeleton and the protein kinase domain To assess retention of nonobvious PK or TF domains we classified domains as PK or TF specific if over of the copies were found in PK or TF halves of the fusion proteins This resulted in copies of different TFspecific domains predicted to exist in the fusions compared to copies of PKspecific domains Most common TF domains were zinc finger C2H2 type KRAB and HLH DNA binding domains present in and copies respectively Many TF domains such as KRAB are involved in both transcriptional activation and repression depending on the molecular contextOn average protein producing fusions in samples of most cancer projects tended to have close to intact full length domain per protein producing OF PKTF fusions on average had more intact domains in all except for projects Fig a03B On average fusions in all projects tended to have between and intact domains while PKTF fusions featured a slightly higher average Although some cancers do appear to have particularly many domains this is mostly due to low count of fusions detected in the project Exception seems to be acute myeloid leukemia with detected protein producing fusions of which contain either a PK or a TF Most striking differences being seen in mesothelioma thyroid carcinoma rectum adenocarcinoma and uveal melanoma with and more retained domains on average in PKTF fusions compared to protein producing fusions respectivelyOn the cancer project level thyroid carcinoma had the highest percentage of PK domains of all domains identified in the samples of the project supplementary table a0S7 supplementary Figure S3 which totaled to only exceeded by breast invasive carcinoma with PK specific domains of all BRCA domains and lung adenocarcinoma LUAD with Proportion of TF domains varied less Kidney renal papillary cell carcinoma had of its intact domains in the TFspecific set followed by acute myeloid leukemia with and rectum adenocarcinoma and prostate adenocarcinoma both at Aside from prostate adenocarcinoma these projects had samples in the TCGA datasetinteractors of fusion partners can point to impact of ofs To understand what kind of implications the functional changes of lost conserved PK or TF specific domains in new combinations could have for the Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cNonprotein producing fusionProtein producing fusionLMEKLASMBRBGTIPMVBKSPRTXNURXNURHPMRYLKNWCREATMKLLEPXOFDNCCNPTPCDCCMUPNAKNICCATRFGFSSRPMTGREPAGPBBREARARLMPGRPTPTRCBLBAKRTNVTEBTBZASARFNZPBPSLEHYTTLDAMIUCMCCSAHDTMBMTPALDNYMZAYEDHSABRLBRSMTONCSRAYMUPSGRHPCMProtein kinaseTranscription factorProtein kinase and transcription factorAsnoisufocno fo rebmuNBIJNNAMDKCCMNBDDZDPONEPIRTSLYCHALAGTSfroCIV IIIIIIACCBLCABRCACESC CHOLCOADDLBC ESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diï¬uselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinomaUCS Uterine carcinosarcomaUVM Uveal melanomaFigure a0 Clinical characterization of protein producing oncofusions by cancer stage A The most common protein producing gene pairs in oncofusions In total protein producing fusions were comprised of unique gene pairs predicted to produce one or more unique protein products The most common pair was RPS6KB1VMP1 with over unique proteins followed by ITGB6RBMS2 and ALKEML4 with each and LYRM9P3H4 and RUNX1RUNX1T1 at Kinase and TF fusions were common in top protein producing gene pairs illustrated by blue shading for the presence of a protein kinase in gene pair red for TF and orange for both B Sunburst diagram of project and stage distribution of PKTF oncofusions The innermost layer represents the number of fusions in each project The layers radiating out are the proportion of fusions detected in Stage I II III and IV samples in order from in to out Total numbers of fusions from each stage is marked under the stage indicatorsScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cProtein kinase or TF containing oncofusionsProtein producing oncofusionsAsniamod fo rebmuNniamodHS staeper niryknAniamod III epyt nitcenorbiFniamodC taeper atebG niamodDW niamod HC ygolomohninoplaC fitomnoitingocer ANRregnfiDHPniamodZDP epyt HC regnfi cniZniamod lanimret Cdevresnoc esacileHfitom ahpla eliretS niamodMAS xobBARKniamodHS niamodZOPBTBniamod HS tnairaV niamodnirehdaC niamodgnidnib lorecylglycaidsretse lobrohP niamodgnidnibANDxilehpoolxileH niamodFEGohR niamodPAGohRtaeper nirtcepSnietorp tropsnart noIniamod nilubolgonummIA ssalc niamod rotpecer LDLniamodomorBniamodAHF taeper hcir enicueL niamod tesVnilubolgonummI niamod epyt nidnopsobmorhTniamodMIL niamodWW niamod ekilFGEriap niamoddnahFEniamod lartnec MREF niamodFGEgnidnibmuiclaC esacilehxobHAEDDAEDniamodstEniamodoemoHfitom ahpla eliretS niamodMAS etis yrotibihni aX rotcaFnoitalugaoCrotpecer enomroh raelcun foDBLniamodYRPS niamod htaeDniamodXP niamod HMretropsnart CBAtaeper editpepocirtarteTniamod FASDGlaR noitaicossa saRylimaf nispodohr rotpecer enarbmemsnartniamodHP esanik enisoryt nietorPniamod esanik nietorPniamod tesI nilubolgonummIBnoisufocno gnidoc nietorp rep sniamoDProtein producing oncofusionsProtein kinase or TF containing oncofusionsCsnoisufocnoFT ro esanik nietorp gnitcaretni fo rebmuNLMALOSEMACHTLOHCDAERMVUPRKIMYHTMBGGGLGPCPTCGTDARPCSULDAOCMCKSCSECACLBDAULCRASCSNHCRKIACSECHILACRBVOCCADATSDAAPSCUCECUCBLDHCKIFound in COSMIC cancer gene censusPK oncofusionsTF oncofusionsPKTF oncofusionsOther protein producing oncofusions Percent of unique interactors in COSMIC canger gene census KRTNGAHWYOPXCBURGERFGEAPSHBAHWYRACADHAPSHPECHTRISALERMCMQAHWYFNRXDDNUJLCDCLBVUREAHWYCRSAPRCDCCDAMSMDMCADHPSLMPACPPPNFSAPRROTMBERCDAMSAPSHDNACCADHPCVCDKRPTATSACRBDNCCBDDABUIMBANCPARAROXBFUPNRNHFigure a0 Domain analysis of protein producing fusions A Intact fulllength domains identified in unique protein producing fusions In total intact domains were detected The protein tyrosine kinase domain was the most prevalent with identifications In addition protein kinase domain was detected with copies each Kinase or TF specific domains included and unique domains respectively copies of kinasespecific domains were seen and of TF specific Kinase domains focused more on the two top kinase domains whereas TF domains were a much more evenly distributed group the top TFspecific domain C2H2 type zinc finger having copies B The number of domains per protein coding oncofusion in the TCGA projects C Most common interactors of protein kinaseTF fusions Yaxis describes numbers of unique protein producing fusions where one or both of the fusion partner WT genes interact with the protein Top right inset Proportion of interactors found in COSMIC cancer gene census is higher in both protein kinase and transcription factor fusions and most common in fusions between protein kinase and transcription factor genesScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cATranscriptional regulationpathwaysRNA Polymerase II transcriptionGeneric transcription pathwaySignaling by Rho GTPasesMAPK family signaling cascadeGPCR signalingSignaling by NotchTGFβ signalingSignal transductionTranscriptional regulationby TP53Hedgehog pathwaySignaling by Receptor tyrosine kinasesIntegrin signalingIntracellular signaling bysecond messengersWnt signalingDeath receptor signalingNode proteins interact withTF fusionsKinase fusionsBCytokine Signaling in Immune systemDevelopmental BiologyAxon guidanceMetabolismPIP3 activates AKT signalingIntracellular signaling by second messengersPTEN RegulationRegulation of PTEN gene transcriptionOxidative Stress Induced SenescenceHDMs demethylate histonesSUMOylation of transcription cofactorsTranscriptional Regulation by E2F6SUMOylation of chromatin anization proteinsTranscriptional regulation by RUNX1Chromatin anizationChromatin modifying enzymesCellular SenescenceSUMO E3 ligases SUMOylate target proteinsSUMOylationInterferon alphabeta signalingEPHEphrin signalingPhospholipid metabolismEPHephrin mediated repulsion of cellsEPHAmediated growth cone collapsePI MetabolismSynthesis of PIPs at the plasma membraneInterferon SignalingMetabolism of lipidsOncogenic MAPK signalingSignaling by moderate kinase activity BRAF mutantsParadoxical activation of RAF signaling by kinase inactive BRAFSignaling downstream of RAS mutantsrRNA modification in the nucleus and cytosolRHO GTPases Activate ForminsMajor pathway of rRNA processing in the nucleolus and cytosolrRNA processing in the nucleus and cytosolrRNA processingProcessing of Capped IntronContaining PremRNAmRNA SplicingmRNA Splicing Major PathwayMacroautophagyAutophagyToll Like Receptor TLR3 CascadeMyD88independent TLR4 cascade TRIFTICAM1mediated TLR4 signaling Tolllike Receptor CascadesToll Like Receptor TLR4 CascadeSignaling by TGFbeta family membersSignaling by Nuclear ReceptorsTCF dependent signaling in response to WNTSignaling by WNTSignaling by

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

Detecting ulcerative colitis from a0colon a0samples a0using a0efficient a0feature selection and machine learningHanieh Marvi Khorasani1 a0Hamid a0Usefi2 Lourdes pe±a‘castillo1Ulcerative a0colitis a0UC a0is a0one a0of a0the a0most a0common a0forms a0of a0inflammatory a0bowel a0disease a0IBD a0characterized a0by a0inflammation a0of a0the a0mucosal a0layer a0of a0the a0colon a0Diagnosis a0of a0UC a0is a0based a0on a0clinical a0symptoms a0and a0then a0confirmed a0based a0on a0endoscopic a0histologic a0and a0laboratory a0findings a0Feature a0selection a0and a0machine a0learning a0have a0been a0previously a0used a0for a0creating a0models a0to a0facilitate a0the a0diagnosis a0of a0certain a0diseases a0In a0this a0work a0we a0used a0a a0recently a0developed a0feature a0selection a0algorithm a0DRPT a0combined a0with a0a a0support a0vector a0machine a0SVM a0classifier a0to a0generate a0a a0model a0to a0discriminate a0between a0healthy a0subjects a0and a0subjects a0with a0UC a0based a0on a0the a0expression a0values a0of a0 a0genes a0in a0colon a0samples a0We a0validated a0our a0model a0with a0an a0independent a0gene a0expression a0dataset a0of a0colonic a0samples a0from a0subjects a0in a0active a0and a0inactive a0periods a0of a0UC a0Our a0model a0perfectly a0detected a0all a0active a0cases a0and a0had a0an a0average a0precision a0of a0 a0in a0the a0inactive a0cases a0Compared a0with a0results a0reported a0in a0previous a0studies a0and a0a a0model a0generated a0by a0a a0recently a0published a0software a0for a0biomarker a0discovery a0using a0machine a0learning a0BioDiscML a0our a0final a0model a0for a0detecting a0UC a0shows a0better a0performance a0in a0terms a0of a0average a0precisionInflammatory bowel disease IBD is a chronic inflammatory condition of the gut with an increasing health burden1 Ulcerative colitis UC and Crohn™s disease are the two most common forms of chronic IBD with UC being more widespread than Crohn™s disease2 There is no cure for UC3 and people with the disease alternate between periods of remission inactive and active inflammation2 The underlying causes of UC are not completely understood yet but it is thought to be a combination of genetic environmental and psychological factors that disrupt the microbial ecosystem of the colon34 Genomewide association studies GWAS have identified risk loci for IBD5 and risk loci specifically associated with UC6 However the lower concordance rate in identical twins of in UC compared with in Crohn™s disease indicates that genetic contribution in UC is weaker than in Crohn™s disease7 Thus using gene expression data for disease diagnostic might be more appropriate for UC than using GWAS data as it has been done for Crohn™s disease8There are several features used for clinical diagnosis of UC including patient symptoms and laboratory endoscopic and histological findings7 Boland et at9 carried out a proofofconcept study for using gene expression measurements from colon samples as a tool for clinical decision support in the treatment of UC The purpose of Boland et a0al™s study was to discriminate between active and inactive UC cases even though they only considered gene expression of eight inflammatory genes instead of assessing the discriminatory power of many groups of genes they concluded that mRNA analysis in UC is a feasible approach to measure quantitative response to therapyMachine learningbased models have a lot of potential to be incorporated into clinical practice10 specially in the area of medical image analysis1112 Supervised machine learning has already proved to be useful in disease diagnosis and prognosis as well as personalized medicine1314 In IBD machine learning has been used to classify IBD paediatric patients using endoscopic and histological data15 to distinguish UC colonic samples from control and Crohn™s disease colonic samples16 and to discriminate between healthy subjects UC patients and Crohn™s disease patients using transcriptional profiles of peripheral blood171Department of Computer Science Memorial University St John™s NL A1B3X5 Canada 2Department of Mathematics and Statistics Memorial University St John™s NL A1C5S7 Canada email usefimunca lourdesmuncaScientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cAccession number of controls of UC cases Description of samplesGSE11521819GSE1122320GSE226192124GSE75214active22GSE75214inactive22Mucosal biopsies from uninflammed colonic tissuesBiopsies from uninflammed sigmoid colonMucosal colonic tissue from discordant twinsMucosal colonic biopsies from active UC patients and from controlsMucosal colonic biopsies from inactive UC patients from controlsPlatformAffymetrix Human Genome U133A Array and Affymetrix Human Genome U133B ArrayAgilent012391 Whole Human Genome Oligo Microarray G4112AAffymetrix Human Genome U133 Plus ArrayAffymetrix Human Gene ST ArrayAffymetrix Human Gene ST Array of genes features UsageModel selectionModel selectionModel selectionModel evaluationModel evaluationTable Summary of datasets used in this studyIn this study our goal was to investigate whether combining machine learning with a novel feature selection algorithm an accurate model using the expression profiles of few genes could be generated from transcriptomewide gene expression data To do this we apply a machine learning classifier on gene expression data to generate a model to differentiate UC cases from controls Unlike previous studies1617 to reduce the effect of technical conditions we combined a number of independent gene expression data sets instead of using a single data set to train our model Additionally by using feature selection we were able to identify genes out of thousands genes for which expression measurements were available The expression values of these genes is sufficient to generate a SVM model to effectively discriminate between UC cases and controls On a gene expression dataset not used during training our proposed model perfectly detected all active cases and had an average precision of in the inactive casesMethodsData a0gathering a0 We searched the NCBI Gene Expression Omnibus database GEO for expression profiling studies using colonic samples from UC subjects in active and inactive state and controls healthy donors We identified five datasets accession numbers GSE11521819 GSE1122320 GSE2261921 GSE7521422 and GSE945216 As healthy and Crohn™s disease subjects were used as controls in GSE945216 this data set was excluded from our study We used three of the datasets for model selection using 5fold crossvalidation and left one dataset for independent validation Table a0 We partitioned the validation dataset into two datasets Active UC vs controls and inactive UC vs controlsAll data sets were obtained from studies where the diagnoses of patients were either based on endoscopical findings GSE7521422 and GSE2261921 followed the criteria described by LennardJones23 GSE1122320 or based on clinical features as well as radiologic endoscopic and laboratory findings GSE115218 Disease state was either assessed during colonoscopy and classified into no signs of inflammation inactive low inflammation and moderatehigh inflammation active GSE22619 defined as active with a Mayo endoscopic subscore ‰¥ GSE75214 or graded by a gastroenterologist or gastrointestinal pathologist GSE11223 GSE1152 The control group had either normal mucosa at endoscopic level GSE75214 no significant pathological findings during endoscopic and histological examinations GSE22619 normal colonoscopies GSE1152 and GSE11223 or tissues abnormalities other than IBD GSE1152 and GSE11223For each dataset GEO2R25 was used to retrieve the mapping between probe IDs and gene symbols Probe IDs without a gene mapping were removed from further processing Expression values for the mapped probe IDs were obtained using the Python package GEOparse26 The expression values obtained were as provided by the corresponding authorsData a0pre‘processing a0 We performed the following steps for data preprocessing i Calculating expression values per gene by taking the average of expression values of all probes mapped to the same gene i Handling missing values with KNearest Neighbours KNN imputation method KNNImputer from the œmissingpy library in Python27 KNNImputer uses KNN to fill in missing values by utilizing the values from nearest neighbours We set the number of neighbours to nneighbours2 and we used uniform weightTo get our final training datasets we merged datasets GSE1152 GSE11223 and GSE22619 by taking the genes present in all of them The merged dataset has UC samples and controls and genes These same genes were selected from GSE75214 for validation As the range of expression values across all datasets were different we normalized the expression values of the final merged dataset and validation dataset by calculating Zscores per sampleModel a0 generation a0 To create a model to discriminate between UC patients from healthy subjects we selected the features genes using the dimension reduction through perturbation theory DRPT feature selection method28 Let D [A b] be a dataset where b is the class label and A is an m — n matrix with n columns genes and m rows samples There is only a limited number of genes that are associated with the disease and as such a majority of genes are considered irrelevant DRPT considers the solution x of the linear system Ax b with the smallest 2norm Hence b is a sum of xi Fi where Fi is the ith column of A Then each component xi of x is viewed as an assigned weight to the feature Fi So the bigger the xi the more important Fi is in connection with b DRPT then filters out features whose weights are very small compared to the average of local maximums over Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cSubsetSubset Subset Subset Subset Subset Subset Subset Subset Subset Subset AP of FeaturesTable Ten top subsets of genes with the highest crossvalidated average APxi™s After removing irrelevant features DRPT uses perturbation theory to detect correlations between genes of the reduced dataset Finally the remaining genes are sorted based on their entropySelected features were assessed using 5fold crossvalidation and support vector machines SVMs as the classifier First we performed DRPT times on the training dataset to generate subsets of features Then to find the best subsets we performed repetitions of stratified 5fold crossvalidation CV on the training dataset We utilized average precision AP as calculated by the function average_precision_score from the Python library scikitlearn29 version as the evaluation metric to determine the best subset of genes among those generated subsets The four subsets with the highest mean AP over the crossvalidation folds were chosen for creating the candidate models For each of the four selected subset of features we created a candidate SVM model using all samples in the training dataset To generate the models we used the SVM implementation available in the function SVC with parameter kernel™linear™ from the Python library scikitlearn To evaluate the prediction performance of each of the ten models we validated it on the GSE75214active and GSE75214inactive datasets In this step we utilized the precisionrecall curve PRC to assess the performance of the candidate models on unseen data An additional candidate model was created using the most frequently selected genesBioDiscML a0 BioDiscML30 is a biomarker discovery software that uses machine learning methods to analyze biological datasets To compare the prediction performance of our models with BioDiscML we ran the software on our training dataset of the samples N52 were utilized for training and the remaining N25 for testing Since the software generates thousands of models and we required only one model we specified the number of best models as in the config file numberOfBestModels1 One best model out of all models was created based on the 10fold crossvalidated Area Under PrecisionRecall Curve numberOfBestModelsSortingMetric TRAIN10CVAUPRC on the train set We used Weka “ to evaluate the performance of the model generated by BioDiscML on the GSE75214active and GSE75214inactive datasets Selected features by BioDiscML are C3orf36 ADAM30 SLS6A3 FEZF2 and GCNT3 In order to be able to use the model in Weka we loaded the training dataset as it was created by BioDiscML which was one of the outputs of the software This dataset has six features including selected genes and class labels and samples We also modified our validation datasets by extracting BioDiscML selected features After loading the training and test dataset in Weka explorer we loaded the model and we entered the classifier configuration as œwekaclassifiersmiscInputMappedClassifier I trim W wekaclassifierstreesRandomTree “ K M V S  which is the classifier™s set up in the generated model by BioDiscMLUse a0of a0experimental a0animals a0and a0human a0participants a0 This research did not involve human participants or experimental animalsResultsFeature a0selection a0reduced a0significantly a0the a0number a0of a0genes a0required a0to a0construct a0a a0classifica‘tion a0model a0 We performed DRPT times on the training dataset to select subsets of features Then we performed 5fold crossvalidation to find the subsets with the highest mean average precision AP over the folds The range of AP for the subsets is between and with an average of ± Table a0 shows the ten subsets with the highest crossvalidated AP and the number of selected features genes on each subset On average DRPT selected ± genes per subsetTop a0 five a0 models a0 are a0 able a0 to a0 perfectly a0 discriminate a0 between a0 active a0 UC a0 patients a0 and a0 con‘trols a0 We selected the four top subsets with the highest mean AP which are subsets and Table a0 and created candidate models based on them Each candidate model was created using all samples on the training dataset and the features of the corresponding subset To identify the genes most relevant to discriminate between healthy and UC subjects we looked at the number of times each gene was selected by DRPT On DRPT runs genes were selected at least once The upper plot on Fig a0 shows the number of times each gene was selected and the lower plot shows the normal quantilequantile QQ plot Based on this plot we Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Identifying the most frequently selected genes Top Number of times each gene was selected Genes were sorted based on the number of times they were selected by DRPT Bottom Normal QQplot Horizontal line at indicates the threshold selected to deem a gene as frequently chosensaw that the observed distribution of the number of times a gene was selected deviates the most from a Gaussian distribution above times We considered the genes selected by DRPT more than times as highly relevant and created a fifth model using genes selected by DRPT at least times over runsIn order to evaluate the prediction performance of the candidate models each model was tested on the validation datasets and PRC was plotted for model assessment Figs a0 and As the AP approximates the AUPRC34 we used AP to summarize and compare the performance of these five models All five candidate models achieved high predictive performance on the validation dataset GSE75214active with an average AP of ± while the average AP of these five models on the validation dataset GSE75214inactive was ± The models with the best performance were the model created with the most frequently selected genes and subset with an AP of and on GSE75214active and GSE75214inactive respectively However based on a Friedman test35 p ˆ’ value all five models have comparable performance on the validation datasets We chose the model generated with the most frequently selected genes as our final modelOur a0top a0models a0outperformed a0the a0model a0generated a0by a0BioDiscML a0 The average AUPRC achieved by the model created by BioDiscML on both GSE75214active and GSE75214inactive datasets was and respectively Comparing the performance of our candidate models and the model created by BioDiscML on the two validation datasets we observed that we achieved better AUPRC on both datasets AUPRC on the active dataset AUPRC on the inactive dataset In terms of running time subset selection by DRPT and final model creation and validation took minutes while the running time of BioDiscML to create all the models and output the best final model was minutesScientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Precisionrecall curve of top selected subsets on GSE75214activeFigure a0 Precisionrecall curve of top selected subsets on GSE75214inactiveLinks a0between a0the a0most a0frequently a0selected a0genes a0and a0UC a0 We used Ensembl REST API Version to find the associated phenotypes with each gene belonging to the subset of the most frequently selected genes Table a0 Among these genes FAM118A is the only one with a known phenotypic association with IBD and its subtypes The evidence supporting the association of some of the other genes with UC based on phenotype is more indirect For example long term IBD patients are more susceptible to develop colorectal cancer37 and one of the genes TFRC is associated with colorectal cancer IBD patients are more prone to develop cardio vascular disease which is associated with blood pressure and cholesterol38 and four of the most frequently selected genes LIPF MMP2 DMTN and PPP1CB are associated with blood pressure and cholesterolWe looked at whether some of the most frequently selected genes contained any of the known IBDassociated SNPs5 To do this we utilized Ensembl™s BioMart39 website Ensembl Release version September to retrieve the genomic location of the genes We then used the intersectBed utility in BEDtools40 to find any overlap between the IDB risk loci and the genomic location of the genes None of the IBDassociated SNPs was located on our genes Similarly gene set enrichment analysis found no enriched GO term or pathway among these genes Additionally these genes are not listed as top differentially expressed genes in previous studies on UC4142We searched the literature for links between the genes and UC and we found the following MMP2 expression has been found significantly increased in colorectal neoplasia in a mouse model of UC43 and MMP2 levels are elevated in IBD44 TFRC has been found to have an antiinflammatory effect on a murine colitis model45 KRT8 genetic variants have been observed in IBD patients and it was suggested that these variants are a risk factor for IBD46 DUOXA2 has been shown to be critical in the production of hydrogen peroxide within the colon and to be upregulated in active UC47Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cGene symbolCWF19L1FCER2MMP2PPP1CBRPL23AP32ZNF624REG1BTFRCFAM118ACFHR2KRT8PRELID1ZNF92ABHD2C16orf89CAB39LSPATC1LDUOXA2MESP1MAML3PITX2DMTNASF1BPGFBEX4ODF1PTGR1ZNF35LIPFSLC25A13BARX2C2orf42Associated phenotypesSpinocerebellar ataxia autosomal recessive depressive disorder MajorBlood protein levels post bronchodilator FEV1Multicentric OsteolysisNodulosisArthropathy MONA spectrum disorders cholesterol HDL lip and oral cavity carcinoma body height winchester syndromeNoonan Syndromelike disorder with loose anagen hair Heel bone mineral density Blood pressure basophils asopathy with developmental delay short stature and sparse slowgrowing hairAttention deficit disorder with hyperactivity body HeightNoneContrast sensitivity Body Mass IndexBreast ductal adenocarcinoma esophageal adenocarcinoma thyroid carcinoma clear cell renal carcinoma prostate carcinoma pancreatic cancer gastric adenocarcinoma hepatocellular carcinoma lung adenocarcinoma rectal adenocarcinoma basal cell carcinoma colorectal adenocarcinoma squamous cell lung carcinoma head and neck squamous cell carcinoma colon adenocarcinoma iron status biomarkers transferrin levels mean corpuscular hemoglobin concentration red cell distribution width combined immunodeficiency red blood cell traits high light scatter reticulocyte percentage of red cells reticulocyte fraction of red cells Immunodeficiency Chronic inflammatory diseases ankylosing spondylitis Crohn™s disease psoriasis primary sclerosing cholangitis ulcerative colitis Glucose Peanut allergy maternal genetic effects Heel bone mineral densityMacular degeneration blood protein levels feeling miserable alanine aminotransferase ALT levels after remission induction therapy in acute lymphoblastic leukaemia ALL asthmaCirrhosis familial cirrhosis hepatitis C virus susceptibility to cirrhosis cryptogenic cirrhosis noncryptogenic cirrhosis susceptibility to gamma glutamyl transferase levels cancer pleiotropyBody fat distribution heel bone mineral density activated partial thromboplastin timeNoneItch intensity from mosquito bite adjusted by bite size gut microbiota Obesityrelated traits coronary artery disease advanced age related macular degeneration squamous cell lung carcinoma pulse pressureNoneHemoglobin S erythrocyte count pancreatic neoplasmsNoneFamilial thyroid dyshormonogenesis thyroglobulin synthesis defectNoneSocial science traits intelligence MTAG chronic mucus hypersecretion borderline personality disorder congenital heart malformationAxenfeldRieger syndrome ring dermoid of cornea iridogoniodygenesis type peters anomaly familial atrial fibrillation rieger anomaly stroke ischemic stroke cataract PITX2related eye abnormalities phosphorus cognitive decline rate in late mild cognitive impairment creatinine intraocular pressure incident atrial fibrillation wolffparkinsonwhite pattern parkinson disease early onset atrial fibrillation anterior segment sygenesis Total cholesterol levels LDL cholesterolNoneMood instability blood protein levelsNoneBody weight body mass index glucose IgA nephropathy Chronic lymphocytic leukaemia type diabetes erythrocyte indicesBody height menarche monocyte count blood protein levelsNoneMaximal midexpiratory flow rate blood protein levels respiratory function tests blood pressureCitrullinemia type II neonatal intrahepatic cholestasis due to citrin deficiency citrin deficiency citrullinemia type I bone mineral densityType diabetes breast cancer night sleep phenotypes response to cyclophosphamide in systemic lupus erythematosus with lupus nephritis strokeNone of times selectedTable Phenotypes associated with the most frequently selected genes by DRPT as obtained from Ensembl REST API Version DiscussionIn this study we showed the feasibility of using machine learning and feature selection to identify a reduced number of genes from microarray data to aid in the diagnosis of UC One might argue that distinguishing UC patients from Crohn™s disease CD patients has more clinical relevance than distinguishing UC patients from controls However we were limited on the choice of groups to classify by data availability as we could only find three gene expression data sets obtained from colonic samples of UC and CD patients in GEO GSE1152 GSE75214 and GSE126124 As children samples were transcriptionally profiled for GSE12612448 instead of adults ones we decided that the age difference could introduce extra biological variation in the expression data unrelated to UC That left us with only two data sets which were not enough to train the model with multiple data sets and have at least one holdout data set for validationAnother limitation of this study is that we used gene expression profiles of colonic samples Further research is required to assess the accuracy of our 32gene model in gene expression profiles of blood samples A recent study48 found a similar transcriptional profile between blood and colon tissue from patients with IBD If indeed Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cour 32gene model is found accurate in blood samples then a less invasive procedure such as a blood test could be used to diagnose UC instead of a colonoscopy or sigmoidoscopyIn a previous study where machine learning was employed to perform a risk assessment for CD and UC using GWAS data49 a twostep feature selection strategy was used on a dataset containing Crohn™s disease cases UC cases and controls with SNPs In that study Wei et a0al reduced the number of features by filtering out SNPs with pvalues greater than ˆ’ and then applied a penalized feature selection with L1 penalty to select a subset of SNPs We decided against filtering out genes based on an arbitrary pvalue of statistical significance of differential expression as researchers are strongly advised against the use of pvalues and statistical significance in relation to the nullhypothesis5051Our 32gene model achieved AP of and discriminating active UC patients from healthy donors and inactive UC patients from healthy donors respectively We found direct or indirect links to UC for about a quarter of the most frequently chosen genes The remaining genes should be further investigated to find associations with UC To put the performance of our 32gene model into perspective we looked at previous studies applying machine learning to create models for the diagnostic of UC Maeda et a0al52 extracted features from endocystoscopy images to train a SVM to classify UC patients as active or healing This approach achieve precision at recall which is lower than the one achieved by our 32gene model Figs a0 and Yuan et a0al17 applied incremental feature selection and a SMO classifier a type of SVM on gene expression data from blood samples to discriminate between healthy subjects UC patients and Crohn™s disease patients The 10fold crossvalidation accuracy of their best model using the expression values of genes to classify UC patients was while our method obtained better accuracy than this with substantially less number of genes In terms of potential for clinical translation of a machine learningbased model a model requiring to quantify the gene expression levels of fewer genes is more suitable for the development of a new diagnostic test than one requiring the quantification of the expression levels of thousands of genesUsing an efficient feature selection method such as DRPT and a SVMclassifier on gene expression data we generated a model that could facilitate the diagnosis of UC from expression measurements of genes from colonic samples To avoid systematic experimental bias on the training data we used three transcriptomic datasets from three separated studies Our top model was validated with promising results on a data set not used for training however additional research is required to evaluate the genes as potential biomarkers on a external set of subjectsReceived March Accepted July References Kaplan G G The global burden of IBD from to Nat Rev Gastroenterol Hepatol “ 101038 Ord¡s I Eckmann L Talamini M Baumgart D C Sandborn W J Ulcerative colitis Lancet “ nrgas tro2015150 101016S0140 Eisenstein M Ulcerative colitis towards remission Nature S33 101038d4158 Khan I et al Alteration of gut microbiota in inflammatory bowel disease IBD cause or consequence IBD treatment targeting the gut microbiome Pathogens a0 103390patho gens8 de Lange K M et al Genomewide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease Nat Genet “ 101038ng3760 Anderson C A et al Metaanalysis identifies additional ulcerative colitis risk loci increasing the number of confirmed associations to Nat Genet “ 101038ng764 Conrad K Roggenbuck D Laass M W Diagnosis and classification of ulcerative colitis Autoimmun Rev “ 101016jautre v201401028 Romagnoni A et al Comparative performances of machine learning methods for classifying Crohn disease patients using genomewide genotyping data Sci Rep 101038s4159 z Boland B S et al Validated gene expression biomarker analysis for biopsybased clinical trials in ulcerative colitis Aliment Pharmacol Ther “ 101111apt12862 Shah P et al Artificial intelligence and machine learning in clinical development a translational perspective NPJ Digit Med 101038s4174 Esteva A et al Dermatologistlevel classification of skin cancer with deep neural networks Nature “ McKinney S M et al International evaluation of an AI system for breast cancer screening Nature “ Molla M Waddell M Page D Shavlik J Using machine learning to design and interpret geneexpression microarrays AI 101038natur e2105 101038s4158 Mag perspectives Hum Genet “ 101038s4159 Xu J et al Translating cancer genomics into precision medicine with artificial intelligence applications challenges and future Mossotto E et al Classification of paediatric inflammatory bowel disease using machine learning Sci Rep Olsen J et al Diagnosis of ulcerative colitis before onset of inflammation by multivariate modeling of genomewide gene expression data Inflamm Bowel Dis “ 101002ibd20879 Yuan F Zhang YH Kong XY Cai YD Identification of candidate genes related to inflammatory bowel disease using minimum redundancy maximum relevance incremental feature selection and the shortestpath approach Biomed Res Int 101155201757419 Moehle C et al Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease J Mol Med Berl “ 101007s0010 Zahn A et al Aquaporin8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis World J Gas Noble C L et al Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis Gut “ troenterol Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0c Lepage P et al Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis Gastroenterology “ Inflamm Bowel Dis “ Vancamelbeke M et al Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease LennardJones J E Classification of inflammatory bowel disease Scand J Gastroenterol Suppl “ 10310900365 discussion “ H¤sler R et al A functional methylome map of ulcerative colitis Genome Res “ Barrett T et al NCBI GEO archive for functional genomics data sets“update Nucleic Acids Res D991“D995 Gumienny R GEOparse pypiproje ctGEOpa rse Troyanskaya O et al Missing value estimation methods for DNA microarrays Bioinformatics “ 101093bioin forma tics176520 abs200212104 Afshar M Usefi H HighDimensional Feature Selection for Genomics Datasets KnowledgeBased Systems arxiv Pedregosa F et al Scikitlearn machine learning in Python J Mach Learn Res “ Leclercq M et al Largescale automatic feature selection for biomarker discovery in highdimensional omics data Front Genet Burlington Holmes G Donkin A Witten I a0H Weka A machine learning workbench In Proceedings of ANZIIS ™ Australian New Zealand Intelligent Information Systems Conference “ Hall M et al The weka data mining software an update ACM SIGKDD Explor Newsl “ Witten I H Frank E Hall M A Pal C J Data Mining Practical Machine Learning Tools and Techniques Man Kaufmann M¼ller A C et al Introduction to Machine Learning with Python A Guide for Data scientists O™Reilly Media Inc California DemÅ¡ar J Statistical comparisons of classifiers over multiple data sets J Mach Learn Res “ Yates A et al The Ensembl REST API Ensembl data for any language Bioinformatics “ Kim E R Chang D K Colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis World J Gastroenterol diovasc Dis “ Schulte D et al Small dense LDL cholesterol in human subjects with different chronic

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

Thyroid_Cancer

"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine œLa Sapienza University Viadi Grottarossa Rome ItalyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a “7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders [“]its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[“] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [“] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures “ °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no “ revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITO“Luc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kgˆ’ STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of “ g·kgˆ’ ·dayˆ’ [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at ˆ’ °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect„¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect„¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of Sant™Andrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withœAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of field™s area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect„¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe œResults sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturer™s protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fisher™s exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and “ animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr    P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see œResults section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fisher™s exact test P vsCtr  P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see œResults section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ±     ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr   P or  P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fisher™s exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKras“driven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in

Thyroid_Cancer

Detecting ulcerative colitis from a0colon a0samples a0using a0efficient a0feature selection and machine learningHanieh Marvi Khorasani1 a0Hamid a0Usefi2 Lourdes pe±a‘castillo1Ulcerative a0colitis a0UC a0is a0one a0of a0the a0most a0common a0forms a0of a0inflammatory a0bowel a0disease a0IBD a0characterized a0by a0inflammation a0of a0the a0mucosal a0layer a0of a0the a0colon a0Diagnosis a0of a0UC a0is a0based a0on a0clinical a0symptoms a0and a0then a0confirmed a0based a0on a0endoscopic a0histologic a0and a0laboratory a0findings a0Feature a0selection a0and a0machine a0learning a0have a0been a0previously a0used a0for a0creating a0models a0to a0facilitate a0the a0diagnosis a0of a0certain a0diseases a0In a0this a0work a0we a0used a0a a0recently a0developed a0feature a0selection a0algorithm a0DRPT a0combined a0with a0a a0support a0vector a0machine a0SVM a0classifier a0to a0generate a0a a0model a0to a0discriminate a0between a0healthy a0subjects a0and a0subjects a0with a0UC a0based a0on a0the a0expression a0values a0of a0 a0genes a0in a0colon a0samples a0We a0validated a0our a0model a0with a0an a0independent a0gene a0expression a0dataset a0of a0colonic a0samples a0from a0subjects a0in a0active a0and a0inactive a0periods a0of a0UC a0Our a0model a0perfectly a0detected a0all a0active a0cases a0and a0had a0an a0average a0precision a0of a0 a0in a0the a0inactive a0cases a0Compared a0with a0results a0reported a0in a0previous a0studies a0and a0a a0model a0generated a0by a0a a0recently a0published a0software a0for a0biomarker a0discovery a0using a0machine a0learning a0BioDiscML a0our a0final a0model a0for a0detecting a0UC a0shows a0better a0performance a0in a0terms a0of a0average a0precisionInflammatory bowel disease IBD is a chronic inflammatory condition of the gut with an increasing health burden1 Ulcerative colitis UC and Crohn™s disease are the two most common forms of chronic IBD with UC being more widespread than Crohn™s disease2 There is no cure for UC3 and people with the disease alternate between periods of remission inactive and active inflammation2 The underlying causes of UC are not completely understood yet but it is thought to be a combination of genetic environmental and psychological factors that disrupt the microbial ecosystem of the colon34 Genomewide association studies GWAS have identified risk loci for IBD5 and risk loci specifically associated with UC6 However the lower concordance rate in identical twins of in UC compared with in Crohn™s disease indicates that genetic contribution in UC is weaker than in Crohn™s disease7 Thus using gene expression data for disease diagnostic might be more appropriate for UC than using GWAS data as it has been done for Crohn™s disease8There are several features used for clinical diagnosis of UC including patient symptoms and laboratory endoscopic and histological findings7 Boland et at9 carried out a proofofconcept study for using gene expression measurements from colon samples as a tool for clinical decision support in the treatment of UC The purpose of Boland et a0al™s study was to discriminate between active and inactive UC cases even though they only considered gene expression of eight inflammatory genes instead of assessing the discriminatory power of many groups of genes they concluded that mRNA analysis in UC is a feasible approach to measure quantitative response to therapyMachine learningbased models have a lot of potential to be incorporated into clinical practice10 specially in the area of medical image analysis1112 Supervised machine learning has already proved to be useful in disease diagnosis and prognosis as well as personalized medicine1314 In IBD machine learning has been used to classify IBD paediatric patients using endoscopic and histological data15 to distinguish UC colonic samples from control and Crohn™s disease colonic samples16 and to discriminate between healthy subjects UC patients and Crohn™s disease patients using transcriptional profiles of peripheral blood171Department of Computer Science Memorial University St John™s NL A1B3X5 Canada 2Department of Mathematics and Statistics Memorial University St John™s NL A1C5S7 Canada email usefimunca lourdesmuncaScientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cAccession number of controls of UC cases Description of samplesGSE11521819GSE1122320GSE226192124GSE75214active22GSE75214inactive22Mucosal biopsies from uninflammed colonic tissuesBiopsies from uninflammed sigmoid colonMucosal colonic tissue from discordant twinsMucosal colonic biopsies from active UC patients and from controlsMucosal colonic biopsies from inactive UC patients from controlsPlatformAffymetrix Human Genome U133A Array and Affymetrix Human Genome U133B ArrayAgilent012391 Whole Human Genome Oligo Microarray G4112AAffymetrix Human Genome U133 Plus ArrayAffymetrix Human Gene ST ArrayAffymetrix Human Gene ST Array of genes features UsageModel selectionModel selectionModel selectionModel evaluationModel evaluationTable Summary of datasets used in this studyIn this study our goal was to investigate whether combining machine learning with a novel feature selection algorithm an accurate model using the expression profiles of few genes could be generated from transcriptomewide gene expression data To do this we apply a machine learning classifier on gene expression data to generate a model to differentiate UC cases from controls Unlike previous studies1617 to reduce the effect of technical conditions we combined a number of independent gene expression data sets instead of using a single data set to train our model Additionally by using feature selection we were able to identify genes out of thousands genes for which expression measurements were available The expression values of these genes is sufficient to generate a SVM model to effectively discriminate between UC cases and controls On a gene expression dataset not used during training our proposed model perfectly detected all active cases and had an average precision of in the inactive casesMethodsData a0gathering a0 We searched the NCBI Gene Expression Omnibus database GEO for expression profiling studies using colonic samples from UC subjects in active and inactive state and controls healthy donors We identified five datasets accession numbers GSE11521819 GSE1122320 GSE2261921 GSE7521422 and GSE945216 As healthy and Crohn™s disease subjects were used as controls in GSE945216 this data set was excluded from our study We used three of the datasets for model selection using 5fold crossvalidation and left one dataset for independent validation Table a0 We partitioned the validation dataset into two datasets Active UC vs controls and inactive UC vs controlsAll data sets were obtained from studies where the diagnoses of patients were either based on endoscopical findings GSE7521422 and GSE2261921 followed the criteria described by LennardJones23 GSE1122320 or based on clinical features as well as radiologic endoscopic and laboratory findings GSE115218 Disease state was either assessed during colonoscopy and classified into no signs of inflammation inactive low inflammation and moderatehigh inflammation active GSE22619 defined as active with a Mayo endoscopic subscore ‰¥ GSE75214 or graded by a gastroenterologist or gastrointestinal pathologist GSE11223 GSE1152 The control group had either normal mucosa at endoscopic level GSE75214 no significant pathological findings during endoscopic and histological examinations GSE22619 normal colonoscopies GSE1152 and GSE11223 or tissues abnormalities other than IBD GSE1152 and GSE11223For each dataset GEO2R25 was used to retrieve the mapping between probe IDs and gene symbols Probe IDs without a gene mapping were removed from further processing Expression values for the mapped probe IDs were obtained using the Python package GEOparse26 The expression values obtained were as provided by the corresponding authorsData a0pre‘processing a0 We performed the following steps for data preprocessing i Calculating expression values per gene by taking the average of expression values of all probes mapped to the same gene i Handling missing values with KNearest Neighbours KNN imputation method KNNImputer from the œmissingpy library in Python27 KNNImputer uses KNN to fill in missing values by utilizing the values from nearest neighbours We set the number of neighbours to nneighbours2 and we used uniform weightTo get our final training datasets we merged datasets GSE1152 GSE11223 and GSE22619 by taking the genes present in all of them The merged dataset has UC samples and controls and genes These same genes were selected from GSE75214 for validation As the range of expression values across all datasets were different we normalized the expression values of the final merged dataset and validation dataset by calculating Zscores per sampleModel a0 generation a0 To create a model to discriminate between UC patients from healthy subjects we selected the features genes using the dimension reduction through perturbation theory DRPT feature selection method28 Let D [A b] be a dataset where b is the class label and A is an m — n matrix with n columns genes and m rows samples There is only a limited number of genes that are associated with the disease and as such a majority of genes are considered irrelevant DRPT considers the solution x of the linear system Ax b with the smallest 2norm Hence b is a sum of xi Fi where Fi is the ith column of A Then each component xi of x is viewed as an assigned weight to the feature Fi So the bigger the xi the more important Fi is in connection with b DRPT then filters out features whose weights are very small compared to the average of local maximums over Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cSubsetSubset Subset Subset Subset Subset Subset Subset Subset Subset Subset AP of FeaturesTable Ten top subsets of genes with the highest crossvalidated average APxi™s After removing irrelevant features DRPT uses perturbation theory to detect correlations between genes of the reduced dataset Finally the remaining genes are sorted based on their entropySelected features were assessed using 5fold crossvalidation and support vector machines SVMs as the classifier First we performed DRPT times on the training dataset to generate subsets of features Then to find the best subsets we performed repetitions of stratified 5fold crossvalidation CV on the training dataset We utilized average precision AP as calculated by the function average_precision_score from the Python library scikitlearn29 version as the evaluation metric to determine the best subset of genes among those generated subsets The four subsets with the highest mean AP over the crossvalidation folds were chosen for creating the candidate models For each of the four selected subset of features we created a candidate SVM model using all samples in the training dataset To generate the models we used the SVM implementation available in the function SVC with parameter kernel™linear™ from the Python library scikitlearn To evaluate the prediction performance of each of the ten models we validated it on the GSE75214active and GSE75214inactive datasets In this step we utilized the precisionrecall curve PRC to assess the performance of the candidate models on unseen data An additional candidate model was created using the most frequently selected genesBioDiscML a0 BioDiscML30 is a biomarker discovery software that uses machine learning methods to analyze biological datasets To compare the prediction performance of our models with BioDiscML we ran the software on our training dataset of the samples N52 were utilized for training and the remaining N25 for testing Since the software generates thousands of models and we required only one model we specified the number of best models as in the config file numberOfBestModels1 One best model out of all models was created based on the 10fold crossvalidated Area Under PrecisionRecall Curve numberOfBestModelsSortingMetric TRAIN10CVAUPRC on the train set We used Weka “ to evaluate the performance of the model generated by BioDiscML on the GSE75214active and GSE75214inactive datasets Selected features by BioDiscML are C3orf36 ADAM30 SLS6A3 FEZF2 and GCNT3 In order to be able to use the model in Weka we loaded the training dataset as it was created by BioDiscML which was one of the outputs of the software This dataset has six features including selected genes and class labels and samples We also modified our validation datasets by extracting BioDiscML selected features After loading the training and test dataset in Weka explorer we loaded the model and we entered the classifier configuration as œwekaclassifiersmiscInputMappedClassifier I trim W wekaclassifierstreesRandomTree “ K M V S  which is the classifier™s set up in the generated model by BioDiscMLUse a0of a0experimental a0animals a0and a0human a0participants a0 This research did not involve human participants or experimental animalsResultsFeature a0selection a0reduced a0significantly a0the a0number a0of a0genes a0required a0to a0construct a0a a0classifica‘tion a0model a0 We performed DRPT times on the training dataset to select subsets of features Then we performed 5fold crossvalidation to find the subsets with the highest mean average precision AP over the folds The range of AP for the subsets is between and with an average of ± Table a0 shows the ten subsets with the highest crossvalidated AP and the number of selected features genes on each subset On average DRPT selected ± genes per subsetTop a0 five a0 models a0 are a0 able a0 to a0 perfectly a0 discriminate a0 between a0 active a0 UC a0 patients a0 and a0 con‘trols a0 We selected the four top subsets with the highest mean AP which are subsets and Table a0 and created candidate models based on them Each candidate model was created using all samples on the training dataset and the features of the corresponding subset To identify the genes most relevant to discriminate between healthy and UC subjects we looked at the number of times each gene was selected by DRPT On DRPT runs genes were selected at least once The upper plot on Fig a0 shows the number of times each gene was selected and the lower plot shows the normal quantilequantile QQ plot Based on this plot we Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Identifying the most frequently selected genes Top Number of times each gene was selected Genes were sorted based on the number of times they were selected by DRPT Bottom Normal QQplot Horizontal line at indicates the threshold selected to deem a gene as frequently chosensaw that the observed distribution of the number of times a gene was selected deviates the most from a Gaussian distribution above times We considered the genes selected by DRPT more than times as highly relevant and created a fifth model using genes selected by DRPT at least times over runsIn order to evaluate the prediction performance of the candidate models each model was tested on the validation datasets and PRC was plotted for model assessment Figs a0 and As the AP approximates the AUPRC34 we used AP to summarize and compare the performance of these five models All five candidate models achieved high predictive performance on the validation dataset GSE75214active with an average AP of ± while the average AP of these five models on the validation dataset GSE75214inactive was ± The models with the best performance were the model created with the most frequently selected genes and subset with an AP of and on GSE75214active and GSE75214inactive respectively However based on a Friedman test35 p ˆ’ value all five models have comparable performance on the validation datasets We chose the model generated with the most frequently selected genes as our final modelOur a0top a0models a0outperformed a0the a0model a0generated a0by a0BioDiscML a0 The average AUPRC achieved by the model created by BioDiscML on both GSE75214active and GSE75214inactive datasets was and respectively Comparing the performance of our candidate models and the model created by BioDiscML on the two validation datasets we observed that we achieved better AUPRC on both datasets AUPRC on the active dataset AUPRC on the inactive dataset In terms of running time subset selection by DRPT and final model creation and validation took minutes while the running time of BioDiscML to create all the models and output the best final model was minutesScientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Precisionrecall curve of top selected subsets on GSE75214activeFigure a0 Precisionrecall curve of top selected subsets on GSE75214inactiveLinks a0between a0the a0most a0frequently a0selected a0genes a0and a0UC a0 We used Ensembl REST API Version to find the associated phenotypes with each gene belonging to the subset of the most frequently selected genes Table a0 Among these genes FAM118A is the only one with a known phenotypic association with IBD and its subtypes The evidence supporting the association of some of the other genes with UC based on phenotype is more indirect For example long term IBD patients are more susceptible to develop colorectal cancer37 and one of the genes TFRC is associated with colorectal cancer IBD patients are more prone to develop cardio vascular disease which is associated with blood pressure and cholesterol38 and four of the most frequently selected genes LIPF MMP2 DMTN and PPP1CB are associated with blood pressure and cholesterolWe looked at whether some of the most frequently selected genes contained any of the known IBDassociated SNPs5 To do this we utilized Ensembl™s BioMart39 website Ensembl Release version September to retrieve the genomic location of the genes We then used the intersectBed utility in BEDtools40 to find any overlap between the IDB risk loci and the genomic location of the genes None of the IBDassociated SNPs was located on our genes Similarly gene set enrichment analysis found no enriched GO term or pathway among these genes Additionally these genes are not listed as top differentially expressed genes in previous studies on UC4142We searched the literature for links between the genes and UC and we found the following MMP2 expression has been found significantly increased in colorectal neoplasia in a mouse model of UC43 and MMP2 levels are elevated in IBD44 TFRC has been found to have an antiinflammatory effect on a murine colitis model45 KRT8 genetic variants have been observed in IBD patients and it was suggested that these variants are a risk factor for IBD46 DUOXA2 has been shown to be critical in the production of hydrogen peroxide within the colon and to be upregulated in active UC47Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cGene symbolCWF19L1FCER2MMP2PPP1CBRPL23AP32ZNF624REG1BTFRCFAM118ACFHR2KRT8PRELID1ZNF92ABHD2C16orf89CAB39LSPATC1LDUOXA2MESP1MAML3PITX2DMTNASF1BPGFBEX4ODF1PTGR1ZNF35LIPFSLC25A13BARX2C2orf42Associated phenotypesSpinocerebellar ataxia autosomal recessive depressive disorder MajorBlood protein levels post bronchodilator FEV1Multicentric OsteolysisNodulosisArthropathy MONA spectrum disorders cholesterol HDL lip and oral cavity carcinoma body height winchester syndromeNoonan Syndromelike disorder with loose anagen hair Heel bone mineral density Blood pressure basophils asopathy with developmental delay short stature and sparse slowgrowing hairAttention deficit disorder with hyperactivity body HeightNoneContrast sensitivity Body Mass IndexBreast ductal adenocarcinoma esophageal adenocarcinoma thyroid carcinoma clear cell renal carcinoma prostate carcinoma pancreatic cancer gastric adenocarcinoma hepatocellular carcinoma lung adenocarcinoma rectal adenocarcinoma basal cell carcinoma colorectal adenocarcinoma squamous cell lung carcinoma head and neck squamous cell carcinoma colon adenocarcinoma iron status biomarkers transferrin levels mean corpuscular hemoglobin concentration red cell distribution width combined immunodeficiency red blood cell traits high light scatter reticulocyte percentage of red cells reticulocyte fraction of red cells Immunodeficiency Chronic inflammatory diseases ankylosing spondylitis Crohn™s disease psoriasis primary sclerosing cholangitis ulcerative colitis Glucose Peanut allergy maternal genetic effects Heel bone mineral densityMacular degeneration blood protein levels feeling miserable alanine aminotransferase ALT levels after remission induction therapy in acute lymphoblastic leukaemia ALL asthmaCirrhosis familial cirrhosis hepatitis C virus susceptibility to cirrhosis cryptogenic cirrhosis noncryptogenic cirrhosis susceptibility to gamma glutamyl transferase levels cancer pleiotropyBody fat distribution heel bone mineral density activated partial thromboplastin timeNoneItch intensity from mosquito bite adjusted by bite size gut microbiota Obesityrelated traits coronary artery disease advanced age related macular degeneration squamous cell lung carcinoma pulse pressureNoneHemoglobin S erythrocyte count pancreatic neoplasmsNoneFamilial thyroid dyshormonogenesis thyroglobulin synthesis defectNoneSocial science traits intelligence MTAG chronic mucus hypersecretion borderline personality disorder congenital heart malformationAxenfeldRieger syndrome ring dermoid of cornea iridogoniodygenesis type peters anomaly familial atrial fibrillation rieger anomaly stroke ischemic stroke cataract PITX2related eye abnormalities phosphorus cognitive decline rate in late mild cognitive impairment creatinine intraocular pressure incident atrial fibrillation wolffparkinsonwhite pattern parkinson disease early onset atrial fibrillation anterior segment sygenesis Total cholesterol levels LDL cholesterolNoneMood instability blood protein levelsNoneBody weight body mass index glucose IgA nephropathy Chronic lymphocytic leukaemia type diabetes erythrocyte indicesBody height menarche monocyte count blood protein levelsNoneMaximal midexpiratory flow rate blood protein levels respiratory function tests blood pressureCitrullinemia type II neonatal intrahepatic cholestasis due to citrin deficiency citrin deficiency citrullinemia type I bone mineral densityType diabetes breast cancer night sleep phenotypes response to cyclophosphamide in systemic lupus erythematosus with lupus nephritis strokeNone of times selectedTable Phenotypes associated with the most frequently selected genes by DRPT as obtained from Ensembl REST API Version DiscussionIn this study we showed the feasibility of using machine learning and feature selection to identify a reduced number of genes from microarray data to aid in the diagnosis of UC One might argue that distinguishing UC patients from Crohn™s disease CD patients has more clinical relevance than distinguishing UC patients from controls However we were limited on the choice of groups to classify by data availability as we could only find three gene expression data sets obtained from colonic samples of UC and CD patients in GEO GSE1152 GSE75214 and GSE126124 As children samples were transcriptionally profiled for GSE12612448 instead of adults ones we decided that the age difference could introduce extra biological variation in the expression data unrelated to UC That left us with only two data sets which were not enough to train the model with multiple data sets and have at least one holdout data set for validationAnother limitation of this study is that we used gene expression profiles of colonic samples Further research is required to assess the accuracy of our 32gene model in gene expression profiles of blood samples A recent study48 found a similar transcriptional profile between blood and colon tissue from patients with IBD If indeed Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cour 32gene model is found accurate in blood samples then a less invasive procedure such as a blood test could be used to diagnose UC instead of a colonoscopy or sigmoidoscopyIn a previous study where machine learning was employed to perform a risk assessment for CD and UC using GWAS data49 a twostep feature selection strategy was used on a dataset containing Crohn™s disease cases UC cases and controls with SNPs In that study Wei et a0al reduced the number of features by filtering out SNPs with pvalues greater than ˆ’ and then applied a penalized feature selection with L1 penalty to select a subset of SNPs We decided against filtering out genes based on an arbitrary pvalue of statistical significance of differential expression as researchers are strongly advised against the use of pvalues and statistical significance in relation to the nullhypothesis5051Our 32gene model achieved AP of and discriminating active UC patients from healthy donors and inactive UC patients from healthy donors respectively We found direct or indirect links to UC for about a quarter of the most frequently chosen genes The remaining genes should be further investigated to find associations with UC To put the performance of our 32gene model into perspective we looked at previous studies applying machine learning to create models for the diagnostic of UC Maeda et a0al52 extracted features from endocystoscopy images to train a SVM to classify UC patients as active or healing This approach achieve precision at recall which is lower than the one achieved by our 32gene model Figs a0 and Yuan et a0al17 applied incremental feature selection and a SMO classifier a type of SVM on gene expression data from blood samples to discriminate between healthy subjects UC patients and Crohn™s disease patients The 10fold crossvalidation accuracy of their best model using the expression values of genes to classify UC patients was while our method obtained better accuracy than this with substantially less number of genes In terms of potential for clinical translation of a machine learningbased model a model requiring to quantify the gene expression levels of fewer genes is more suitable for the development of a new diagnostic test than one requiring the quantification of the expression levels of thousands of genesUsing an efficient feature selection method such as DRPT and a SVMclassifier on gene expression data we generated a model that could facilitate the diagnosis of UC from expression measurements of genes from colonic samples To avoid systematic experimental bias on the training data we used three transcriptomic datasets from three separated studies Our top model was validated with promising results on a data set not used for training however additional research is required to evaluate the genes as potential biomarkers on a external set of subjectsReceived March Accepted July References Kaplan G G The global burden of IBD from to Nat Rev Gastroenterol Hepatol “ 101038 Ord¡s I Eckmann L Talamini M Baumgart D C Sandborn W J Ulcerative colitis Lancet “ nrgas tro2015150 101016S0140 Eisenstein M Ulcerative colitis towards remission Nature S33 101038d4158 Khan I et al Alteration of gut microbiota in inflammatory bowel disease IBD cause or consequence IBD treatment targeting the gut microbiome Pathogens a0 103390patho gens8 de Lange K M et al Genomewide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease Nat Genet “ 101038ng3760 Anderson C A et al Metaanalysis identifies additional ulcerative colitis risk loci increasing the number of confirmed associations to Nat Genet “ 101038ng764 Conrad K Roggenbuck D Laass M W Diagnosis and classification of ulcerative colitis Autoimmun Rev “ 101016jautre v201401028 Romagnoni A et al Comparative performances of machine learning methods for classifying Crohn disease patients using genomewide genotyping data Sci Rep 101038s4159 z Boland B S et al Validated gene expression biomarker analysis for biopsybased clinical trials in ulcerative colitis Aliment Pharmacol Ther “ 101111apt12862 Shah P et al Artificial intelligence and machine learning in clinical development a translational perspective NPJ Digit Med 101038s4174 Esteva A et al Dermatologistlevel classification of skin cancer with deep neural networks Nature “ McKinney S M et al International evaluation of an AI system for breast cancer screening Nature “ Molla M Waddell M Page D Shavlik J Using machine learning to design and interpret geneexpression microarrays AI 101038natur e2105 101038s4158 Mag perspectives Hum Genet “ 101038s4159 Xu J et al Translating cancer genomics into precision medicine with artificial intelligence applications challenges and future Mossotto E et al Classification of paediatric inflammatory bowel disease using machine learning Sci Rep Olsen J et al Diagnosis of ulcerative colitis before onset of inflammation by multivariate modeling of genomewide gene expression data Inflamm Bowel Dis “ 101002ibd20879 Yuan F Zhang YH Kong XY Cai YD Identification of candidate genes related to inflammatory bowel disease using minimum redundancy maximum relevance incremental feature selection and the shortestpath approach Biomed Res Int 101155201757419 Moehle C et al Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease J Mol Med Berl “ 101007s0010 Zahn A et al Aquaporin8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis World J Gas Noble C L et al Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis Gut “ troenterol Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0c Lepage P et al Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis Gastroenterology “ Inflamm Bowel Dis “ Vancamelbeke M et al Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease LennardJones J E Classification of inflammatory bowel disease Scand J Gastroenterol Suppl “ 10310900365 discussion “ H¤sler R et al A functional methylome map of ulcerative colitis Genome Res “ Barrett T et al NCBI GEO archive for functional genomics data sets“update Nucleic Acids Res D991“D995 Gumienny R GEOparse pypiproje ctGEOpa rse Troyanskaya O et al Missing value estimation methods for DNA microarrays Bioinformatics “ 101093bioin forma tics176520 abs200212104 Afshar M Usefi H HighDimensional Feature Selection for Genomics Datasets KnowledgeBased Systems arxiv Pedregosa F et al Scikitlearn machine learning in Python J Mach Learn Res “ Leclercq M et al Largescale automatic feature selection for biomarker discovery in highdimensional omics data Front Genet Burlington Holmes G Donkin A Witten I a0H Weka A machine learning workbench In Proceedings of ANZIIS ™ Australian New Zealand Intelligent Information Systems Conference “ Hall M et al The weka data mining software an update ACM SIGKDD Explor Newsl “ Witten I H Frank E Hall M A Pal C J Data Mining Practical Machine Learning Tools and Techniques Man Kaufmann M¼ller A C et al Introduction to Machine Learning with Python A Guide for Data scientists O™Reilly Media Inc California DemÅ¡ar J Statistical comparisons of classifiers over multiple data sets J Mach Learn Res “ Yates A et al The Ensembl REST API Ensembl data for any language Bioinformatics “ Kim E R Chang D K Colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis World J Gastroenterol diovasc Dis “ Schulte D et al Small dense LDL cholesterol in human subjects with different chronic

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

Thyroid_Cancer

"Marek™s disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Marek™s disease p53 P53 antibodyBackgroundMarek™s disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Marek™s disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to “ billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the œguardian of the genome [] andthe œcellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame ™ and ™ untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between “ []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [“] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMarek™s disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to “ h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE — b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE — 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE — b Spleen lymphoid tumor cells with cytoplasm expression HE — c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE —d Liver Negative controls were incubated with PBS HE —which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as theœhotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the “ bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequence™GTGGCCGTCTATAAGAAATCAGA3™™AAAAAGGGGGCGTGGTCAGT3™p532Annealing temperature „ƒSize of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturer™s instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript„¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions „ƒ for min followed by cycles of „ƒfor s „ƒ for s „ƒ for s and a final „ƒTable Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA“ delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere “ pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Student™s Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors™ contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe People™s Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Marek™s disease virus lytic replicationoncogenesis and control Expert Rev Vaccines “Reddy MS Book Review Marek™s Disease An Evolving Problem Vet Pathol“Boodhoo N Gurung A Sharif S Behboudi S Marek™s disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature “Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMarek™s Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep “Levine AJ p53 the cellular gatekeeper for growth and division Cell “Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist Updat“K¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res“ Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med “ Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther Med“Sabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol “Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res“ Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol “ Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer “ Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol “ 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Marek™s diseasetumorderived cell lines Leuk Res “ Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics “Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research “Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett “Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

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

Thyroid_Cancer

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

Thyroid_Cancer

EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food This risk assessment coversedible parts of potato plants and other food plants containing GAstomato andaubergine In humans acute toxic effects of potato GAs asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea For these effects the CONTAMPanel identified a lowestobservedadverseeffect level of mg total potato GAskg body weight bwper day as a reference point for the risk characterisation following acute exposure In humans noevidence of health problems associated with repeated or longterm intake of GAs via potatoes hasbeen identified No reference point for chronic exposure could be identified from the experimentalanimal studies Occurrence data were available only for asolanine and achaconine mostly forpotatoes The acute dietary exposure to potato GAs was estimated using a probabilistic approach andapplying processing factors for food Due to the limited data available a margin of exposure MOEapproach was applied The MOEs for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the P95 exposure in all surveysFor adult age groups the MOEs indicate a health concern only for the food consumption surveys withthe highest P95 exposures For tomato and aubergine GAs the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data For horses farm andcompanion animals no risk characterisation for potato GAs could be performed due to insufficient dataon occurrence in feed and on potential adverse effects of GAs in these species European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityKeywords glycoalkaloids GAs solanine chaconine potato margin of exposure MOE food feedRequestor European CommissionQuestion number EFSAQ201600811Correspondence contamefsaeuropaeu Leon Brimer was a member of the Working Group on Glycoalkaloids in food and feed until August wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodPanel members Margherita Bignami Laurent Bodin James Kevin Chipman Jes 13us del Mazo BettinaGraslKraupp Christer Hogstrand Laurentius Ron Hoogenboom JeanCharles Leblanc Carlo StefanoNebbia Elsa Nielsen Evangelia Ntzani Annette Petersen Salomon Sand Dieter Schrenk TanjaSchwerdtle Christiane Vleminckx and Heather WallaceAcknowledgements The Panel wishes to thank the following for the support provided to thisscientific output Kelly Niermans The Panel wishes to acknowledge all European competentinstitutions Member State bodies and other anisations that provided consumption and occurrencedata for this scientific outputSuggested citation EFSA CONTAM Panel EFSA Panel on Contaminants in the Food Chain Schrenk DBignami M Bodin L Chipman JK del Mazo J Hogstrand C Hoogenboom LR Leblanc JC Nebbia CSNielsen E Ntzani E Petersen A Sand S Schwerdtle T Vleminckx C Wallace H Brimer L Cottrill BDusemund B Mulder P Vollmer G Binaglia M Ramos Bordajandi L Riolo F Rold 13anTorres R and GraslKraupp B Scientific Opinion “ Risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products EFSA Journal pp 102903jefsa20206222ISSN European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityThis is an access under the terms of the Creative Commons AttributionNoDerivs Licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodifications or adaptations are madeReproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderFigure Elsevier Figure Springer Figure American Chemical Society SpringerThe EFSA Journal is a publication of the European FoodSafety Authority an agency of the European UnionwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodSummaryThe European Commission asked EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food in particular in potatoes andpotatoderived products This risk assessment covers edible parts of potato plants and other foodplants containing GAs in particular tomato and aubergine Nonedible parts of GA containing plantshave not been considered with the exception of potato sprouts The Panel developed the draftscientific opinion which underwent a public consultation from February to April Thecomments received and how they were taken into account when finalising the scientific opinion werepublished in an EFSA Technical Report EFSA GAs are present in many plants of the family of Solanaceae and contribute to plant resistanceagainst pests and pathogens GAs are composed of a steroidal aglycone and an oligosaccharide sidechain In commercial potato cultivars S tuberosum the main GAs are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively The aubergine fruit S melongena contains primarily the GAs asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively Inlycopersicum atomatine and adehydrotomatine are the major GAs withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelySHuman risk assessmentIn experimental animals the potato GAs asolanine and achaconine show a relatively low oralbioavailability with differences between species Hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats Due to the limited information themetabolic profiles of potato GAs in experimental animals could not be characterisedIn humans asolanine and achaconine are systemically absorbed following ingestion For bothsubstances relatively long serum halflives were reported suggesting a possible accumulation The bloodclearance of the respective aglycone solanidine appears to be slow Accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of GAs Nofurther information is available on metabolism and excretion of potato GAs in humansThere are no toxicokinetic data on tomato and aubergine GAs and their aglycones in experimentalanimals and humansIn acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice Reliable data on other potatoGAs or tomato and aubergine GAs and their aglycones are missingIn repeated oral dose studies on potato GAs rodents showed nonspecific effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine Hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw Solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the GAsThe tomato GA atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day At higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats In mice a to 2weektreatment with the aubergine GA asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per dayDevelopmental studies have been performed mainly in hamsters treated with potato GAs and theiraglycones for only one day or for a short very restricted time period during gestation Outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia These malformations occurred at dosesof mgkg bw per day and above for GAs and of mgkg bw per day and above for theaglycones No noobservedadverseeffectlevelLOAEL could be identified from these studies Reduced postnatal survival of pups due to insufficientmilk production was reported when pregnant Holtzman rats had been exposed to mg of asolaninekg bw per day Studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine Decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectNOAEL orlevelwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodfor month Similar effects were observed in Rhesus monkeys exposed to mgkg bw per day for monthsFrom the limited number of studies available there was no evidence for genotoxicity of the potatoGAs asolanine and achaconine and the aglycone solanidine as well as for the aubergine GA asolamargine However there is not sufficient information to conclude on the genotoxic potential ofthese GAsNo longterm chronic toxicitycarcinogencity study for potato tomato or aubergine GAs or for therespective aglycones could be identifiedIn humans acute toxic effects following ingestion of potato GAs include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato GAs potato TGA intake of mgkg bw or more Further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeaIn severe cases paralysis respiratory insufficiency cardiac failure coma and death have beenreported Doses in the range of “ mg potato TGAskg bw are considered to be potentially lethal forhumans Results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato GAsRegarding the mode of action adverse effects of GAs may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesAfter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeaGAs inhibit acetylcholinesterase AChE and serum butyrylcholinesterase BuChE by a reversiblecompetitive mode of action The relative potency of inhibition of asolanine and achaconine appearsto be similar The aglycones exert weak or no inhibitory effects The excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with GAsAt high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol As a consequence blood cholesterollevels were lowered in rodentsThe CONTAM Panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato GAs in humans The CONTAM Panel selectedthe LOAEL of mg potato TGAkg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers The available data onacute toxicity were considered insufficient to establish a healthbased guidance value Instead thePanel used the margin of exposure MOE approach to assess a possible health concern from acuteexposure to potato TGAs via foodAssuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of AChE activity the Panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics Accordingly anMOE higher than indicates that there is no health concern This MOE of takes into account theextrapolation from a LOAEL to a NOAEL a factor of and the interindividual variability intoxicodynamics a factor of The experimental data available for repeated dose toxicity are not sufficient to identify a referencepoint for chronic exposure to potato GAs In humans no evidence of health problems associated withrepeated or longterm intake of GAs via potatoes has been identifiedRegarding GAs or aglycones occurring in edible parts of food plants other than S tuberosum nosuitable study for determining a reference point for tomato or aubergine GAs or aglycones wasidentifiedOccurrence data were only available for asolanine and achaconine and mostly for ˜Maincroppotatoes™ and ˜New potatoes™ Few data were available for processed food No data on the occurrenceof tomato and aubergine GAs and their aglycones were submitted to EFSASince the occurrence data on potato GAs did not cover all the food categories containing potatoesin the Consumption Database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities RPC maincrop potatoes andnew potatoes and the RPC Consumption Database The Panel decided to combine the occurrence of˜New potatoes™ with that of ˜Maincrop potatoes™ and the mean upper bound UB occurrence sum ofwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the P95 occurrence was mgkg The minimum and maximum reported concentrations were and mgkg respectivelyThe acute dietary exposure to potato TGAs was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes As no occurrence data wereavailable for GAs in tomato and aubergine these foods were not included in the exposure assessmentProcessing of potatoes has been reported to reduce the content of GAs in the final processedproduct In general and according to the literature the peeling of potatoes reduced the GA contentby “ boiling in water and blanching of peeled potatoes by “ and frying in oil of peeledpotatoes by “ Microwave and oven baking of unpeeled potatoes may cause a reduction in theGA content by “ and by “ respectively No information has been found about thechemical nature of the GA degradation products For the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsInformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled Where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedThe mean UB exposure to potato TGAs across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers The 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the confidence intervalComparing the LOAEL for potato TGAs of mgkg bw per day with the acute exposure estimatesthe MOEs for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the P95 exposure in all surveys For adult age groups theMOEs indicate a health concern only for the food consumption surveys with the highest P95exposuresThe CONTAM Panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato TGA intake may be below the MOE of The highestnumber of survey days with intake of potatoes below the MOE of was estimated for toddlers followed by children For the other age groups the estimated TGA intake was below the MOEof in up to “ of the survey daysFor tomato and aubergine GAs the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansThe CONTAM Panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato GAs in food is moderate and that overall the identified uncertainties may eithercause an over or underestimation of the riskFarm animals horses and companion animals risk assessmentInformation on the toxicokinetics of GAs was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows™ milkNo data on the potential adverse effects of potato GAs in horses companion animals cats anddogs or fur animals were identified Due to an insufficient database on the adverse effects of GAs inruminants pigs poultry rabbits and fish an acute reference dose could not be derivedPotatoes are not grown specifically as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs Some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsData on potato GAs in feed were insufficient to perform an exposure assessmentThus no risk characterisation could be performed due to insufficient occurrence data of GAs forfeed and the lack of or limited data on the adverse effects of GAs in farm animals horses orcompanion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodRecommendationsThe following needs have been identified to improve the risk assessment for humans and reducethe uncertaintiescid129Research on the occurrence of GAs and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 Occurrence data on GAs and their aglycones in potato processed products including foods forinfantscid129 Occurrence data on GAs and their aglycones in tomato and aubergine and products thereofcid129 Data on the toxicokinetics of potato tomato and aubergine GAs and aglycones in experimentalanimals and humanscid129 Data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine GAs and aglycones in experimental animalsStudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129The following needs have been identified to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 Occurrence data on potato GAs and their aglycones in feedcid129Studies on the kinetics and the potential adverse effects from feed material containing GAs ofpotato GAs in farm animals horses and companion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodTable of contentsAbstractSummaryIntroduction Background and Terms of Reference as provided by the requestor Interpretation of the Terms of Reference Supporting information for the assessment Chemistry Analytical methods Sources Potatoes Tomatoes Aubergine Previous risk assessments Legislation and other standards Data and methodologies Methodology for data collection selection of evidence and study appraisal Food and feed occurrence data submitted to EFSA Data collection and validation Data analysis Food and feed consumption data Food consumption data Feed consumption data Food classification Methodology for Exposure assessment Methodology for Risk characterisation Assessment Hazard identification and characterisation Toxicokinetics Experimental animals aSolanine aChaconine Humans Mixtures of asolanine and achaconine Solanidine Biomarkers of exposure Farm animals horses and companion animals Summary on toxicokinetics Toxicity in experimental animals Acute toxicity studies GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Summary on acute toxicity studies Repeated dose toxicity studies GAs and aglycones from edible parts of S tuberosum GAs and aglycones from edible parts of food plants other than S tuberosum Developmental and reproductive toxicity studies Developmental effects Reproductive effects Immunotoxicity studies Studies on cardiovascular effects Neurotoxicity studies Genotoxicity GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Carcinogenicity studies Studies on metabolic effects GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Observations in humans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and food GAs from S tuberosum Reports on intoxications Studies in human volunteers Epidemiological studies Summary GAs from food plants other than S tuberosum Case Reports Adverse effects in farm animals horses and companion animals Ruminants Pigs Poultry Rabbits Fish Horses Companion animals cats and dogs Fur animals Reports on intoxications Mode of action Membrane effects with implications for the gastrointestinal tract Inhibition of cholinesterases ChEs Comparative determination of inhibition of ChEs in vitro Determination of inhibitory constants Ki for GAs on inhibition of ChEs in vitro Inhibition of ChEs in vivo Developmental and reproductive effects of GAs and their aglycones Inhibition of cholinesterases and effects in the immune system Interference with metabolism Considerations of critical effects and doseresponse analysis for the human risk assessment GAs from edible parts of S tuberosum Considerations of critical effects and doseresponse analysis Derivation of a healthbased guidance value HBGV or margin of exposure MOE approach GAs from edible parts of food plants other than S tuberosum Considerations of critical effects and doseresponse analysis Consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment Occurrence data Occurrence data submitted to EFSA Previously reported occurrence data in the literature Literature on occurrence data on food Occurrence data on GAs in potatoes Occurrence data on GAs in tomatoes Occurrence data on GAs in aubergines Occurrence data on GAs in other food products Literature occurrence data in feed Influence of storage and processing on the content of GAs GAs from S tuberosum Storage of potatoes Processing of potatoes for food consumption Processing of potatoes for feed GAs from food plants other than S tuberosum Summary on the influence of storage and processing on the levels of GAs Exposure assessment Current acute dietary exposure assessment for humans Previously reported dietary exposure assessments Current dietary exposure assessment for farm animals horses and companion animals Risk characterisation Human health risk characterisation GA from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Farm animals horses and companion animal risk characterisation Uncertainty analysis Assessment objectives Exposure scenarioexposure model wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodHazard identification and characterisation Summary of uncertainties Conclusions Hazard identification and characterisation Toxicokinetics Toxicity in experimental animals Observations in humans Adverse effects in farm animals horses and companion animals Mode of action Margin of exposure MOE approach Occurrence and exposure Food Feed Risk characterisation Human health risk characterisation Farm animals horses and companion animal health risk characterisation Recommendations Documentation provided to EFSA References Abbreviations Appendix A “ Major glycoalkaloids and their aglycones present in Solanum species Appendix B “ Identification and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food Appendix C “ Details of the study design of the toxicokinetic studies Appendix D “ Comparison of developmental toxicity of single dose studies Appendix E “ Inhibition of cholinesterases by GAs Appendix F “ Rapid Alert System for Food and Feed RASFF reports on the presence of Solanum nigrum infood products Appendix G “ Studies on the toxicity of Glycoalkaloids not considered in the risk assessment Appendix H “ Additional scenario for the human risk characterisation Annex A “ Occurrence data in food and feed submitted to EFSA and dietary exposure assessment forhumans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodIntroductionBackground and Terms of Reference as provided by the requestorBackgroundMany plants in the family Solanaceae contain glycoalkaloids and they are considered to be naturaltoxins The plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine Their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcAmongst the most widely cultivated food crops aubergines tomatoes and potatoes are in theSolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowThe glycoalkaloids of most relevance to food safety are those occurring in the potato Thepredominant toxic steroidal glycosides in potato are asolanine and achaconine They occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors Concentrations ofglycoalkaloids are “ times greater in the peel than in the flesh There is considerable variation inglycoalkaloid content among potato cultivars Storage conditions especially light and temperature aremainly responsible for increases in solanine Although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light Increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel Thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsBitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include flulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea More severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision There are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriesPotatoes and potatoderived products are listed in the Catalogue of feed materials1Terms of ReferenceIn accordance with Art of Regulation EC No the European Commission asks theEuropean Food Safety Authority for a scientific opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsInterpretation of the Terms of ReferenceThe CONTAM Panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids GAs eg tomato and aubergine Nonedible parts ofGA containing plants have not been considered with the exception of potato sprouts In particular theCONTAM Panel concluded this Opinion should comprise thea evaluation of the toxicity of GAs in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid profile ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to EFSAc estimation of the dietary exposure of the European population to GAs in food in particular inpotatoes and potatoderived products including the consumption patterns of specific groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to GAs in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the European population including specific groupsof the population if appropriate as the consequence of the estimated dietary exposure Commission Regulation EU No of January on the Catalogue of feed materials OJL p wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in Europe as the consequence ofthe estimated dietary exposure Exposure to GAs from weeds containing GA is only addressedin this Opinion in the context of accidental intake by farm animalsWhen referring to GAs in potatoes the term total GAs TGA refers to a material comprising asolanineand achaconine as major fraction with no specification on the occurrence of minor GAs as well as band cforms of solanine and chaconine Similarly when referring to tomato and aubergine the termTGA refers to the GAs from the corresponding species and forms thereofSupporting information for the assessment ChemistrySolanine is one of the first alkaloids that has been isolated from nature by Desfosses in Friedman et al In Zwenger and Kind reported that solanine contains a glycoside sidechain Zwenger and Kind Only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids GAs asolanine and achaconine that share the same solanidineaglycone Kuhn and L‚¬ow Since then at least different GAs have been isolated and fullystructurally elucidated from over species of the Solanaceae family S 13anchezMata et al AlSinani and Eltayeb The chemical structures and some physical properties of the most importantones are listed in Appendix AGAs are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see Figure Friedman et al Friedman Milner et al The GAs of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone GAs often contain a double bond between C5 and C6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents The stereochemistry at carbons C22 and C25 is well definedtheconfiguration is 22R 25Stheitconfiguration is 22S 25S Friedman et al in solanidineis 22R 25R and in tomatidenoltomatidinein solasodineFurther diversification is generated by the composition of the glycoside sidechain Most GAscontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate In commercial potato cultivars Solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentFigure Wild S tuberosum varieties may contain a much wider range of GAs Friedman et al Distl and Wink The aubergine fruit derived from S melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose Friedman derived from SIn tomato fruitThe prefix alpha a refers to the intact glycoside while the prefixe

Thyroid_Cancer

case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies P“LGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine„¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete„¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and “ of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in “ and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patient™s glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors™ contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci “ https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol “ https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol “ https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg “ https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully 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doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol “ https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med “ https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol “ https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology “ https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other epileptogenic brain neoplasms J Neurol Neurosurg Psychiatry “ https doi101136jnnp200405160 Ryall S Tabori U Hawkins C Pediatric lowgrade glioma in the era of molecular diagnostics Acta Neuropathol Commun https doi101186s4047 z Ryall S Zapotocky M Fukuoka K Nobre L Guerreiro Stucklin A Bennett J Siddaway R Li C Pajovic S Arnoldo A et al Integrated molecular and clinical analysis of pediatric lowgrade gliomas Cancer Cell “583e565 https doi101016jccell Salisbury TB Arthur S The regulation and function of the ltype amino acid transporter LAT1 in cancer Int J Mol Sci https doi103390ijms1 Schindler G Capper D Meyer J Janzarik W Omran H HeroldMende C Schmieder K Wesseling P Mawrin C Hasselblatt M et al Analysis of BRAF V600E mutation in nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma ganglioglioma and extracerebellar pilocytic astrocytoma Acta Neuropathol “ https doi101007s0040 Sumdani H Shahbuddin Z Harper G Hamilton L Case report of rarely described polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg “ https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol “ https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery “ discussion “ https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med “ https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging “ https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg “ https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol “ https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol “ https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol “ https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol “ https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res “ https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and tumorigenesis Cell Rep “ https doi101016jcelre p201712002 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res “ https doi101038sjcr72901 Zhao C Zhang Y Wang J A metaanalysis on the diagnostic performance of 18FFDG and 11Cmethionine PET for differentiating brain tumors AJNR Am J Neuroradiol “ https doi103174ajnrA3718 Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

Rheumatoid arthritis RA is a systemic chronic inflammatory disease that affects synovial joints and has various extraarticular manifestations including atherosclerotic cardiovascular disease CVD Patients with RA experience a higher risk of CVD leading to increased morbidity and mortality Inflammation is a common phenomenon in RA and CVD The pathophysiological association between these diseases is still not clear and thus the risk assessment and detection of CVD in such patients is of clinical importance Recently artificial intelligence AI has gained prominence in advancing healthcare and therefore may further help to investigate the RACVD association There are three aims of this review to summarize the three pathophysiological pathways that link RA to CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators useful for RA patients and to understand the role of artificial intelligence in CVD risk assessment in RA patients Our search strategy involves extensively searches in PubMed and Web of Science databases using search terms associated with CVD risk assessment in RA patients A total of peerreviewed s were screened for this review We conclude that a two of the three pathways directly affect the atherosclerotic process leading to heart injury b carotid ultrasound imagebased calculators have shown superior performance compared with conventional calculators and c AIbased technologies in CVD risk assessment in RA patients are aggressively being adapted for routine practice of RA patientsKeywords Arthritis a0· Rheumatoid a0· Atherosclerosis a0· Cardiovascular disease a0· Inflammation a0· Carotid artery diseases a0· Carotid intimamedia thickness a0· Risk assessmentIntroductionRheumatoid arthritis RA is a chronic inflammatory disease that not affects only synovial joints but also has several extraarticular involvements including those related to the skin eyes heart lungs kidneys and other ans [ ] It affects of the global population with a higher prevalence in females when compared with males [ ] Cardiovascular disease CVD is a common manifestation in RA patients with a two to threefold higher risk of cardiovascular events and mortality compared with a normal population [] However this increased risk is not entirely jasjitsuriatheropointcom Jasjit S Suri Extended author information available on the last page of the explained by conventional risk factors [] Current statistically derived CVD risk calculators use conventional risk factors alone [“] are not suitable for RA patients and they either underestimate or overestimate the risk [“] This may be because of the paradoxical behavior of some of the conventional risk factors such as body mass index lowdensity lipoprotein highdensity lipoprotein and total cholesterol in RA [ ] Despite this lack of clarity the guidelines by the European League Against Rheumatism EULAR recommend aggressive control of these conventional risk factors [ ] Recent attempts were made to improve the CVD risk assessment in the RA population including the development of œRAspecific risk factors in the CVD risk calculators [“] However such calculators could not provide adequate improvement in risk Vol01234567891 0c Rheumatology Internationalprediction and reportedly still underestimated or overestimated CVD risk in RA patients [ ]To provide a better CVD risk assessment in RA a pathophysiological association between these diseases should be understood as this would help in refining CVD risk predictors in RA patients [] Atherosclerosis a common phenomenon in RA [ ] can be adequately monitored using imaging modalities such as magnetic resonance imaging [] computed tomography [] optical coherence tomography [] and ultrasound [] Each of these imaging modalities offers unique information about morphological variations in atherosclerotic plaque Ultrasound imaging specifically in carotid arteries is a comparatively lowcost noninvasive radiationfree and easytouse imaging modality that is widely adopted in preventive cardiovascular and clinical vascular practices [ ] The imagebased phenotypes of carotid ultrasound such as carotid intimamedia thickness cIMT and carotid plaque are considered surrogate markers of coronary artery disease and have been used for preventive CVD risk assessments in several studies [“] These imagebased phenotypes indicate the morphological variations in the atherosclerotic plaque and are associated with the inflammatory markers of RA [“] Patients with RA have elevated cIMT and have more plaque area PA when compared with nonRA patients [“] Thus the inclusion of these imagebased phenotypes in risk prediction models may improve the CVD risk assessments of RA patients Recent studies have combined the effect of these imagebased phenotypes with conventional risk factors including proinflammatory markers like erythrocyte sedimentation rate ESR to perform CVD risk assessment [“] Such integrated risk calculators have demonstrated better CVD risk stratification when compared to traditional CVD risk calculators in nonRA patients [ ]Besides these statistically derived CVD risk calculators artificial intelligence AIbased techniques are also penetrating several medical imaging and risk assessment applications [“] AIbased algorithms such as machine learning ML methods provide a better CVD risk assessment when compared with statistically derived conventional risk calculators [ ] So far AI algorithms have been used for CVD risk assessment in the nonRA population and their potential still needs to be evaluated in RA cohorts However AI is well adapted for RA screening and diagnosis [“] This review provides an insight into how the AIbased algorithms can be used for CVD risk assessment in RA patients There are three aims of this review to summarize the pathophysiological pathways that link RA with CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators for RA patients and to provide an understanding of the role of artificial intelligence in CVD risk assessment in RA patientsSearch strategyFigure a0 shows a flow diagram indicating the search strategy for this narrative review To write a comprehensive narrative review it is essential to select at least two credible databases that provide highquality peerreviewed s [] This review is the outcome of several searches in the PubMed and Web of Science databases using keywords such as œcardiovascular diseases AND œrisk assessment AND œrheumatoid arthritis œcarotid atherosclerosis AND œrheumatoid arthritis œnoninvasive imaging AND œrheumatoid arthritis œcarotid ultrasound AND œrheumatoid arthritis œcarotid intimamedia thickness OR œcarotid plaque AND œinflammatory markers œcarotid atherosclerosis AND œerythrocyte sedimentation rate OR œC reactive protein œmachine learning AND œrheumatoid arthritis and œmachine learning AND œcardiovascular risk assessment AND œrheumatoid arthritis The availability of all these keywords in the and the full text was investigated to select the relevant s Peerreviewed s published in the last a0years were then given priority Citations from the published s were also shortlisted for this review All these s were subsequently filtered by the expert coauthors to select only those that met the objectives of this review leading to sPathophysiology of a0RA leading to a0CVDThe pathophysiological association between RA and CVD can be explained in two stages the role of traditional risk factors and direct vascular damage Inflammation plays a pivotal role in both of these stages []The role of a0traditional risk factors in a0the a0pathophysiology of a0RA‘driven atherosclerotic CVDThe righthand panel of Fig a0 explains the pathophysiological association between RA and CVD via four pathways [Ia“Id] governed by traditional risk factors such as hypertension proatherogenic dyslipidemia insulin resistance and obesity Patients with RA are generally found with proinflammatory cytokines such as interleukin IL IL6 and tumor necrosis factor α TNFα [] These proinflammatory cytokines are found in the synovium which triggers a systemic inflammatory response and may result in damage to the vascular endothelial cells [] Nitric oxide NO and cyclooxygenase1 are two essential components required 0cRheumatology International Fig Flow diagram for the search strategyto maintain the healthy endothelium which is inhibited by TNFα and IL6 thereby resulting in endothelial cell damage [ ] Inhibiting endothelial NO leads to arterial stiffness [] and is further associated with an increase in peripheral vascular resistance PVR [] thus leading to hypertension in RA patients Additionally several medications used to treat RA such as diseasemodifying antirheumatic drugs DMARDs leflunomide and cyclosporine glucocorticoids nonsteroidal antiinflammatory drugs NSAIDs and cyclooxygenase II inhibitors Cox IBs may also be involved in the development of hypertension in RA patients [ ]Another pathophysiological link between RA and CVD is proatherogenic dyslipidemia [] Nearly “ of RA patients have proatherogenic dyslipidemia [] In nonRA patients increased CVD risk is associated with elevated levels of lowdensity cholesterol LDLc total cholesterol and reduced highdensity lipoprotein cholesterol HDLc However in RA patients low levels of total cholesterol TC low levels of LDLc and suppressed levels of HDLc have been reported This condition is known as œthe lipid paradox [] Highly suppressed HDL levels in RA patients are œproatherogenic [] Furthermore RA patients show high atherogenic index levels despite low lipid levels The atherogenic index is calculated as a ratio of TC HDLc and it may vary according to their levels [] Apolipoprotein B Apo B is a major apolipoprotein in LDL and several studies have indicated an increase in the ratio of Apo B Apo A in RA patients [] A combination of low TC LDLc and suppressed HDLc levels with a high atherogenic index and a high ApoBApoA ratio behaves as proatherogenic dyslipidemia [ ] Longstanding proatherogenic dyslipidemia causes atherosclerosis and eventually CVDRheumatoid cachexia is another important RAspecific characteristic that increases CVD risk [] It is characterized by significantly increased adiposity and reduced muscle mass while one maintains their bodyweight [] The pathophysiology [shown in œpathwayI c] behind R cachexia can be explained in two ways It is characterized by the reduction of muscle mass that is largely due to increased inflammatory cytokines particularly TNFα by activating the transcriptional nuclear factorkappa B cells NFkB pathway and promoting the ubiquitin pathway which causes catabolismproteolysis muscle protein breakdown [ ] Central obesity or abdominal obesity is present in “ of women and “ of men This causes visceral adiposity in RA which has an additional adverse impact on CVD [] On the other hand increased adiposity also induces the production of inflammatory cytokines in RA which further worsens this 0c Rheumatology InternationalFig Pathophysiological association between rheumatoid arthritis and cardiovascular disease IL1 interleukin IL6 interleukin TNFα tumor necrosis factor α EC endothelial cells SMC smooth muscle cells MCP1 monocyte chemoattractant protein MCSF macrophage colonystimulating factor VCAM vascular cell adhesion molecule ICAM intercellular adhesion molecule NSAIDs nonsteroidal antiinflammatory drugs CoxIBs cyclooxygenase inhibitors HTN hypertension PVR peripheral vascular resistance TC total cholesterol HDL highdensity lipoprotein LDL lowdensity lipoprotein APOB apolipoprotein B APOA apolipoprotein A NFkB nuclear factorkappa B cellsscenario [] This syndrome may be explained in the triad of increased adiposity reduced muscle mass and low body mass index BMIEpidemiological studies have suggested a strong association between insulin resistance IR metabolic syndrome and RA [ ] [shown in œpathwayI d in the dark greendotted box] Inflammation plays a crucial role in these three conditions [] In patients with RA IR serves as an independent prognostic risk factor that signifies the presence of subclinical atherosclerosis it is determined by carotid intimal thickness cIMT and is measured by carotid ultrasonography [] Longstanding inflammation due to RA promotes oxidative stress endothelial dysfunction and atherosclerosis in this population []Progression of a0atherosclerosis and a0direct vessel damage in a0RAIn RA the activation of Tcells and mast cells increases the production of proinflammatory cytokines such as IL1 IL6 and TNFα These proinflammatory cytokines stimulate endothelial cells ECs and smooth muscle cells SMCs in subendothelium [] by expressing cell adhesion molecules such as vascular cell adhesion molecule VCAM and the œintercellular adhesion molecule ICAM [] and by producing chemokines including monocyte chemoattractant protein MCP and macrophage colonystimulating factor MCSF The activation of endothelial cells allows the migration of LDLc into the subendothelial layer where 0cRheumatology International it becomes oxidized and triggers the inflammatory response for the recruitment of immune cells such as T lymphocytes and monocytes in the intimal layer Once they enter the intimal layer monocytes are transformed into macrophages and they then take up the oxidized LDLc to become foam cells The completion of this complex process then leads to the formation of atherosclerotic plaque Macrophages also trigger the migration of smooth muscle cells from tunica media to tunica intima and initiate their proliferation The SMCs form a thin fibrous cap to prevent the encroachment of atherosclerotic plaque towards the lumen However over time proinflammatory cytokines enzymes and free radicals cause fibrous cap erosion and make the plaque vulnerable for rupture The amplification of the inflammatory response results in the acceleration of plaque formation eventually leading to plaque rupture and thrombotic events which damage the blood vessels Pathway II of Fig a0 represents this processCurrent conventional CVD risk prediction models for a0RAOver the last decade several CVD risk assessment calculators have been developed very few of which are recommended by the cardiovascular risk management guidelines [ ] Some standard cardiovascular risk prediction algorithms are the Framingham risk score FRS [] Systematic Coronary Risk Evaluation SCORE [] American College of CardiologyAmerican Heart Association ACCAHA risk score [] World Health anization WHO risk charts [] and Reynolds™s risk score RRS [] These risk calculators use traditional risk factors such as patient demographics age gender ethnicity blood biomarkers lowdensity lipoprotein cholesterol highdensity lipoprotein cholesterol and total cholesterol behavioral markers smoking and alcohol consumption and physiological markers height weight body mass index All these risk calculators were initially developed for nonRA populations therefore when used in RA cohorts CVD risk is substantially underestimated [“] The use of traditional risk factors alone while not considering RAspecific inflammatory markers could be another reason for such underestimation However RRS included an RAspecific inflammatory marker called œhigh sensitivity Creactive protein hsCRP [] for CVD risk prediction but did not report any significant improvement in the CVD risk assessment [] Rajagopalan et a0al [] also reported a small improvement in area under the curve in females and in males when C reactive protein CRP or erythrocyte sedimentation rate ESR was added to the FRSOver the past few years several efforts have been made to improve the cardiovascular risk assessment in RA patients The EULAR guidelines recommended the use of a modified SCORE mSCORE in RA patients positive with rheumatoid factor RF or anticitrullinated protein antibodies ACPA and RA duration of more than a0years [ ] Cox et a0al [ ] developed the QRISK2 and QRISK3 algorithms which use the presence of RA as a CVD risk predictor hazard ratio confidence interval “ Solomon et a0al [] also developed an RAspecific CVD risk calculator called expanded risk score or ERS by including RAspecific biomarkers [such as disease activity disease duration a modified health assessment questionnaire HAQ disability index and daily prednisone use] with the traditional biomarkers used in the Coxbased model The authors reported an improvement of in cindex when validating the risk score on the reserved dataset Recently Curtis et a0al [] also proposed a CVD risk prediction tool for RA patients by combining conventional and RAspecific risk factors The authors predicted the risk of composite CVD events such as MI stroke and death during the followup period of a0years The area under the curve AUC for cardiovascular risk stratification for this model was All these RAspecific CVD risk scores reported a better risk assessment on the proprietary databases Still when compared with other risk calculators in different RA cohorts these calculators have demonstrated mixed results [“] Crowson et a0al [] reported an underestimation of CVD risk by FRS and RRS in RA patients The observed risk was twice the predicted risk Furthermore the authors did not report any improvement in cardiovascular risk prediction when CRP was added to their model Arts et a0al [] investigated the roles of SCORE FRS RRS and QRISK2 in RA patients Out of these four models SCORE FRS and RRS underestimated CVD risk in RA patients whereas the QRISK2 reported an overestimation The AUC ranged between and for the four risk models A similar study by Arts et a0al [] investigated the performance of the original recalibrated and improved version of SCORE calculators to predict the CVD risk in RA patients The AUC values for these scores were CI “ “ and “ All these three scores underestimated the CVD risk in RA patients In short even after the SCORE was redesigned using the RAbased risk factors it did not result in an adequate CVD risk assessment In another study by Crowson et a0al [] of RA patients a CVDrisk prediction model was developed that reported better performance AUC compared with conventional risk calculators such as FRS AUC ACCAHA AUC SCORE AUC and QRISK2 AUC Furthermore conventional risk calculators either overestimated or underestimated CVD risk in RA patients Wahlin et a0al [] compared the expanded risk score ACCAHA risk score and a modified version of ACCAHA with a multiplier of for a CVD risk assessment of 0c Rheumatology International RA patients The authors also reported an underestimation of CVD risk by all calculators However the discrimination ability was slightly better since AUC for ERSRA risk was compared to AUC of for two variants of ACCAHAThe overall trend of all these risk prediction algorithms developed for general and RA cohorts indicates a œpoor CVD risk assessment in patients with RA One possible reason for such poor performance is the paradoxical behavior of some of the risk factors such as lipids and body mass index Another potential reason for this outcome is the inclusion of risk factors that do not provide complete information about the CVD risk profile in RA patients [] Corrales et a0al [] indicated a high prevalence of carotid atherosclerosis plaque in patients with lowCVD risk This observation demonstrated the limited ability of conventional risk factorbased algorithms to improve the CVD risk assessment process which may be improved using imaging modalities Therefore there is still room to develop more accurate automated and reliable risk calculators for RA patients by exploring and including nontraditional risk factors such as genetic biomarkers inflammatory biomarkers or imagebased atherosclerotic plaque phenotypes in the risk prediction algorithmCarotid ultrasound atherosclerosis imaging for a0CVD risk assessment in a0RA patientsImaging modalities are becoming essential for the visualization of atherosclerotic plaque and CVD risk assessment in RA patients [] Noninvasive imaging modalities such as computed tomography magnetic resonance imaging ultrasound and positron emission tomography are currently used to assess carotid atherosclerosis in RA patients [] MRI is used to measure the plaque composition including calcification lipidrich necrotic core and fibrous cap thickness [] Computed tomography is generally used to determine carotid artery stenosis [] Ffludeoxyglucose“positron emission tomography FDGPET is a nuclear imaging modality that quantifies the inflammation in carotid atherosclerotic plaque [] Noninvasive carotid ultrasound is a commonly adopted imaging modality that can capture morphological variations in the atherosclerotic plaque quantified using carotid intimamedia thickness cIMT carotid intimamedia thickness variability IMTV and plaque area [] When compared with other noninvasive counterparts carotid ultrasound is less expensive and easier to use [ ] Therefore the scope of this review is restricted to the use of carotid ultrasound for CVD risk assessment in RA patients The automated cIMT and carotid PA are considered surrogate markers of coronary artery disease and widely used for CVDstroke risk assessment [“]Several studies have shown a high prevalence of increased cIMT and carotid plaque in RA patients [“] Studies have also demonstrated the significant association between these carotid atherosclerosis biomarkers and RAspecific markers of inflammation such as ESR CRP and IL6 [“] Table a0 provides some of such studies that link both carotid atherosclerosis and RA using two sets of biomarkers ie imagebased phenotypes and inflammatory biomarkers One common observation from these studies is that patients with RA show an elevated cIMT and carotid plaque area compared with nonRA cohorts row R2“R4 of Table a0 [ ] This association between carotid atherosclerosis and RA also seems independent of the three carotid artery segments common carotid artery carotid bulb and internal carotid artery from where the cIMT or plaque was measured [ ] However several studies have reported more aggressive atherosclerotic plaque formation in the carotid bulb segment when compared to other arterial segments [] The higher plaque prevalence in the carotid bulb is a consequence of turbulent blood flow and reduced shear stress which leads to endothelial dysfunction [ ] This observation of higher plaque in a bulb has also been confirmed in RA patients [] Figure a0 shows carotid ultrasound scans for RA Fig a03a b and nonRA patients Fig a03c d The lefthand side panel of Fig a03a c shows the raw carotid ultrasound scans measured using a Bmode ultrasound scanner The broad usage of carotid ultrasoundbased phenotypes and their significant association with RAspecific Similarly the righthand side panels of Fig a03b d show the processed scans tracking morphological variations in the carotid atherosclerotic plaque for the quantification of cIMT and plaque area The cIMT and plaque area are both greater in RA patients than in nonRA patientsAnother important observation from Table a0 is the significant association between carotid atherosclerotic biomarkers and RAspecific inflammatory markers such as ESR CRP and IL6 [ ] ESR is a relatively inexpensive measure of inflammation in RA patients”therefore several studies have used ESR for CVD risk assessment [“] Some of such studies are listed in Table a0 All these studies indicated a substantially higher CVD event rate in patients with elevated ESR levels Besides ESR studies have also suggested the use of other popular RAspecific inflammatory markers such as CRP or hsCRP and IL6 for the improvement in the CVD risk assessment [ ] Furthermore these RAspecific inflammatory markers are also associated with the annual progression of cIMT [ “] which is a prominent surrogate marker of cardiovascular events In a study with RA patients Kaseem et a0al [] demonstrated the association of ESR CRP and IL6 with carotid atherosclerosis with 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Cytivitisnesnietorpopil ytisnedhgih oitar sddoRO erusserp cRheumatology Internationalinflammatory markers has also enabled their inclusion in the CVD risk prediction calculators [ ] Recently several CVDstroke risk prediction models have been developed that have combined the effect of conventional risk factors and the automated carotid atherosclerosis biomarkers [ ] These risk prediction models reported a better performance in identifying high CVD risk patients compared with current standardofcare risk calculators However such socalled integrated risk prediction models were developed for the general population They were based on the annual progression rates of carotid atherosclerotic biomarkers and conventional risk factors [“] Therefore given the progression rates of cIMT and PA due to the RAspecific inflammatory markers such models can be updated and might be useful for CVD risk assessment in RA patientsArtificial intelligence in a0CVDstroke risk assessmentArtificial intelligence AI is expeditiously changing the landscape of the global healthcare system and assisting the healthcare workforce in clinical decisionmaking [] Machine learning ML and deep learning DL are the two common branches of AI that have broad ranges of applications in almost every medical imaging sector eg classification and plaque characterization for stroke risk assessment [] thyroid cancer characterization [] liver cancer diagnosis [] prostate cancer diagnosis ovarian cancer diagnosis [] lung cancer detection [] brain tumor classification [] and heart disease prediction and disease classification [ ] During the recent global pandemic of coronavirus diseases AI is providing promising results in the diagnosis of patients with the help of several imaging techniques such as computed tomography [] and Xrays []Since this review is on CVDstroke risk assessment we have summarized several studies that have used MLbased algorithms for CVDstroke risk assessment Table a0 All of these studies follow a supervised learning approach in which the MLbased classifier is trained to identify the correct output labels using input risk factors or features and predefined gold standards or labels Figure a0 shows the generalized framework of supervised MLbased CVD risk assessment In the case of CVD risk assessment the gold standard can be the primary endpoints such as presence or absence of cardiovascular events or surrogate endpoints such as cIMT PA and CAC score or a combination of these risk factors [ ] Several types of input features can be used to train the AIbased algorithms They can be traditional risk factors imagebased phenotypes grayscale image features or statistically derived features Once the offline ML classifier is trained using these features and gold DAC rof RO roF gnikomS MD NTH PBD PBS IMB egAaimedipilrepyH thgiehPCdna TM Ic ± ][ nosetnavSR 0cRheumatology International Fig Carotid ultrasound image of the common carotid artery for control patientsstandard its coefficients are then used in the online ML system to predict the out risk labels Online ML systems do not require a gold standard to make the final risk classification All the studies provided in Table a0 used this approach for CVD risk assessment Unlike MLbased algorithms DLbased models such as convolutional neural networks do not require input features beforehand Instead such algorithms automatically learn their offline coefficients from the input image datasets [] Currently AIbased techniques are used in the diagnosis of RA [] the identification of RA disease severity [] the classification of several RA synovial tissues [] and mortality prediction due to RA [] Although MLbased algorithms are used in the RA field no efforts have been made to assess the CVD risk in RA patients using such automated intelligencebased paradigms MLbased algorithms have been used to perform CVD risk assessments in nonRA populations and reported a better performance in identifying highrisk CVD patients when compared with the current standard of care conventional risk calculators [ ] Patients with RA experience more atherosclerotic plaque in the carotid artery which might lead to cardiovascular events [“] In recent years several studies have demonstrated a better stroke risk assessment using MLbased strategies [] and DLbased strategies [] Besides all these studies attempts can be made to develop more accurate CVD risk prediction tools for RA patients using AI techniques Figure a0 conceptualizes several components required for CVD risk assessment in RA patients The AIbased CVD risk assessment for RA patients can be made possible by combining several types of risk factors such as patients™ demographics physiological parameters behavioral risk factors imagebased phenotypes and most importantly RAspecific inflammatory markers This combined set of features can be used as inputs along with the gold standard to identify what CVD risk category RA patients belong to As such both ML and DLbased systems can be employed to performed CVD risk assessment in patients Because of the significant association between carotid atherosclerosis and RA researchers can conduct a pilot study with cIMT and plaque areas as the surrogate markers for CVD risk assessmentSummaryIn this review we provided several

Thyroid_Cancer

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

Thyroid_Cancer

Microbial colonisation of the gastrointestinal tract of newly hatched chicks starts at hatch seeded from the immediate hatching environment and quickly results in dense colonisation The role of ecological factors in gut colonisation has been extensively investigated as well as the role of micro and macronutrients in supporting and selecting for bacterial species highly adapted for utilising those nutrients However the microbial community contained in poultry feed and its influence on colonisation and maturation of gut microbiota has not been directly addressed In this study we compared the microbiota found in poultry feed with the microbiota of ileum cecum and excreta to identify substantial overlap in core microbiotas of the compared groups We then investigated the microbiota present in raw feedstuffs meat and bone meal wheat corn canola barley soybean millrun shum poultry oil oats limestone and bloodmeal from four geographically distinct feedstuff suppliers Each of the feedstuffs had diverse microbial communities The meat and bone meal and bloodmeal samples had the most complex and distinct microbial populations There was substantial overlap in the phylogenetic composition found in the grain and seed samples barley canola corn millrun oats shum soybean meal and wheat Issues related to methodology viability of microbial communities in the gut and feed and the implications for biosecurity are discussedKeywords Feed Microbiota Colonisation ChickenKey points¢ Rapid microbiota colonisation starts from birth or hatch in poultry¢ Feed carries rich microbial community within and seeds the host during colonisation¢ In poultry feed ingredients grains have similar microbiota¢ The meat and bone meal and bloodmeal had the most complex and distinct microbial populationsCorrespondence DStanleycqueduau Sarah Haberechta and Yadav S Bajagai contributed equally to this work Institute for Future Farming Systems Central Queensland University Rockhampton QLD AustraliaFull list of author information is available at the end of the IntroductionUntil recent advances in technology allowed us to sequence total DNA from any environmental sample and to identify almost all bacteria including uncultured our knowledge was limited to a small proportion of bacteria we could grow and investigate using classic microbiology growth methods Nowadays instead of taking a fecal sample and pulling out only targeted bacteria on specially selected microbiological plates we could see thousands of species in a sample and investigate their role in the gut This revolutionised our knowledge of the intestinal microbiota and its role in health and digestion We now know that the number of gut bacteria outnumber our own cells up to ten times and contribute around of unique genetic material to our genetic pool Cebra Fujimura et a0al Joyce and Gahan The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cHaberecht a0et a0al AMB Expr Page of The poultry intestinal microbiota has evolved into its present form incorporating many different communities from the environment and the animals and humans they contact This means that the phylogenetic composition of chicken gut microbiota strongly but not entirely overlaps with the microbiota of humans and other farmed animals However recent research into chicken gut microbiota has suggested that industrialization of chicken production has transformed the chicken gut microbiota to such an extent that modern commercial chicken microbiota is probably very different in composition to that which would be found in native jungle fowl the wild precursors of the highly selected modern chicken due to the unnatural hatching practices with separation of chicks from hens and natural nest environments In hatcheries chicks are immediately exposed to bacteria different from bacterial communities to those that were selected in chicken guts and historically adapted to chicken as host Stanley et a0al The practices of commercial poultry production expose newly hatched chicks to microbes from the hatchery environment from human handlers transport boxes and transport vehicles prior to arrival at the farm Stanley et a0 al This process is typically carried out in the first days of life during the period when there is a rapid increase in bacterial diversity and load in the gut These environmental sources of bacteria appear to have a significant influence on the establishment of intestinal microbiota given that most significant colonisation in chickens occurs within the first few days posthatch Lu et a0al In the absence of the natural chicken feeding brooding and nesting habits CrisolMartinez et a0al the chicken gut bacterial community becomes susceptible to the influences of human and environmental sources Apajalahti et a0al Stanley et a0al Until recently it was believed that chicks are sterile in ovo and that colonisation begins posthatch The application of recent technological advances has suggested that at least in some circumstances there may be very lowlevel bacterial colonisation in ovo Castaneda et a0al There have been attempts to deliver probiotics in ovo AbdelMoneim et a0al Wilson et a0al however bacterial load in ovo is very moderate and has production significance mainly in case of in ovo infection Allan et a0 al Bradbury and Howell Bacterial colonization of the gut is likely to be a competitive process whereby the initial bacterial colonizers inhibit or promote the establishment of subsequent bacterial invaders by modifying the gut environment eg pH andor crossfeeding metabolites that support or retard growth of other bacteria The formation of the microbial community in chickens is very rapid with and bacteria per gram of contents in the ileum and the ceca respectively oneday posthatch Numbers increase to and respectively by day three and remain high while continuously adapting and responding to environmental changes and host stressors Baldwin et a0al Lu et a0al Stanley et a0al This indicates that the first days posthatch are critical for controlled and pathogen restricted microbial exposure In this study we investigated the potential role of microbiota from poultry feed in the establishment and development of chicken gut microbiotaThe food consumed by an animal has an important impact on the composition of gut microbiota it supplies nutrients that the microbiota can use directly or which are derived from the host processing of the feed input Fujimura et a0 al Digestible and simple carbohydrates in the gut are quickly absorbed and used by both host and microbiota in the small intestine where they have the strongest influence on microbiota composition However dietary fibre in the form of nondigestible carbohydrates NDC nonstarch polysaccharides NSP resistant nondigestible oligosaccharides RO and resistant starch RS survive the passage through the small intestine largely unprocessed to reach the ceca and large intestine where they promote the growth of beneficial bacteria such as Bifidobacterium Costabile et a0 al Holscher et a0 al Koecher et a0 al Lecerf et a0al Whelan et a0al Lactobacillus sp Costabile et a0al Walton et a0al Akkermansia muciniphila Fruge et a0 al and Faecalibacterium prausnitzii Benus et a0al Roychowdhury et a0al Some of the metabolic products derived from the bacterial digestion of dietary fibre have beneficial effects on intestinal and general health Holscher et a0 al Koecher et a0al Unfortunately unlike fibredigesting bacteria which are beneficial to the host proteolytic intestinal bacteria in the chicken such as E coli are often pathogenic Tolckmitt King et a0 al identified Enterococcus faecalis Enterococcus gallinarum and Proteus mirabilis as frequently observed proteasesecreting bacterial species in chicken Bacterial metabolism of protein results in toxic metabolites High protein content used in poultry diets may contribute to gut damage and is a predisposing factor in necrotic enteritis Stanley et a0al Herring et a0al demonstrated that in humans high maternal dietary protein intake results in intrauterine growth reduction and embryonic death due to the toxicity of ammonia homocysteine and H2S that are generated from amino acid catabolism Recently proteolytic species like Bacillus subtilis Chen et a0 al have become popular protein digestion probiotics that aid proteolysis without pathogenic effects AbdelMoneim et a0al however the toxicity of the metabolites produced 0cHaberecht a0et a0al AMB Expr Page of requires careful consideration when deciding on the protein content in poultry feedMost of the current knowledge on the role of microbiota in fat metabolism comes from human studies on the effects of highfat diet and obesity Highfat diets have not yet been extensively researched in poultry nutrition from a microbiota perspective Highfat diet consumption generally leads to an increase in Firmicutes and causes microbiota alterations clearly associated with obesity and intestinal diseases A highfat diet increases the number of fatloving bacteria such as Verrucomicrobia Deltaproteobacteria Ruminococcus Lachnospiraceae and Bacteroidaceae Hussain et a0al Despite these bacterial groups being predominantly nonpathogenic or even beneficial to the host under normal diet circumstances under high fat intake conditions cumulative metabolic products of these bacteria can result in multiple negative effects High fat intake results in microbiota and host products that enhance gut permeability and result in chronic gut inflammation and predisposition to food allergy This effect is mediated by fatinduced changes in the gut microbiota Once a high fat diet increases fatloving bacteria the host retains the community with increased ability to extract energy from food as shown in human high fat diet and obesity studies reviewed in Murphy et a0al Many studies have investigated the role of particular micro and macronutrients on microbiota development in chickens and other animals as well as the role of exogenous enzymes and other metabolites found in the feed Other than the nutrientdriven influence of feed on microbiota development the role of feed in contributing to the colonization of the gut with indigenous feed bacteria is underexplored In this manuscript we present evidence of the presence of diverse bacterial population in poultry feed rations that is continuously from the first to the last day of life seeding the poultry intestine with bacteria that are naturally present in feed and thus already adapted to digest and utilise components from that feedMaterials and a0methodsAnimal trialThe study was approved by the Animal Ethics Committee of Central Queensland University under the approval number A1409318 The animal trial used in this study was performed with a range of treatments with different probiotic supplementations However here we present a subsection of that data obtained from the control birds and data from the feed that was provided to the birds from the hatchThe birds were hatched in the Central Queensland University research facility hatchery using Ross Broiler eggs provided by Bond Enterprises Hatchery Toowoomba Queensland The eggs were hatched under relatively clean conditions and immediately moved to the poultry room where the feed was provided immediately posthatch The feed used was a Chicken Starter Diet Red Hen Laucke Mills Australia with no antimicrobials or coccidiostats and was used throughout the trial for weeks The feed was formulated to meet or exceed the National Research Council standards for broiler chickens NRC All birds were fed ad a0libitum and had unrestricted access to drinking water Birds were individually tagged by leg bands and weekly weights demonstrated weights equal or above the Ross performance standards Birds were euthanised at day posthatch CO2 BOC Australia and dissected Ileum and caecum contents were collected for microbiota analysis flashfrozen in liquid nitrogen and stored at ˆ’ a0 a0 a0 a0°C until further processing Excreta samples were also collected by placing a specially made transparent divider into the pen without removing the bird from the pen The clean paper towel was placed under the bird and excreta collected immediately after voiding Fortyeight samples from birds were successfully collected and sequenced for microbiota phylogenetic analysisFeedstuff microbiota experimentAfter confirming the presence of microbial communities in the feed used in the animal trial we further investigated specific feedstuff components used in the poultry feed The original poultry feed ingredients that were sampled included meat and bone meal wheat corn canola barley soybean millrun shum poultry oil oats limestone bloodmeal acid oil and tallow Samples of each type of feed ingredient were sourced from four different suppliers from four distinct regions of Australia three from the state of Victoria which has a temperate climate and one supplier from Queensland a state with a warmer subtropical climateDNA extractionDNA was extracted from the feed feed ingredients ileal content caecal content and excreta samples using the same method previously described Bauer et a0 al 2019a b Briefly a0g of samples were lysed and purified using a DNA spin purification column Enzymax LLC Cat EZC101 Kentucky US The DNA quality and quantity were estimated using a NanoDrop spectrophotometer a0s rRNA gene sequencingThe V3V4 region of a0 s rRNA genes were amplified using ACT CCT ACG GGA GGC AGC AG forward and GGA CTA CHVGGG TWT CTAAT reverse primers containing barcodes spacers and Illumina sequencing linkers Fadrosh et a0al The sequencing library was 0cHaberecht a0et a0al AMB Expr Page of prepared following the manufacturer™s protocol Illumina Inc San Diego CA USA The a0 s rRNA amplicon sequencing was completed on the Illumina MiSeq platform using × a0bp pairedend sequencingThe data was analysed using Quantitative Insights Into Microbial Ecology QIIME v191 Caporaso et a0al The FastqJoin algorithm was used to combine the pairedend sequences allowing no mismatches within the region of overlap Phred quality threshold had a minimum score of The Uclust Edgar was used to pick the OTUs at similarity and chimeric sequences were filtered using Pintail Ashelford et a0 al Taxonomic assignments were performed against the GreenGenes database v2013_8 using QIIME default parameters DeSantis et a0al The OTU abundance table was rarefied to calculate a UniFrac matrix Calypso Zakrzewski et a0al was used for further downstream analysis and visualisation of the data using Hellinger transformed Legendre and Gallagher OTU tableThe sequence data is publicly available at the MGRAST database under a project ID mgp455839ResultsFeed microbiotaThe composition of microbiota in the feed was compared to the structure of the microbiota in the different gut sections in the birds fed the same feed Feed microbial composition comprised of phyla Actinobacteria of all reads Proteobacteria of reads Firmicutes and Bacteroidetes spread across genera including in alphabetic order Arthrobacter Acinetobacter Aerococcus Bacillus Bifidobacterium Blautia Brachybacterium Brevibacterium Clostridium Comamonas Coprococcus Corynebacterium Dietzia Enterobacter Enterococcus Facklamia Frigoribacterium Jeotgalicoccus Lactobacillus Lactococcus Leuconostoc Microbacterium Oscillospira Paenibacillus Proteus Pseudochrobactrum Pseudomonas Ruminococcus Sphingobacterium Sporosarcina Staphylococcus Streptococcus Trichococcus Turicibacter Wautersiella and unknown generaThe microbial composition of the feed was most similar to the microbiota of the ileum and excreta Fig a01a and most distant from the cecal community The core microbiota at genus level Fig a0 1b showed the overlap in the genera present in the feed and both ileum and cecum as well as with the excreta The linear discriminant analysis LDA effect size method LEfSe was used to determine the genera most likely to explain differences between the feed and gut sections microbiota via coupling standard tests for statistical significance with additional tests encoding biological consistency and effect relevance Fig a01c The Chao estimated richness of feed microbiota was very low compared to cecum and excreta samples Fig a01d but comparable to some ileal samplesIndividual feed ingredients carry distinctive microbial communityTo determine the potential origins of the microbiota found in the whole formulated feed ration the microbiota composition of the component ingredients of the feed were analysed Independentsamples of each of the ingredients were sourced from four different locations in Australia Fig a0 Figure a02a and b show clear differences in genus level composition of feed ingredients as well as in their estimated richness with bloodmeal and meat and bone meal showing more complex microbial richness compared to other feed ingredient groupsThe similarities and differences in microbiota compositions were further investigated by Discriminant Analysis of Principal Components DAPC and NonMetric Multidimensional Scaling NMDS multivariate analysis Fig a03ab This showed that bloodmeal meat and bone meal and limestone had the most distinct microbial communities followed by poultry oil whereas barley canola corn millrun oats shum soybean meal and wheat clustered together into almost entirely overlapping groupsBased on LEfSe the genera most likely to explain differences between each type of feed ingredient indicate that some groups of pathogens populating the bird GIT may originate from specific feedstuff with Clostridium and Streptococcus identified as representatives of bloodmeal Fig a0The influence of the feed ingredients supplier environmental differences andor climate conditions on the microbiota in feedstuffs is presented in a DAPC plot Fig a0 Surprisingly the differences in feedstuffs microbiota presented in the DAPC plot resemble the geographical position of suppliers and may be influenced by the differences between the processing facilities but also climate and geographic region from which the ingredients have been grown and producedDiscussionIt is now well established that an appropriate healthy microbiota provides individuals with numerous nutritional benefits intestinal mucosa development pathogen protection and immune system maturation Stecher and Hardt Young Zhu et a0al Although the mainstream knowledge on gut microbiota came from human research it is understood that intestinal bacterial inhabitants of chickens play both similar and poultryspecific influential roles Stecher and Hardt Young Zhu et a0al Although in some cases there may be small populations of bacteria in the gut 0cHaberecht a0et a0al AMB Expr Page of Fig Comparison of feed microbiota with ileum cecum and excreta microbiota The Fig presents 3D PCoA plot based on unweighted UniFrac distance a core microbiota at a genus level b LEfSe analysis of the top genera c and Chao microbial richness estimator d In all plots feed samples are represented in grey cecum in red ileum yellow and excreta in blueof embryos in ovo the main events of the establishment of the chicken intestinal microbiota community starts immediately posthatch Based on the current literature on poultry gut colonisation the environment rather than parental influence plays the major role in chicken gut maturation Stanley et a0al The microbiota in chickens is considered fully formed within the first weeks of life but it continues to mature and to respond to stressors and environmental challenges throughout the life of birdsThe type of the production system defines the environment and type of feed that birds are exposed to and hence has a major influence on the microbiota especially in freerange systems that replace strict biosecurity with exposure to the soil grass and other plant microbiota but most concerning microbiota of rodents wild birds and other animals and increases pathogen load in birds all presenting challenges for freerange production Biasato et a0al Islam et a0al Ocejo et a0al Until now the feed has been usually discussed in poultry gut maturation as a growth medium that will support a nutrient determined cohort of intestinal microbes This study now brings a new dimension to the role of feed in microbiota formation as a source of colonizing bacteria Fig a0The microbial community contained in poultry feed may play an important role in the colonisation of poultry gut microbiota as the study found a large overlap in the core microbiota of the feed and the birds™ ileum caecum and excreta Our results also indicate that there is a substantial overlap in the microbial composition of grains and seeds barley canola corn millrun oats shum soybean meal and wheat almost entirely overlap while blood meal meat and bone meal and limestone stand quite apart with more distinct microbiotas Each batch of feed ingredient differs in the composition of the microbiota it carries and hence each batch of formulated feed will also differ in microbiota composition Some of the ingredients carry bacteria which are potentially pathogenic These findings suggest that it would be prudent 0cHaberecht a0et a0al AMB Expr Page of Fig The feed ingredients microbiota composition and diversity Genus level barchart shows the top most abundant genera a and a Chao richness estimator b MBM meat and bone meal BM blood meal PO poultry oil Feed finished mixed feedFig Multivariate presentation of feed ingredients microbiota similarities via DAPC a and NMDS b plotsto consider and monitor feed microbial community particularly in starter diets used in the period when the core microbiota is formed and stabilisedIt is worth noting how poorly researched the field of microbial colonisation of feed ingredients used in poultry and human feed is A Google Scholar search on keywords 0cHaberecht a0et a0al AMB Expr Page of Fig Linear discriminant analysis effect size method LEfSe of the top genera in feedstuffsFig Discriminant Analysis of Principal Components DAPC of feedstuffs from the different suppliers a and a GoogleMap b view of their geographical regionœgrain and œmicrobiota January returned only manuscripts all investigating the role of grains consumed in the diet on microbiota structure Similar outputs are found when using specific grains On the other hand the feed can be easily contaminated with microbes as feed spoilage is not uncommon out in field situations 0cHaberecht a0et a0al AMB Expr Page of Fig Feed contains a diverse microbial community and is an active bacterial coloniser in poultryWhile biosecurity measures are implemented to variable degrees feed ingredients in milling environments are not always well protected from birds rodents insects and other potential microbial exposures It was believed that the process of pelletising will remove most of the bacteria However in addition to the fact that brief heating of feed during pelletising cannot remove microbial spores the pelletising process is far from sterilisation and will remove most but not all viable bacteria Its antimicrobial efficiency will be varying between the different processing systems It is also notable that feed can be easily colonized postpelletising from external and pelletising surviving microbiota This will strongly depend on the packaging humidity temperature and other finished feed storage conditionsAnother question that needs to be addressed is how much of the microbial signal identified by a0 s rRNA gene analysis represents viable bacteria DNA from the dead bacteria would be subjected to natural degradation but still depending on time frames and storage conditions significant amounts of amplifiable DNA from dead bacteria could be present Of course this can also be an issue with microbiota analysis of gut samples although in that case it is well established that DNA is efficiently digested and degraded by gastric juice and pepsin Liu et a0al We were able to grow bacteria on rich brainheart infusion media from all of the feed samples we investigated however culturing is strongly restricted by the media and anaerobicaerobic environment used Therefore it would certainly not be readily possible to identify culturable bacteria of all the genera identified in feed ingredients Although there are other methods capable of determining bacterial viability via sequencing and vital staining methodologies Young et a0al they are not commonly in use and face different challenges and methodological issuesThis study shows that further research into feed as a source of random or as the means of targeted colonisation of poultry gut could be productive Investigating feed microbiota will bring new challenges unlike the diverse and dense microbial populations present in intestinal samples many of the feed ingredients are hard to process for DNA isolation and for some only small amounts of bacterial DNA can be recovered Amplicon and whole metagenome sequencing methods are limited in resolution and cannot reliably detect bacteria that make up only a small fraction of the microbiota Resolution depends on sequencing depth but in most studies a presence at less than is unlikely to be reliably detected Therefore for some significant bacteria for example pathogens such as Clostridium perfringens or Salmonella it may be necessary to use more sensitive methods such as culturing or specific PCR to detect in feed ingredients Control of viable pathogens in feed 0cHaberecht a0et a0al AMB Expr Page of should be considered as a standard part of production system biosecurityAcknowledgementsThe data was analysed using the Marie Curie HighPerformance Computing System at Central Queensland University We wish to acknowledge and appreciate help from Jason Bell provided in all aspects of HighPerformance ComputingAuthors™ contributionsSH performed research YB performed research analysed data RM TTHV performed research DS conceived the study analysed data wrote the paper All authors edited the manuscript and agreed with its final form All authors read and approved the final manuscriptFundingThis research did not receive any specific grant from funding agencies or the commercial sector The project was funded internally by Central Queensland University Office of Research Merit Grant fund At our request undisclosed poultry feed company kindly provided annotated feedstuffs but had no further involvement Availability of data and materialsSequencing data is publically available on MGRAST Metagenomics Analysis Server Database https wwwmgrast with full sample annotation under project ID mgp455839Ethics approval and consent to participateAnimal ethics approvals were obtained from the Animal Ethics Committee at Central Queensland University with the Approval Number A1409318Competing interestsThe authors have no conflict of interest to declareAuthor details University of New England Armidale NSW Australia Institute for Future Farming Systems Central Queensland University Rockhampton QLD Australia School of Science RMIT University Bundoora VIC Australia Department of Microbiology Monash University Clayton VIC Australia Received May Accepted August ReferencesAbdelMoneim AE Elbaz AM Khidr RE Badri FB Effect of in ovo inoculation of Bifidobacterium spp on growth performance thyroid activity ileum histomorphometry and microbial enumeration of broilers Probiotics Antimicro doihttps doi101007s1260 xAbdelMoneim AE Selim DA Basuony HA Sabic EM Saleh AA Ebeid TA Effect of dietary supplementation of Bacillus subtilis spores on growth performance oxidative status and digestive enzyme activities in Japanese quail birds Trop Anim Health Prod “ https doi101007s1125 Allan B Wheler C Koster W Sarfraz M Potter A Gerdts V Dar A In ovo administration of innate immune stimulants and protection from early chick mortalities due to yolk sac infection Avian Dis “ doihttps doi10163711840 8Reg1Apajalahti J Kettunen A Graham H Characteristics of the gastrointestinal microbial communities with special reference to the chicken Worlds Poult Sci J “ doi 101079nVPS2004 Ashelford KE Chuzhanova NA Fry JC Jones AJ Weightman AJ At least in 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies Appl Environ Microbiol “ doi Doi https doi101128Aem7112772477362005Baldwin S Hughes RJ Hao Van TT Moore RJ Stanley D Athatch administration of probiotic to chickens can introduce beneficial changes in gut microbiota PloS One 133e0194825 doihttps doi101371journ alpone01948 Bauer BW Gangadoo S Bajagai YS Van TTH Moore RJ Stanley D 2019a Oregano powder reduces Streptococcus and increases SCFA concentration in a mixed bacterial culture assay PloS One 1412e0216853 doihttps doi101371journ alpone02168 Bauer BW Radovanovic A Willson NL Bajagai YS Hao Van TT Moore RJ Stanley D 2019b Oregano A potential prophylactic treatment for the intestinal microbiota Heliyon 510e02625 doihttps doi101016jheliy on2019e0262 Benus RF van der Werf TS Welling GW Judd PA Taylor MA Harmsen HJ Whelan K Association between Faecalibacterium prausnitzii and dietary fibre in colonic fermentation in healthy human subjects Br J Nutr “ doihttps doi101017S0007 Biasato I Ferrocino I Biasibetti E Grego E Dabbou S Sereno A Gai F Gasco L Schiavone A Cocolin L Capucchio MT Modulation of intestinal microbiota morphology and mucin composition by dietary insect meal inclusion in freerange chickens BMC Vet Res doihttps doi101186s1291 70181690yBradbury JM Howell LJ The response of chickens to experimental infection ˜em ovo™ with Mycoplasma synoviae Avian Pathol “ doihttps doi10108003079 Caporaso JG Kuczynski J Stombaugh J Bittinger K Bushman FD Costello EK Fierer N Pena AG Goodrich JK Gordon JI Huttley GA Kelley ST Knights D Koenig JE Ley RE Lozupone CA McDonald D Muegge BD Pirrung M Reeder J Sevinsky JR Tumbaugh PJ Walters WA Widmann J Yatsunenko T Zaneveld J Knight R QIIME allows analysis of highthroughput community sequencing data Nat Methods “ doihttps doi101038nmeth f303Castaneda CD McDaniel CD Abdelhamed H Karsi A Kiess AS Evaluating bacterial colonization of a developing broiler embryo after in ovo injection with a bioluminescent bacteria Poult Sci “ doihttps doi103382pspez05 Cebra JJ Influences of microbiot

Thyroid_Cancer

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

Thyroid_Cancer

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

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

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

Thyroid_Cancer

This letter summarizes recommendations from the interdisciplinary working group of renal tumors IAGN of the German Cancer Society for the systemic treatment of advancedmetastatic renal cell carcinoma in the context of the current SARSCoV2 pandemicBackgroundDuring the SARSCoV2 pandemic oncologists faced several new challenges Jones et a0al Hanna et a0al Lai et a0al Data on SARSCoV2 in cancer patients suggested outstanding rates of severe clinical courses “ and mortality up to upon SARSCoV2 infection although methodological limitations of these observations are of concern Desai et a0al Liang et a0al Yang et a0al Zhang et a0al However oncological therapies might raise SARSCoV2related morbidity and mortality Desai et a0al Liang et a0al Yang et a0al Zhang et a0al Checkpoint inhibitors CPI could Jens Bedke and Viktor Gr¼nwald shared senior authorviktruenwaldukessendejensbedkemedunituebingende Jens Bedke Viktor Gr¼nwald Department of a0Hematology Hemostasis Oncology and a0Stem Cell Transplantation Hannover Medical School Hannover GermanyImmunonclogical COoperative Group ICOG of a0the a0Comprehensive Cancer Center Lower Saxoney Hannover CCCN Hannover Germany Genitourinary Oncology Department of a0Urology University Hospital Dresden Dresden Germany Department of a0Urology University Medicine Greifswald Greifswald Germanynegatively interfere with the pathogenesis of SARSCoV2 and an overlap of symptoms of SARSCoV2 and cancer treatment related adverse events can be assumed which could result in a diagnostic challenge Zhang et a0al Bersanelli Rotz et a0al In particular interstitial pneumonia from SARSCoV2 cannot be differentiated from CPIassociated pneumonitis with certainty using CT images and may present with overlapping features Zhang et a0al Bersanelli Pneumonitis in CPItreated patients are rare with an incidence von “ but accounts with treatmentrelated death rate of Wang et a0al Naidoo et a0al Effects of immune suppression due to supportive measure has also to be considered although only preliminary data exist on effects of steroids in cancer patients during the SARSCoV2 pandemic Yang et a0al UrologieAltstadtquartier Magdeburg Magdeburg Germany Department of a0Radiation Oncology West German Cancer Center University Hospital Essen of a0the a0University DuisburgEssen Essen Germany Urology and a0Urooncology Practice Berlin Vice Chairman duo Deutsche UroOnkologen Berlin Germany Department of a0Urology Eberhard Karls University Tuebingen HoppeSeylerStr a0T¼bingen GermanyInterdisciplinary Genitourinary Oncology WestGerman Cancer Center Essen Clinic for a0Medical Oncology and a0Clinic for a0Urology OZ1 University Hospital Essen A¶R Room HufelandstraŸe a0Essen Germany Vol01234567891 0c Journal of Cancer Research and Clinical Oncology As a result use of CPI in advancedmetastatic renal cell carcinoma patients mRCC may be associated with a specific risk and requires a critical risk“benefit assessment Therefore choosing the appropriate therapeutic regimen in mRCC is more challenging than everSeveral recommendations focus on reducing the risk of SARSCoV2 exposures for patients as well as for medical staff as part of oncological routine and addresses mainly questions of resource allocation Discussion about treatmentspecific considerations remains scare Herein we report a consensus of the interdisciplinary working group on renal tumors IAGN of the German cancer society towards treatment of mRCCDuring the process of treatment evaluation the assessment of treatment indication must be made on an individual basis However once selecting treatment patient and tumorspecific parameters patient´s comorbidity and the availability of local caregiving facilities should be taken into account In our opinion two major scenarios according to systemic treatment have to be considered initiation or change medication of a systemic treatment upon progressing diseases and measures taken during ongoing systemic treatmentConsidering urgency once a0initiating treatment upon a0advanced or a0metastatic RCC For systemic treatment the appropriate treatment should be selected based on patientspecific factors that take the overall picture into account when searching for treatment Escudier et a0al The indication for the systemic treatment of mRCC should be made strictly taking active surveillance and deferred medical treatment into consideration Escudier et a0al This should minimize patient exposure to the atrisk medical care environment as well potentially towards particular treatmentassociated risk in terms of SARSCoV2 infection Patients who do not require urgent systemic treatment should therefore primarily be offered active surveillance and deferred medical treatment No validated tool that identifies indolent disease is established However various parameters are used by clinicians to identify the appropriate patient population for active surveillance Table a0 represents a number of such variables which are used by the authorsAlso considering the CARMENAdiscussion on palliative nephrectomy in asymptomatic synchronous metastasized patients cytoreductive nephrectomy should be considered critically respecting the fact of reported nosocomial SARSCoV2 infection in cancer patients as well as a surprising high rate of SARSCoV2 diseases among cancer patients receiving surgery Liang et a0al Zhang et a0al Table Clinical parameters to estimate the urgency of treatment need in patients with metastasized renal cell carcinoma UnfavorableParametersHigh tumor load polytopic Patientrelatedbone metastases andor multiple an systemsECOG Risk factors for secondary complicationsbFavorableOligometastasis glandular involvementa an system involved asymptomaticECOG “Clear cell RCC TumorrelatedSarcomatoid subtype medullary subtype nonclear cell subtypesIntermediate and poor riskFavorable riskPrognosis according to the IMDCa Pancreas thyroid gland saliva adrenal glandsab Impending complications in the event of further progression eg bronchial compression pathological fracture cross section vascular erosionocclusionHow should I treat a a0patient with a0favorable riskSuitable systemic treatment is selected based on its marketing authorization principle recommendations as well as on published data The strengths and weaknesses of a treatment regime should be weighed against its suitability for the best possible application In order to minimize the treatment risk and if the clinical course is indolent the necessity for systemic therapy is low and active surveillance is our preferred option Restaging is recommended in “ a0months™ time Based on these considerations the feasibility of an active monitoring strategy or initiation of a therapeutic measure should be evaluatedIn light of CheckMate214 JAVELINRenal and Keynote426 studies no significant overall survival OS benefit was gained by any of the immune combinations for patients with a favorable risk profile HR for OS ipilimumabnivolumab vs sunitinib CI “ axitinibpembrolizumab vs sunitinib CI “ axitinibavelumab vs sunitinib CI “ Motzer et a0al KeytrudaEMEA Choueiri et a0al A major limitation of these data are the short followup duration which limits the data interpretation However at the current state there is no signal that a specific combination is superior in OS expectations when compared to single agent sunitinib This is supported by molecular findings wherein IMDC favorable risk is associated with proangiogenic dependency McDermott et a0al However a proportion of patients exert an inflamed tumor type which may identify a patient population with potential clinical benefit from 0cJournal of Cancer Research and Clinical Oncology Table Summary of the treatment recommendations for firstline treatment of metastatic RCC during the SARSCoV2 pandemicIMDC risk groupsFavorableIntermediateUnfavorable SelectionActive surveillanceAxitinib avelumabAxitinib pembrolizumabAxitinib avelumabAxitinib pembrolizumabIpilimumab nivolumabOptionTKIActive surveillance restaging in a0months cabozantinibIpilimumab nivolumabCabozantinibThe list is shown in alphabetical order When selecting treatment the individual patient and tumor characteristics listed in Table a0 should be consideredCPI treatment Today there is no clinical test available to identify such patients The best treatment strategy in these patients™ remains has not been defined and data is scarce The identification of indolent disease and the absence of a survival signal for combinations in this patient population should caution treatment intensification and puts active monitoring and single agent TKI as preferred treatment options in the treatment algorithm of this patient cohortHow should I treat a a0patient at a0intermediate or a0poor riskIn cases who exert favorable clinical parameters depicted Table a0 patients with intermediate risk may receive active monitoring and deferred medical treatment as an alternative strategy to upfront medical treatment However most patients require systemic treatment and clinical benefits outweighs risk of immunerelated adverse eventsSARS CoV2Patients with an intermediate or unfavorable risk profile had longer OS when taking immune combinations than patients taking TKI alone which is why they should be used preferentially [HR for OS ipilimumabnivolumab vs sunitinib CI “ axitinibpembrolizumab intermediate prognosis CI “ poor prognosis CI “ axitinibavelumab intermediate prognosis CI “ poor prognosis CI “ Cabozantinib vs sunitinib “] Motzer et a0al KeytrudaEMEA2020 Choueiri et a0al To minimize the risk of immunemediated adverse events as well as high dose steroids application due to immune related adverse events a careful patients based valuing is demanded to choose the best treatment regimen Table a0 Overall if clinical parameters in this cohort are favorable the TKI cabozantinib reflects a therapeutic option Otherwise an immune combination represents the preferred form of treatment in particular in poor prognosis patients wherein the clear harm of the mRCC and the oncological efficacy overweight™s the risk of pandemic associated concernsWhat to a0conclude in a0SARS‘CoV‘ pandemic for a0mRCC Although precise guidelines according to mRCC treatment reflects best efficacy and QoL data within the SARSCoV2 pandemic a patientcentered treatment choice which is adapted to the local pandemic situation is warranted s Table a0 Reflecting SARSCoV2related comorbidity patient´s and tumor characteristic´s adverse events and hospitalization rates seem to be useful parameters to adjust riskbenefit ratio pandemicadapted mRCC treatment choice However expanding realworld register data will answer the question which concerns where the right one over or undertreatmentAcknowledgements Open Access funding provided by Projekt DEALCompliance with ethical standards Conflict of interest PI Receipt of grantsresearch supports Astra Zeneca BMS Bayer Lilly Merck Novartis EISAI Pfizer MSD AstraZeneca Roche Ipsen Receipt of honoraria or consultation fees Arbeitsgemeinschaft interdiziplin¤re Medizin Altana Health Service AstaZeneca Budosy BMS Bayer DKGOnkoweb ESAI EUSA IDInstitut Ipsen Impulze MedKom Acadamy Merck Medac MediSeminar MSD MTEAcadamy New Concept Oncology NGAAcadamy Novartis Pfizer Roche Roche StreamedUP Solution Acadamy Onkowissen Think Wired Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense BMS Byer DKG EUSA Ipsen Novartis Merck PharmaMar CG Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astellas Astra Zeneca BMS EISAI IPSEN MSD Merck Serono Pfizer Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Not applicable NK Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astellas BMS EISAI EUSA IPSEN Jansen MSD Novartis Pfizer Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Aristo BMS Janssen EUSA Pharma MSD TG Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astra Zeneca BMS EISAI IPSEN MSD Merck Serono Novartis 0c Journal of Cancer Research and Clinical OncologySanofiAventis Pfizer Roche Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Not applicable MJ Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees BMS Bayer EISAI EUSA Hexal Jansen Merck Serono Medac MSD Pfizer Roche Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Astellas Bayer Pfizer JB Receipt of grantsresearch supports inst Eisai Ipsen MSD Novartis Roche Pfizer Receipt of honoraria or consultation fees AstraZeneca Astellas BMS Eisai Ipsen MSD Novartis Roche EUSA Pharma Nektar Pfizer Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Inst BMS Ipsen Pfizer Roche VG Receipt of grantsresearch supports Astra Zeneca BMS MSD Ipsen Novartis Pfizer Receipt of honoraria or consultation fees Astra Zeneca BMS MSD Ipsen Novartis pfizer Merck Serno EUSAPharm EISAI Roche Bayer JanssenVilag Lilly PharmaMar Participation in a company sponsored speaker™s bureau Not applicable Stock shareholder Astra Zeneca BMS MSD Spousepartner Not applicable Other support please specify Travel Expense Astra Zeneca BMS Ipsen Pfizer BayerOpen Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40ReferencesBersanelli M Controversies about COVID19 and anticancer treatment with immune checkpoint inhibitors Immunotherapy “ https doi102217imt20200067Choueiri TK Hessel C Halabi S Sanford B Michaelson MD Hahn O et a0al Cabozantinib versus sunitinib as initial therapy for metastatic renal cell carcinoma of intermediate or poor risk Alliance A031203 CABOSUN randomised trial PROGRESSIONfree survival by independent review and overall survival update Eur J Cancer [Internet] “Choueiri TK Motzer RJ Rini BI Haanen J Campbell MT Venugopal B et a0al Updated efficacy results from the JAVELIN Renal trial firstline avelumab plus axitinib versus sunitinib in patients with advanced renal cell carcinoma Ann Oncol “ https doi101016jannon c202004010Desai A Sachdeva S Parekh T Desai R COVID19 and cancer lessons from a pooled metaanalysis JCO Glob Oncol “Escudier B Porta C Schmidinger M RiouxLeclercq N Bex A Khoo V et a0al Renal cell carcinoma ESMO clinical practice guidelines for diagnosis treatment and followup Ann Oncol [Internet] “Hanna TP Evans GA Booth CM Cancer COVID19 and the precautionary principle prioritizing treatment during a global pandemic Nat Rev Clin Oncol “Jones D Neal RD Duffy SRG Scott SE Whitaker KL Brain K Comment Impact of the COVID19 pandemic on the symptomatic diagnosis of cancer the view from primary care Lancet Oncol [Internet] “Lai AG Pasea L Banerjee A Denaxas S Katsoulis M Chang WH et a0al Estimating excess mortality in people with cancer and multimorbidity in the COVID19 emergency https doi1011012020052720083 Liang W Guan W Chen R Wang W Li J Xu K et a0al Cancer patients in SARSCoV2 infection a nationwide analysis in China Lancet Oncol “ https doi101016S1470 McDermott DF Huseni MA Atkins MB Motzer RJ Rini BI Escudier B et a0al Clinical activity and molecular correlates of response to atezolizumab alone or in combination with bevacizumab versus sunitinib in renal cell carcinoma Nat Med “Naidoo J Wang X Woo KM Iyriboz T Halpenny D Cunningham J et a0al Pneumonitis in patients treated with antiprogrammed death1programmed death ligand therapy J Clin Oncol “Motzer RJ Escudier B McDermott DF Ar©n Frontera O Melichar B Powles T et a0al Survival outcomes and independent response assessment with nivolumab plus ipilimumab versus sunitinib in patients with advanced renal cell carcinoma 42month followup of a randomized phase clinical trial J Immunother Cancer 82e000891 https doi101136jitc202000089 Rotz SJ Leino D Szabo S Mangino JL Turpin BK Pressey JG Severe cytokine release syndrome in a patient receiving PD1directed therapy Pediatr Blood Cancer https doi101002pbc26642 Wang DY Salem JE Cohen JV Chandra S Menzer C Ye F et a0al Fatal toxic effects associated with immune checkpoint inhibitors a systematic review and metaanalysis JAMA Oncol “Yang F Shi S Zhu J Shi J Dai K Chen X Clinical characteristics and outcomes of cancer patients with COVID19 J Med Virol https doi101002jmv25972 Zhang L Zhu F Xie L Wang C Wang J Chen R et a0al Clinical characteristics of COVID19infected cancer patients a retrospective case study in three hospitals within Wuhan China Annal oncol “ KeytrudaEMEAHC003820 R0081 https wwwemaeurop aeuendocum entsprodu ctinfor matio nkeytr udaeparprodu ctprodu ctinf ormat ion_enpdf Accessed May Publisher™s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

"primary bone tumors in early adolescence with unsatisfied prognosisAberrant DNA methylation had been demonstrated to be related to tumorigenesis and progression of multiple cancers andcould serve as the potential biomarkers for the prognosis of human cancers In this study identified downregulated hypomethylation genes and upregulated hypomethylation genes in OS by integrating the analysis theGSE97529 and GSE42572 datasets Bioinformatics analysis revealed that OSspecific methylated genes were involved inregulating multiple biological processesincluding chemical synaptic transmission transcription response to drug andregulating immune response KEGG pathway analysis showed that OSspecific methylated genes were associated with theregulation of Hippo cAMP calcium MAPK and Wnt signaling pathways By analyzing R2 datasets this study showed that thedysregulation of these OSspecific methylated genes was associated with the metastasisfree survival time in patients with OSincluding CBLN4 ANKMY1 BZW1 KRTCAP3 GZMB KRTDAP LY9 PFKFB2 PTPN22 and CLDN7 This study provideda better understanding of the molecular mechanisms underlying the progression and OS and novel biomarkers for the prognosisof OS IntroductionOsteosarcoma OS is one of the most common types of primary bone tumors in early adolescence which was characterized by an aggressive osteolytic or osteoblastic appearancewith a periosteal reaction [] Chemotherapy and surgeryare the most important treatments for patients with OS [] The survival rate of primary OS patients after treatmentsremains at “ [] However the prognosis of patientswith progressive or recurrent OS was less than [] Inthe past decades emerging studies reported that multiple factors are associated with the tumorigenesis and progression ofOS including germline genetic variants [] dysregulation ofoncogenes or tumor suppressors [] and the abnormal epigenetics change [ ] A few proteins had been revealed tobe related to the progression of OS For example GFRA1was reported to promote autophagy and cisplatininducedchemoresistance in OS [] The isoform of TMIGD3 suppressed OS progression though downregulating NFκB []Understanding the mechanisms related to OS developmentcould provide new targets for OSDNA methylation could aï¬ectthe gene expressionthough suppressing transcription [] Aberrant DNA methylation had been demonstrated to be involved in regulatingtumorigenesis and progression of multiple cancers [ ]In OS DNA methylationmediated suppression of miR449c could promote cell cycle though inhibiting cMyc inOS [] Hypomethylation of IRX1 was found to promote 0cComputational and Mathematical Methods in Medicineamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEiiiiiiiiiiamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsOOOOOOOOOOOOOOO“““Figure OSspecific methylated genes were identified by using the public dataset GSE97529 a DNA methylation status of CpGsites in Ewing™s sarcoma synovial sarcoma and OS samples were included in this datasetabOS metastasis by activating CXCL14NFκB signaling []Very interestingly recent studies showed that aberrantDNA methylation was associated with the prognosis of OSFor example the DNA methylation level of WNT6 was negatively correlated to the prognosis of children with osteosarcoma [] The hypermethylation of ESR1 was correlated tothe worse overall survival of OS [] These results suggestedthat the DNA methylation status could be potential diagnostic and therapeutic targets for OSThe present study analyzed the GSE97529 [] dataset toidentify OSspecific methylated genes In silico analyses wereperformed to explore the functions of OSspecific methylatedgenes Next the GSE42572 dataset was used to validate theexpression levels of OSspecific methylated genes [] Ofnote we found that these OSspecific methylated genes werecorrelated to the prognosis of patients with OS By thesemethods it is hopeful that novel aberrant methylation genesand pathways will be screened in the OS and an understanding ofthe underlying molecular mechanisms will beenhanced Materials and Methods Microarray Data The present study is aimed at identifying dysregulated OSspecific methylated genes in OS by analyzing public databases with bioinformatics analysis Thuswe screened the GEO databases The candidate databaseswere selected according to standards the candidatedatabase should contain clinical OS samples the numberof clinical samples should be more than cases and thecandidate database was not noncoding RNA datasets Finallyonly the SE97529 and GSE42572 datasets were selected forfurther analysis We have included this information in Materials and Methods The GSE97529 dataset was used to identify OSspecific methylated genes which was downloadedfrom the NCBI GEO database GSE97529 A total of Ewing™s sarcoma synovial sarcoma and OS sampleswere included in this dataset The GSE42572 dataset was analyzed to identify diï¬erently expressed genes in OS comparedto normal samples which was also downloaded from theNCBI GEO database GSE42572 Diï¬erentially expressedgenes DEGs and diï¬erentially methylated genes DMGswere identified by applying GEO2R P and ˆ£foldchange ˆ£ ‰¥ is set as the cutoï¬ criterion Functional and Pathway Enrichment Analyses TheDAVID system was used to predict the potential biologicalprocesses and KEGG pathways involved in target genes inthis study [] P was set as the cutoï¬ criterion ProteinProtein Interaction PPI Network Analysis Inthe present study PPI networks were used to reveal the interactions among diï¬erentially expressed OSspecific methylated genes using the STRING database stringdb PPI was visualized using Cytoscape [] Survival Analysis Survival analysis was performed usingthe OS microarray dataset mixed osteosarcoma mesenchymalKuijjer127vstilmnhwg6v2 from the R2 GenomicsAnalysis and Visualization Platform httpr2amcnl Themedian expression of targets was selected as the cutoï¬ todivide all OS samples into the high or low group 0cComputational and Mathematical Methods in MedicineTranscription from RNA polymerase II promoterNervous system developmentResponse to drugChemical synaptic transmissionCell migrationPotassium ion transmembrane transportNeuron differentiationMovement of cell or subcellular componentAnteriorposterior pattern specificationSkeletal muscle cell differentiationRegulation of synaptic plasticityLongterm memoryHindbrain developmentGland developmentQ valueCountaSignal transductionGprotein coupled receptor signaling pathwayImmune responseInflammatory responseDetection of chemical stimulusDefense response to bacteriumSodium ion transportPositive regulation of PI3K signalingMonocyte chemotaxisAcutephase responseDefense response to fungusCountbPathways in cancerHTLVI infectionMAPK signaling pathwaycAMP signaling pathwayHippo signaling pathwayRap1 signaling pathwayCalcium signaling pathwayTranscriptional misregulation in cancerWnt signaling pathwayEpsteinˆ’Barr virus infectionMelanogenesisColorectal cancerEndometrial cancerBasal cell carcinomaCocaine addictionType I diabetes mellitusThyroid cancerQ valueQ valueCountcFigure Continued 0cComputational and Mathematical Methods in MedicineOlfactory transductionCytokineˆ’cytokine receptor interactionPhagosomeCell adhesion molecules CAMsStaphylococcus aureus infectionComplement and coagulation cascadesInflammatory bowel disease IBDHematopoietic cell lineageBile secretionArrhythmogenic right ventricular cardiomyopathy ARVCViral myocarditisAutoimmune thyroid diseaseType I diabetes mellitusFat digestion and absorptionAllograft rejectionEther lipid metabolismGraftˆ’versusˆ’host diseaseAsthmaFatty acid biosynthesisQ valueCountdFigure Bioinformatics analysis of hypermethylation genes and hypomethylation genes a GO analysis of OSspecific hypermethylationgenes b GO analysis of OSspecific hypomethylation genes c KEGG pathway analysis of OSspecific hypermethylation genes dKEGG pathway analysis of OSspecific hypomethylation genes The gene ratio was present in the Xaxis Results Identification of OSSpecific Methylated Genes The public dataset GSE97529 was used to identify OSspecific methylated genes DNA methylation status of CpG sitesin Ewing™s sarcoma synovial sarcoma and OS samples were included in this dataset Figure 1a Totally weidentified OSspecific methylated genes including hypermethylation genes and hypomethylation genesin OS samples compared to Ewing™s sarcoma or synovial sarcoma samples Figure 1a GO and KEGG Pathway Enrichment Analyses GO analysis showed that hypermethylation genes were significantlyassociated with biological processes BP of the nervous system development chemical synaptic transmission transcription from RNA polymerase II promoter anteriorposteriorpattern specification regulation of synaptic plasticity neurondiï¬erentiation movement of cell or subcellular componentskeletal muscle cell diï¬erentiation response to drug potassium ion transmembrane transport hindbrain developmentgland development and cell migration Figure 2a Hypomethylation genes were significantly related to immuneresponseinflammatory responseacutephase response sodium ion transport monocyte chemotaxis detection of chemical stimulus defense responseto fungus positive regulation of PI3K pathway cell chemotaxis chemotaxis neutrophil chemotaxis innate immuneresponse ion transmembrane transport and cell adhesionFigure 2btransductionsignalKEGG pathway analysis showed that significant pathways of hypermethylation genes in OS included the Hippopathway cAMP signaling thyroid cancer pathways in cancer calcium signaling endometrial cancer Rap1 signalingpathway transcriptional misregulation in cancer MAPK signaling pathway EpsteinBarr virus infection Wnt signalingpathway cocaine addiction and basal cell carcinomaFigure 2c And hypomethylation genes in OS were associated with Staphylococcus aureus infection olfactory transductioninflammatory bowel disease IBD complementand coagulation cascades allograft rejection fat digestionand absorption graftversushost disease phagosome viralmyocarditis and fatty acid biosynthesis Figure 2d OSSpecific Methylated Genes Were Diï¬erentiallyExpressed in OS Subsequently an independent public dataset GSE42572 was used to identify diï¬erentially expressedgenes in OS As shown in Figure 3a we identified upregulated genes and downregulated genes in OS compared to healthy control samples Figure 3a AmongDEGs a total of downregulated hypomethylation geneswere screened out from overlapping hypermethylationand downregulated genes while upregulated hypomethylation genes were screened out from overlapping hypomethylationgenesFigure 3b The diï¬erentially expressed OSspecificmethylated genes were presented by heat map Figure 3cdownregulatedand Construction of PPI Network to Identify HubDiï¬erentially Expressed OSSpecific Methylated Genes Furthermore we constructed a PPI network to identify a hub differentially expressed OSspecific methylated gene using theSTRING database As presented in Figure a total of nodes and edges were included in this network The hubgenes included NPSR1 PTAFR LPAR5 PTGER3 NPY5RKCNK3 KRTDAP HCN4 KRT38 KCNIP2 KCNJ5 andKRTCAP3 Figure The Survival Time Analysis of Diï¬erentially ExpressedOSSpecific Methylated Genes The above analysis was conducted with the GSE97529 and GSE42572 datasets Unfortunately the clinical information about metastasisfree survivaltime was not included in both databases Thus we analyzedan independent database R2 dataset httpr2amcnl to 0cComputational and Mathematical Methods in MedicineDownregulatedLow methylatedHigh methylatedUpregulated“““aFigure Continuedb 0cComputational and Mathematical Methods in MedicineCAMK2AKIAA1211CLDN7CECR1DCCCALCBLY9KLHL32FCRL3KRT38PIK3AP1CR2IFT122KCNJ5SV2CHPSE2NPY5RPTAFRGAS2IL22PCSK6PPP2R2BAQP9PCDH21GADL1ARHGAP9MPP2LILRA4MDM2KRTDAPEPHA10KCNIP2PTGER3C10orf55NOS1NETO1NPSR1MARCOGZMBLPAR5ARSIKCNK3SAMSN1ACSM1FUT6PTPN22BREMRPL39OR2J2PCDHA12PFKFB2STAB2ZAKHCN4NR2E3IQSEC3CBLN4PCDHGB5RHOBTB1RAB3DBZW1ANKMY1MACF1C19orf12PRXCCDC149PLEC1KRTCAP3MEOX1GJA4RHOBTB11ˆ’ˆ’CSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOcCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHFigure GSE42572 was analyzed to identify diï¬erently expressed genes in OS compared to normal samples a induced genes and reduced genes in OS compared to healthy control samples b Among DEGs a total of downregulated hypomethylation genes and upregulated hypomethylation genes were screened out c The diï¬erentially expressed OSspecific methylated genes were presented byheat mapfurther evaluate the prognostic value of OSspecific methylated genes The median expression of candidates in all OSsamples was selectedAs the cutoï¬ is used to divide OS samples into the highand low groups it was shown that higher expression ofCBLN4 P was associated with longer metastasisfree survival time in patients with OS as well as ANKMY1P BZW1 P and KRTCAP3 P However higher expression of GZMB P KRTDAPP LY9 P PFKFB2 P PTPN22P and CLDN7 P was associated withshorter metastasisfree survival time in patients with OSFigure DiscussionThe mechanisms underlying OS progression remainedlargely unclear It has been widely accepted that DNA 0cComputational and Mathematical Methods in MedicineIFT122STAB2FCRL3MARCOCALCBGAS2PLECNPSR1PTPN22LY9NPY5RPTGER3LPAR5PTAFRMPP2NOS12AMDM2RHOBTB1ARHGAP9KRTDAPKRT38KCNK3KCNIP2HCN4KRTCAP3KCNJ5GZMBIL22Figure PPI network analysis PPI network was used to identify a hub diï¬erentially expressed OSspecific methylated gene using theSTRING databasemethylation was involved in regulating the tumorigenesisand developmentthough modulating gene expressionDNA methylation has been shown to play an important rolein gene regulation and implicated in various types of cancerEmerging studies revealed that the cancerspecific CpGhypermethylation could turn oï¬ the expression of tumorsuppressors however cancerspecific CpG hypomethylationcould activate the expression of oncogenes [] Identification of aberrantly methylated genes in OS would be helpfulto identify new diagnostic and therapeutic biomarkers forOS The present study identified OSspecific methylatedgenes from Ewing™s sarcoma or synovial sarcoma samplesBioinformatics analysis revealed that OSspecific methylatedgenes were involved in regulating multiple biological processes including chemical synaptic transmission transcription response to drug and regulating immune responseFurther validation indicated that OSspecific methylatedgenes were dysregulated in OS samples and correlated tothe prognosis of patients with OSOS together with Ewing™s Sarcoma EWS and synovialsarcoma SS was the most common pediatric sarcomas[] These types of sarcomas occur in similar anatomicallocations however the treatments for these sarcomas differed depending on the tumor type The accurate diagnosisof OS remained to be a big challenge Emerging studies demonstrated that aberrant DNA methylation was associatedwith the prognosis of human cancers including OS Forexample DNA methylation level of WNT6 and ESR1 wasrelated to the prognosis of OS The present study is aimedat identifying OSspecific methylated genes A total of OSspecific methylated genes were identified including hypermethylation genes and hypomethylation genesin OS samples compared to Ewing™s sarcoma or synovial sarcoma samples Furthermore GO and KEGG pathway analyses were further used to predict the potential roles of OSspecific methylated genes Of note our predictions showedthat these methylated genes were associated with the Hipposignaling and Wnt signaling Hippo pathway aberrationshad been demonstrated in OS by multiple studies andinvolved in regulating primary tumor growth angiogenesisepithelial to mesenchymal transition and metastatic dissemination [] The Hippo signaling played an important rolecontrolling cancer cell proliferation and apoptosis [] Multiple studies indicated YAP was overexpressed in OS samplesand knockdown of YAP significantly inhibits OS cell growthand invasion [] Sox2 as a YAP upstream regulator wasreported to be required for tumor development and cancercell proliferation in OS [] This study provided a potentialmechanism to elucidate how the Hippo signaling activated inOS Many studies support an aberrant activation of thecanonical Wnt signaling pathway in osteosarcoma cells Forexample two recent studies described a high βcatenin levelin osteosarcoma tissues compared to adjacent healthy tissuesassociated with poor prognosisand lung metastatic 0cComputational and Mathematical Methods in Medicine lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P Bonf P Time monthsCBLN4 highCBLN4 lowaRaw P Bonf P Time monthsGZMB highGZMB lowd lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P Bonf P Time monthsANKMY1 highANKMY1 lowbRaw P 62e03Bonf P Time monthsKRTCAP3 highKRTCAP3 loweFigure ContinuedRaw P Bonf P Time monthsBZW1 highBZW1 lowcRaw P Bonf P Time monthsKRTDAP highKRTDAP lowf 0cComputational and Mathematical Methods in Medicine lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P 71e03Bonf P Time monthsLY9 highLY9 lowgRaw P Bonf P Time monthsPTPN22 highPTPN22 lowiRaw P Bonf P Time monthsPFKFB2 highPFKFB2 lowhRaw P Bonf P Time monthsCLDN7 highCLDN7 lowjFigure The prognostic values of diï¬erentially expressed OSspecific methylated genes were calculated by using the R2 Genomics Analysisand Visualization Platform a“j Higher expression of CBLN4 a was associated with longer metastasisfree survival time in patients withOS as well as ANKMY1 b BZW1 c and KRTCAP3 e However higher expression of GZMB d KRTDAP f LY9 g PFKFB2h PTPN22 i and CLDN7 j was associated with shorter metastasisfree survival time in patients with OSdissemination Wnt signaling pathway played a crucial rolein tumorigenicity and metastasis via regulation oftheimmune system bone remodeling angiogenesis hypoxiaresponse and EMT []Of note this study showed that OSspecific methylatedgenes were significantly diï¬erentially expressed in OS samples A total of downregulated hypomethylation genesand upregulated hypomethylation genes were identifiedin this study PPI network analysis was constructed to revealthe relation among these genes Totally nodes and edges were included in this network By analyzing R2 datasets we found the dysregulation of these OSspecific methylated genes were associated with the metastasisfree survivaltime in patients with OSincluding CBLN4 ANKMY1BZW1 KRTCAP3 GZMB KRTDAP LY9 PFKFB2PTPN22 and CLDN7 Among these regulators BZW1 is atranscription factor related to the regulation of cell cycleand proliferation [] LY9 was a member of SLAM familyof immunomodulatory receptors [] and interacted withthe adaptor molecule signaling lymphocyte activationmoleculeassociated proteins A previous study showed LY9was related to the cancer progression and correlated to overall survival of the patients with breast cancer PFKFB2 is anenzyme involved in regulating the Warburg eï¬ect alsotermed as glycolysis [] PFKFB2 had been found to havea key role in regulating tumor growth and survival in multiple cancer types including gastric cancer gliomas and osteosarcoma [“]Several limitations were also exited in this study Firstour studies revealed several hub OSspecific methylatedgenes However the roles of these genes remained to beunclear The gain or loss of function assays should be performed to further explore their roles in OS Next the expression levels and methylation levels of hub OSspecificmethylated genes in OS samples should be confirmed usingclinical samples Third the direct interaction among thesehub genes has not been confirmed using experimental assays ConclusionIn this study identified downregulated hypomethylation genes and upregulated hypomethylationgenes in OS and a series biological processes and pathwaysregulated by aberrantly methylated genes PPI network analysis revealed the interactions among these genes Moreoverthe present study showed that the dysregulation of OSspecific methylated genes wascorrelated with themetastasisfree time in patients with OS including CBLN4 0cComputational and Mathematical Methods in MedicineANKMY1 BZW1 KRTCAP3 GZMB KRTDAP LY9PFKFB2 PTPN22 and CLDN7 This study provided a betterunderstanding of the molecular mechanisms underlying theprogression and OS and novel biomarkers for the prognosisof OSData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that they have no conflicts of interestAuthors™ ContributionsFei Wang and Guoqing Qin are cofirst authorsAcknowledgmentsThis work was supported by the Special fund for the key laboratory of Science and Technology Department of Jilin Province 20190201282JCReferences[] E Simpson and H L Brown œUnderstanding osteosarcomasJAAPA vol no pp “ [] G Bacci P Picci P Ruggieri œPrimary chemotherapyand delayed surgery neoadjuvant chemotherapy for osteosarcoma of the extremities The Istituto Rizzoli experience in patients treated preoperatively with intravenous methotrexate high versus moderate doses and intraarterial cisplatin Cancer vol no pp “ [] A Luetke P A Meyers I Lewis and H Juergens œOsteosarcoma treatment where do we stand A state of the art reviewCancer Treatment Reviews vol no pp “ [] P Picci œOsteosarcoma osteogenic sarcoma OrphanetJournal of Rare Diseases vol no p [] W Wang H Shen G Cao and J Huang œLong noncodingRNA Xist predicts poor prognosis and promotes malignantphenotypes in osteosarcoma Oncology Letters vol no pp “ [] R E Windsor S J Strauss C Kallis N E Wood and J SWhelan œGermline genetic polymorphisms may influencechemotherapy response and disease outcome in osteosarcomaa pilot study Cancer vol no pp “ [] W Liu G Xu H Liu and T Li œMicrorna4903p regulatescell proliferation and apoptosis by targeting Hmga2 in osteosarcoma FEBS Lett vol no 20PartB pp “[] J Cui W Wang Z Li Z Zhang B Wu and L Zeng œEpigenetic changes in osteosarcoma Bulletin du Cancer vol no pp E62“E68 [] K RaoBindal and E S Kleinerman œEpigenetic regulation ofapoptosis and cell cycle in osteosarcoma Sarcoma vol Article ID pages [] M Kim J Y Jung S Choi œGfra1 promotes cisplatinin osteosarcoma by inducinginduced chemoresistanceautophagy Autophagy vol no pp “ [] S V Iyer A Ranjan H K Elias œGenomewide RNAiscreening identifies TMIGD3 isoform1 as a suppressor ofNF κB and osteosarcoma progression Nature Communications vol no p [] F Neri S Rapelli A Krepelova œIntragenic DNA methylation prevents spurious transcription initiation Naturevol no pp “ [] N Nishida T Nagasaka T Nishimura I Ikai C R Bolandand A Goel œ'Aberrant methylation of multiple tumor suppressor genes in aging liver chronic hepatitis and hepatocellular carcinoma Hepatology vol no pp “ [] C G Ekmekci M I Gutierrez A K Siraj U Ozbek andK Bhatia œAberrant methylation of multiple tumor suppressor genes in acute myeloid leukemia American Journal ofHematology vol no pp “ [] Q Li H Li X Zhao œDNA methylation mediated downregulation of Mir449c controls osteosarcoma cell cycle proCMycgressionInternational Journal of Biological Sciences vol no pp “ oncogenetargetingdirectlyby[] J Lu and J Wang œIrx1 hypomethylation in osteosarcomametastasis Oncotarget vol no pp [] L Li C Xu P Liu and J Huang œCorrelation study of DNAmethylation of Wnt6 gene with osteosarcoma in childrenOncology Letters vol no pp “ [] V Sonaglio A C de Carvalho S R Toledo œAberrantDNA methylation of Esr1 and P14arf genes could be usefulas prognostic indicators in osteosarcoma Oncotargets andTherapy vol pp “ [] S P Wu B T Cooper F Bu œDNA methylationbasedclassifier for accurate molecular diagnosis of bone sarcomasJCO Precision Oncology vol [] E P Buddingh S E N Ruslan C M A Reijnders œMesenchymal stromal cells of osteosarcoma patients do not showevidence of neoplastic changes during longterm cultureClinical Sarcoma Research vol no p [] G Dennis Jr B T Sherman D A Hosack œDavid database for annotation visualization and integrated discoveryGenome Biology vol no p [] P Shannon A Markiel O Ozier œCytoscape a softwareenvironment for integrated models of biomolecular interaction networks Genome Research vol no pp “ [] J C Spainhour H S Lim S V Yi and P Qiu œCorrelationpatterns between DNA methylation and gene expression inThe Cancer Genome Atlas Cancer Informatics vol p [] A Ferrari U Dirksen and S Bielack œSarcomas of soft tissueand bone Progress in Tumor Research vol pp “[] S Morice G Danieau F Redini B BrounaisLeRoyer andF Verrecchia œHippoYap signaling pathway a promisingin bone paediatric cancers Cancerstherapeutic targetBasel vol no p [] S L Teoh and S Das œThe emerging role of the hippo pathway in lung cancers clinical implications Current Drug Targets vol no pp “ [] H Wang Y C Du X J Zhou H Liu and S C Tang œThedual functions of yap1 to promote and inhibit cell growth inhuman malignancy Cancer Metastasis Reviews vol no pp “ 0cComputational and Mathematical Methods in Medicine[] Y A Chen C Y Lu T Y Cheng S H Pan H F Chen andN S Chang œWw domaincontaining proteins yap and Taz inthe hippo pathway as key regulators in stemness maintenancetissue homeostasis and tumorigenesis Frontiers in Oncologyvol p [] P McQueen S Ghaï¬ar Y Guo E M Rubin X Zi and B HHoang œThe Wnt signaling pathway implications for therapyin osteosarcoma Expert Review of Anticancer Therapyvol no pp “ [] S Li Z Chai Y Li œBzw1 a novel proliferation regulatorthat promotes growth of salivary muocepodermoid carcinoma Cancer Letters vol no pp “ [] A Angulo M Cuenca P MartinezVicente and P EngelœViral Cd229 Ly9 homologs as new manipulators of hostimmunity Journal of Leukocyte Biology vol no pp “ [] S C Ozcan A Sarioglu T H Altunok œPfkfb2 regulatesglycolysis and proliferation in pancreatic cancer cells Molecular and Cellular Biochemistry vol no pp “[] Q Cheng and L Wang œLncRNA XIST serves as a ceRNA toregulate the expression of ASF1a BRWD1M and PFKFB2 inkidney transplant acute kidney injury via sponging hsamiR2123p and hsamiR1225p Cell Cycle vol no pp “ [] H Liu K Chen L Wang œmiR613 inhibits Warburgeï¬ect in gastric cancer by targeting PFKFB2 Biochemicaland Biophysical Research Communications vol no pp “ [] M Camargo BarrosFilho L Barreto Menezes de LimaM Bisarro dos Reis œPfkfb2 promoter hypomethylationas recurrence predictive marker in welldiï¬erentiated thyroidcarcinomas InternationalJournal of Molecular Sciencesvol no p [] Z He C You and D Zhao œLong noncoding RNA UCA1miR182Pfkfb2 axis modulates glioblastomaassociated stromal cellsmediated glycolysis and invasion of glioma cellsBiochemical and Biophysical Research Communicationsvol no pp “ [] A Sreedhar P Petruska S Miriyala M Panchatcharam andY Zhao œUcp2 overexpression enhanced glycolysis via activation of PFKFB2 during skin cell transformation Oncotargetvol no pp “ 0c"

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

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

"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

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

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

Thyroid_Cancer

Human pyruvate dehydrogenase phosphatase PDP1 plays an important physiological role in energy metabolism however its expression and function in human pancreatic adenocarcinoma PDAC remain unknown This study aimed to investigate the expression pattern and mechanisms of action of PDP1 in human PDACMethods The expression pattern of PDP1 in PDAC was determined and its correlation with patient survival was analyzed Ectopic expression or knockdown of PDP1 was performed and in vitro proliferation and migration as well as in vivo tumor growth of PDAC were measured The mechanism was studied by biochemical approachesResults PDP1 was overexpressed in human PDAC samples and high expression of PDP1 correlated with poor overall and disease‘free survival of PDAC patients PDP1 promoted the proliferation colony formation and invasion of PDAC cells in vitro and facilitated orthotopic tumor growth in vivo PDP1 accelerated intracellular ATP production leading to sufficient energy to support rapid cancer progression mTOR activation was responsible for the PDP1‘induced tumor cell proliferation and invasion in PDAC AMPK was downregulated by PDP1 overexpression resulting in mTOR activa‘tion and cancer progressionConclusion Our findings suggested that PDP1 could be a promising diagnostic and therapeutic target for anti‘PDAC treatmentKeywords Pancreatic adenocarcinoma Pyruvate dehydrogenase phosphatase ATP mTOR AMPKIntroductionPancreatic adenocarcinoma PDAC which accounts for of pancreatic cancer cases is among the top lifethreatening cancers with a very high death rate and a survival rate of only approximately [] The malignancy of PDAC has been increasing in recent years Correspondence zhaojiangang78163com lianyu_chenhotmailcom Ye Li and Jia Shen contributed equally to this work Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shaoxing Central Hospital Zhejiang ChinaFull list of author information is available at the end of the and this disease is estimated to be the second leading cause of cancerrelated death in the USA [] The dismal prognosis of PDAC could be due to the lack of sensitive detection at its early stage although many biomarkers have been suggested as indicators of PDAC in various experimental studies [] Effective treatments for PDAC are unavailable Although surgical resection is recommended as the only curative treatment [] only a very low number of patients who have small tumors that are detected early are suitable for surgery [] Chemotherapy as the firstline treatment is applied to nonsurgical PDAC patients [] however the response rate is low and these drugs can only prolong survival by a few months even in 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 httpcrea‘tivecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdo‘mainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLi a0et a0al Cell Biosci Page of responding patients [] Identifying new prognostic and therapeutic biomarkers for PDAC patients is importantThe human PDP1 gene encodes pyruvate dehydrogenase phosphatase PDP one of the two PDP isoforms in mammalian cells [] Physiologically PDP1 serves as a critical regulator of the pyruvate dehydrogenases complex PDC PDP1 positively regulates the catalytic activities of PDC by the removal of phosphates from the serine sites on E1α of the complex [] Upon activation by PDP1 the PDC irreversibly triggers oxidative decarboxylation of pyruvate into acetylCoA which is the main substrate for cellular energy production [] Mutation of PDP1 in some patients may cause PDC deficiency a genetic disorder characterized by neurodegeneration and abnormal metabolism [] Loss of PDP1 may also cause intolerance to exercise and mild developmental delay in patients [ ] and may produce a lethal phenotype in infants [] The expression of PDP1 in muscle cells of obese and diabetic subjects was reduced and could be reversed by endurance training [ ] The loss of PDP1 in obesity was found to signal insulin resistance that could be reversed by plasma insulin supplementation [] Overexpression of PDP1 was also found in human prostate cancer and could promote cell proliferation and tumor growth [] The expression pattern and function of PDP1 in PDAC remain unclearIn this study we attempted to identify the expression and functional role of PDP1 in human PDAC The expression pattern of PDP1 in human PDAC samples was illustrated by extracting data from the GEO database The correlation between PDP1 expression and patient survival was profiled to evaluate its prognostic value The functional role of PDP1 in the proliferation growth and invasion of PDAC cells was then evaluated in cellular and animal models The signaling pathway involved in the regulation of PDP1 was elucidated We believe this study will shed light on the prognostic and therapeutic value of PDP1 in PDACMaterials and a0methodsReagents plasmids and a0antibodiesSodium acetate and compound C were obtained from SigmaAldrich USA Human and murine ORF clones of PDP1 and shRNA against human PDP1 were purchased from Origene USA Antibodies against PDP1 phosphomTOR mTOR phosphoAMPKα AMPKα PCNA and actin were purchased from Cell Signaling Technologies USACell line and a0cell cultureThe PDAC cell line KP3 was purchased from the Japanese Collection of Research Bioresources Cell Bank Cells were cultured in RPMI medium supplemented with fetal bovine serum FBS and penicillinstreptomycin under humid conditions of CO2 and a0°C Panc2 cells were obtained from the Frederick National Laboratory for Cancer Research Frederick MD USA Cells were cultured in DMEM supplemented with FBS and penicillinstreptomycin under humid conditions of CO2 and a0°CAnimal studyThe animal study was performed in accordance with the approved protocol by Fudan University Ref No 2019FUSCCJS017 In brief Panc2 cells tagged with a luciferase reporter were mixed with Matrigel Matrix BD Bioscience USA A a0μl mixture containing — Panc2 cells was injected into the pancreas of C57BLJ mice Measurement of orthotopic tumor size was performed once per week from a0week postinjection using the IVIS Spectrum live animal imager PerkinElmer USA with luciferin a0mgkg ip as a substrate At the end of the study the mice were sacrificed and the pancreas was dissectedCell proliferation and a0colony formation assaysCell proliferation was measured by cell counting In brief cells were seeded for growth and were counted at and a0days of cultivation The formation of colonies was measured by colony formation assays A total of cells were seeded for growth for a0days Cells were fixed with paraformaldehyde and stained with crystal violet The number of colonies formed was countedCell invasion assayThe invasiveness of PDAC cells was measured by chamber assays In brief cells were seeded onto the upper chamber of inserts precoated with Matrigel matrix BD Bioscience USA The receiving chamber was filled with DMEM supplemented with FBS Fortyeight hours later the medium on the upper chamber was removed and cells that migrated through the Matrigel matrix were dissociated with calceinAMcontaining buffer The fluorescence intensity representing the number of invaded cells was measured by a fluorescence microplate readerImmunoblottingTotal protein was extracted from the cell pellets using RIPA buffer and separated by SDSPAGE Protein was then transferred onto the PDVF membrane which was blocked in BSA blocking buffer for a0h at room temperature Then the membrane was incubated with primary antibodies overnight at a0°C followed by secondary 0cLi a0et a0al Cell Biosci Page of antibody incubation for a0 h at room temperature The membrane was visualized with chemiluminescence BioRad USA using ECL Select as a substrate GE Healthcare GermanyIntracellular ATP measurementIntracellular ATP measurement was performed with a commercial kit Biovision USA In brief — cells were lysed with a0μl of ATP assay buffer followed by deproteinization through a a0 kDa spin column Then a0μl of flow through was mixed with a0μl of ATP assay buffer a0μl of ATP probe a0μl of ATP converter and a0μl of developmental reagent for reaction in the dark for a0min at room temperature The absorbance was then read at a0 nm The concentration of ATP in the cell samples was calculated with a standard curve plotted using an ATP standardHistologyParaffinembedded pancreatic sections were prepared and 5mm slices were generated For histological measurement of PDAC the slides were stained with hematoxylin eosin HE The image was captured under a light microscopeStatistical analysisData are presented as the mean ± SD Data were analyzed by oneway ANOVA and p was considered statistically significant All experiments were performed in triplicate unless stated otherwiseResultsPDP1 was a0overexpressed in a0PDAC and a0overexpression of a0PDP1 predicted poor prognosis of a0patientsThe expression and function of PDP1 in PDAC have not been reported A recent study showed that the expression of PDP1 was elevated in prostate cancer in which ectopic expression of PDP1 promoted cancer growth and progression [] Another study however observed that phosphorylation of PDP1 at Tyr94 suppressed its activity and reduced tumor growth [] These divergent observations indicated that PDP1 may have different functions in particular types of cancers To determine the role of PDP1 in PDAC we first examined its expression in human PDAC samples Data retrieved from two published datasets of PDAC GSE15471 and GSE28735 revealed that PDP1 was overexpressed in human PDAC tissues compared with the nonmalignant normal pancreatic tissues Fig a01a b Immunohistochemical examination of the tissue blocks confirmed that the protein expression of PDP1 was substantially elevated in the PDAC tissues Fig a01c To determine the clinical significance of this elevation we retrieved patient survival data from TCGA database We found that the expression of PDP1 predicted poor overall survival and diseasefree survival of the PDAC patients p and p respectively which indicates that PDP1 may be a poor prognostic factor in PDAC Fig a0 1d e Furthermore PDP1 may be a marker for patient mortality as its expression was significantly higher in patients who died than those who survived p Fig a01f These findings suggested that PDP1 was overexpressed in PDAC tissues and its overexpression can predict a poor prognosis for patientsPDP1 regulates the a0in a0vitro proliferation and a0invasion of a0PDAC cellsTo further elucidate the functional role of PDP1 in PDAC cells1 we first identified its in a0 vitro function in a set of PDAC cells The expression of PDP1 in different PDAC cells including MiaPaca2 Panc1 Panc2 KP3 and Capan1 was screened to select suitable cell lines for functional studies data not shown Panc2 and KP3 were selected due to their moderate level of PDP1 expression among the cell lines We then overexpressed PDP1 by transfecting a plasmid encoding the ORF of the genes while knocking down its expression by shRNA against PDP1 and established stably transfected cell lines by selection Fig a0 2a A proliferation assay was performed to examine whether PDP1 expression could alter cell growth in PDAC We found that in both cell lines PDP1 overexpression significantly promoted the growth rate of the cells while PDP1 knockdown had the opposite effects Fig a0 2b suggesting that PDP1 expression may promote tumor cell proliferation in PDAC This finding was further confirmed by colony formation assays which showed that PDP1 overexpression potently increased the number of colonies formed by PDAC cells while suppression of PDP1 reduced the ability to form colonies Fig a02c In addition as the in a0vitro invasiveness of tumor cells indicates the in a0vivo locoregional and distant spread of cancer we examined the invasion of PDAC cell lines with a chamber assay Overexpression of PDP1 significantly increased the number of cells invading through the extracellular matrix Fig a0 2d These observations suggested that PDP1 expression could promote the in a0vitro proliferation and invasion of PDAC cellsPDP1 overexpression accelerated the a0in a0vivo tumor growth of a0PDACTo further elucidate the in a0vivo functional role of PDP1 we established an orthotopic model of PDAC by injecting Panc2 cells into the pancreas of C57BL6J mice For noninvasive monitoring of tumor growth Panc2 cells with or without PDP1 overexpression were stably infected with a luciferase reporter and luciferin could be catalyzed to 0cLi a0et a0al Cell Biosci Page of Fig PDP1 was overexpressed in PDAC Data were retrieved from the GEO database and PDP1 was found to be overexpressed in PDAC tissues compared with normal pancreatic tissues in a GSE15471 and b GSE28735 c Immunohistochemical staining of PDP1 was retrieved from the Human Protein Atlas which suggested that PDP1 protein expression was upregulated in PDAC tissues Survival of PDAC patients was retrieved from TGCA database and patients with PDP1 expression above the median level had poor d overall survival and e disease‘free survival f Surviving PDAC patients had lower expression of PDP1 than the censored patientsFig PDP1 regulated PDAC cell proliferation and migration in vitro a Transfection of the PDP1 ORF clone or shRNA regulated endogenous expression of PDP1 in PDAC cells b Overexpression of PDP1 promoted PDAC cell proliferation while PDP1 suppressed PDAC cell proliferation c Overexpression of PDP1 promoted colony formation of PDAC cells while PDP1 suppressed PDAC cell colony number d Overexpression of PDP1 promoted PDAC cell migration while PDP1 suppressed PDAC cell migration p 0cLi a0et a0al Cell Biosci Page of emit bioluminescent signals Under an in a0vivo imager we observed that overexpression of PDP1 could significantly increase the signal intensity of tumor cellderived luciferase indicating a larger tumor formed by PDP1overexpressing PDAC cells than the control cells Fig a0 3a Endpoint measurement of the dissected tumors showed that tumor growth was accelerated by PDP1 overexpression Fig a03b Histological analysis by HE staining showed that the PDP1overexpressing PDAC cells had a more aggressive phenotype eg an unclear border between pancreatic tissues and the tumor tissues than the control cells Fig a03c This result was further proven by the immunoblotting of PCNA acellular marker of proliferation in PDAC tissues the expression was significantly higher in the PDP1overexpressing PDAC tumors than the control tumors Fig a03d These findings suggested that PDP1 overexpression could substantially accelerate the in a0vivo tumor growth of PDACPDP1 overexpression accelerated ATP‘associated tumor cell growth in a0PDACMitochondrial PDP1 is an important regulator in energy production and metabolism in mammalian cells PDP1 catalyzes the dephosphorylation and concomitant reactivation of the α subunit of the E1 component of the pyruvate dehydrogenase complex PDC whose activity is critical for energy metabolism through the TCA cycle and oxidative phosphorylation [] A previous study suggested that suppression of PDP1 expression in embryonic stem cells is associated with reduced production of ATP the product of energy metabolism that is essential for many cellular processes [] Therefore we examined the cellular content of ATP in PDAC cells Overexpression of PDP1 could significantly increase the cellular ATP content in PDAC cells while knockdown of the protein showed the opposite effects Fig a04a To determine whether cellular ATP content plays an important role in mediating PDP1induced cell proliferation we supplied acetate as a substrate for ATP production by alternative mechanisms [] in PDAC cells with PDP1 knockdown The reconstitution of cellular ATP by acetate supplementation significantly reversed tumor cell proliferation in the cells with PDP1 knockdown Fig a0 4b Colony formation and cell invasion assays proved that the suppressive effect of PDP1 knockdown was attenuated by ATP supplementation in PDAC cells Fig a0 4c d These observations suggested that PDP1regulated cell proliferation and invasion in PDAC cells may be associated with the alteration of cellular ATP content and its related cellular activitiesmTOR is a0a a0downstream signaling pathway that a0mediates PDP1‘driven tumor cell proliferation in a0PDACmTOR signaling is often aberrantly hyperactivated in various types of cancer cells in response to the abundant cellular ATP to promote tumor cell proliferation [] In our study we examined the expression and activity of the mTOR pathway in PDAC cells with PDP1 knockdown Fig PDP1 overexpression promoted PDAC growth in vivo a PDP1 overexpression significantly increased the luciferase signal in the orthotopic tumors of PDAC in mice b PDP1 overexpression increased the tumor size of the orthotopic PDAC model c HE staining suggested that more cells were undergoing mitotic proliferation in the PDP1‘overexpressing tumors than the control tumors d PDP1‘overexpressing tumors expressed a higher level of PCNA than the control tumors p 0cLi a0et a0al Cell Biosci Page of Fig PDP1 induced PDAC cell proliferation and migration by inducing intracellular ATP a PDP1 overexpression induced intracellular ATP content while its knockdown reduced ATP levels Supplementation of an alternative substrate of ATP production acetate restored cell proliferation b colony formation c and migration of PDP1 knockdown PDAC cells d p Upon knockdown of PDP1 mTOR phosphorylation was significantly downregulated without notable changes in total protein expression Fig a0 5a To identify whether mTOR activity is crucial for PDP1mediated tumor cell proliferation we transfected a plasmid encoding the protein with a mutation that leads to constitutive activation of mTOR in PDAC cells with PDP1 knockdown Fig a05b Constitutive activation of mTOR restored the proliferation rate in the PDAC cells with PDP1 knockdown Fig a05c suggesting that mTOR activation is essential for PDP1driven tumor cell growth in PDAC In addition reactivation of mTOR in PDAC cells with PDP1 knockdown reconstituted the capacity of PDAC cells to form colonies and pass through the ECM Fig a0 5d e These findings indicate that mTOR activation mediates PDP1driven cell growth and invasion in PDAC as the downstream effectorAMPKα inhibition mediates PDP1‘induced mTOR activation in a0PDAC cellsWhen the cellular content of ATP is insufficient cells tend to activate AMPKα as a stress response pathway to limit growthrelated signaling [] Activation of AMPKα in PDAC was found to restrict cancer cell proliferation and aggressiveness in previous studies [ ] As PDP1 inhibition reduced ATP content as we observed in the present study we investigated whether AMPKα activity could be altered by PDP1 Overexpression of PDP1 potently suppressed the basal level of phosphorylated AMPKα without changing its total protein level while repressing PDP1 could activate AMPKα Fig a0 6a Inhibition of AMPKα in PDAC cells by its pharmacological inhibitor compound C CC restored mTOR activity Fig a0 6b suggesting that AMPKα acts as the upstream regulator of mTOR in the PDAC cells with PDP1 knockdown Furthermore inhibition of AMPKα significantly reactivated the proliferation colony formation and invasion of PDAC cells Fig a06c“e A previous study showed that an increased AMPATP ratio would activate AMPK activity [] We then added AMP to the PDP1overexpressing cells and found that an increased AMPATP ratio led to the restoration of AMPK in the these cells Fig a0 6f An opposite trend in mTOR activity was observed as mTOR is a downstream effector of AMPK These observations together with the other findings in this study indicated that PDP1 regulates PDAC growth by modulating the ATPAMPKαmTOR pathwayDiscussionIn this study we observed that PDP1 was overexpressed in human PDAC Overexpression of PDP1 in PDAC cells promoted cell proliferation and migration in a0 vitro and stimulated tumor growth in a murine model of PDAC which could be related to the increase in intracellular energy production Fig a0 Expression of PDP1 was reported to be associated with ATP generation in previous studies for instance PDP1 expression and activity were shown to contribute to a higher aerobic capacity 0cLi a0et a0al Cell Biosci Page of Fig mTOR was responsible for PDP1‘mediated PDAC cell proliferation and migration a The phosphorylation of mTOR in PDAC cells representing its activity was measured by immunoblotting PDP1 overexpression activated mTOR signaling while its knockdown suppressed mTOR phosphorylation b Transfection of constitutively activated mTORC reactivated mTOR signaling in PDP1 knockdown PDAC cells mTORC represents a plasmid encoding a mutated mTOR with a change from arginine to proline which leads to constitutive activation of mTOR regardless of intracellular signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e p of the muscle suggesting a sustainable level of oxidative phosphorylationrelated ATP production [] Another study also suggested that activation of PDP1 in cancer cells could induce a switch from cellular glycolysis to oxidative phosphorylation the more efficient method of ATP production [] The depletion of cellular ATP abolished PDP1induced PDAC cell proliferation migration and invasion suggesting that ATP generation was the essential step by which PDP1 triggers these cellular processes PDP1 expression was also found to be critical in some other cellular processes such as recovery from injury and cell differentiation [ ] Given the important regulatory role of PDP1 in the physiological function of PDC our observations may suggest that overexpression of PDP1 in PDAC cells functions to hyperactivate the PDC system overloading the cellular substrate of the tricarboxylic acid cycle which in turn accelerates the oxidative phosphorylation process that can generate more ATP essential for cellular processesWe used acetate as an alternative carbon source in tumor cells with PDP1 knockdown PDP1 is a critical enzyme involved in the pathway of ATP production by consuming glucose PDP1 is involved the reaction of pyruvate to acetylCoA the direct source of the TCA cycle This facilitates the production of reducing equivalents in the form of NADH FADH2 etc by TCA cycles which can be used by oxidative phosphorylation for the production of ATP Acetate has been reported as an additional source for ATP production in cancer cells especially when glucoseglutamine is depleted [] The direct ligation of acetate to CoA by acetylCoA synthetases can yield acetylCoA in the TCA cycle [] The TCA cycle can therefore produce reducing equivalents in the form of NADH FADH2 etc which were used for ATP production by oxidative phosphorylation in PDP1 knockdown cells As we assumed that suppression of ATP production in PDP1 knockdown cells is responsible for inhibition of tumor growth we used acetate as an alternative source of ATP production in these cells and our observations proved that ATP restoration in PDP1 knockdown cells recovered tumor cell proliferation and growth supporting our claims We also observed that the addition of acetate had a minimal effect on tumor growth in mock tumor cells Mock cells can consume glucose to 0cLi a0et a0al Cell Biosci Page of Fig AMPK inactivation was responsible for PDP1‘mediated PDAC cell proliferation and migration a PDP1 overexpression suppressed AMPK signaling while its knockdown induced activation via phosphorylation b The presence of compound C mM inactivated AMPK signaling in PDP1 knockdown PDAC cells which in turn reactivated mTOR signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e f AMP was supplemented in the PDP1‘overexpressing cells to adjust the intracellular AMPATP ratio Increased AMPATP ratio in the PDP‘overexpressing cells resulted in the restoration of AMPK activity but mTOR repression p Fig Schematic regulation of PDP1 in PDAC Overexpression of PDP1 may activate PDH which in turn accelerates the TCA cycle which provides reducing equivalents in the form of NADH FADH2 etc for the ATP production by oxidative phosphorylation The abundance of cellular ATP inhibits AMPK activation restoring the mTOR signaling that can promote proliferation and invasion of PDAC cellsproduce acetate which is then directly ligated by acetylCoA synthetases in the TCA cycle The reason for these results after addition of acetate to cells with an intact pathway of glucose metabolism is not fully understood however as the TCA cycle has a strict regulatory mechanism that involves production of reducing equivalents as 0cLi a0et a0al Cell Biosci Page of sources of ATP production this complicated process has ratelimiting steps even when the acetylCoA amount is overwhelming In this regard the addition of acetate may not further accelerate cell proliferation and growth in mock cells which can normally consume glucose to produce ATPWe observed that AMPKmTOR signaling was involved in PDP1 regulation of PDAC progression As an energy sensor AMPK is activated when the intracellular ATP level is insufficient and triggers a series of downstream responses that inhibit rapid cell proliferation hence AMPK has been frequently identified as a potential target in anticancer treatment [] PDP1 overexpression could induce activation of PDC which in turn generates more substrate for the production of ATP In this case overexpression of PDP1 in our study resulted in sustained repression of AMPK signaling due to the abundance of intracellular ATP that led to suppression of multiple downstream proliferationassociated mechanisms mTOR is a key molecule that mediates rapid protein synthesis during cancer progression [] Phosphorylation of mTOR signaling activates translational control by initiating the formation of the ribosome complex as well as the association of mRNA at its ™cap [] This process was halted under nutrientdeprived conditions in which mTOR was dephosphorylated and could not initiate capdependent translation [] The activation of AMPK as a nutrient sensor in some previous studies has been found to primarily downregulate mTOR activity in cancers [ ] PDP1mediated AMPK repression therefore could be a possible mechanism to facilitate mTORassociated PDAC progression which was confirmed by our observation that suppression of AMPK in PDP1 knockdown cells revoked mTOR signalingAMPK signaling which in turn suppressed mTOR activity Repression of AMPK in PDP1 knockdown cells restored the proliferation and migration of PDAC Our study suggested that PDP1 may be an attractive target for the diagnosis and treatment of PDACAcknowledgementsNot applicableAuthors™ contributionsLC and JZ conceived the idea designed the experiments analyzed the data and drafted the manuscript YL and JS performed the experiments and ana‘lyzed the data CC performed the experiment HG revised the manuscript All authors read and approved the final manuscriptFundingThis work was financially supported by the National Natural Science Foundation of China and Wu JiePing Medical Foundation Availability of data and materialsThe datasets used andor analysed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateThe animal study was performed in accordance with the approved protocol by Fudan University Ref No ‘FUSCC‘JS‘Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shanghai Medical College Fudan University Shanghai China Department of Oncol‘ogy First People™s Hospital of Pinghu Zhejiang China Department of Oncology Shaoxing Central Hospital Zhejiang China Received April Accepted July ConclusionIn summary in this study we identified the expression pattern and function of PDP1 in PDAC PDP1 was overexpressed in PDAC samples compared with normal pancreatic samples and high expression of PDP1 was correlated with a poor prognosis of PDAC patients Overexpression of PDP1 promoted the in a0 vitro proliferation colony formation and migration of PDAC cells while knockdown of PDP1 had the opposite effects Overexpression of PDP1 stimulated the in a0 vivo growth of orthotopic PDAC tumors in a murine model PDP1 regulated the production of intracellular ATP and supplementation with ATP recovered tumor cell proliferation and migration in PDP1 knockdown PDAC cells This result could be related to the alteration of mTOR activity in PDAC cells and recovery of mTOR activity in PDP1 knockdown PDAC cells restored cell proliferation and migration Mechanistically PDP1 knockdown activated References Collaborators GBDPC The global regional and national burden of pancreatic cancer and its attributable risk factors in countries and ter‘ritories “ a systematic analysis for the Global Burden of Disease Study Lancet Gastroenterol Hepatol “ Rahib L Smith BD Aizenberg R Rosenzweig AB Fleshman JM Matrisian LM Projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the United States Cancer Res “ Kim J Bamlet WR Oberg AL Chaffee KG Donahue G Cao XJ Chari S Garcia BA Petersen GM Zaret KS Detection of early pancreatic ductal adenocarcinoma with thrombospondin‘ and CA19‘ blood markers Sci Transl Med Hackert T Surgery for Pancreatic Cancer after neoadjuvant treatment Ann Gastroenterol Surg “Satoi S Yamamoto T Yamaki S Sakaguchi T Sekimoto M Surgical indica‘tion for and desirable outcomes of conversion surgery in patients with initially unresectable pancreatic ductal adenocarcinoma Ann Gastroen‘terol Surg “ Chandana S Babiker HM Mahadevan D Therapeutic trends in pan‘creatic ductal adenocarcinoma PDAC Expert Opin Investig Drugs “ 0cLi a0et a0al Cell Biosci

Thyroid_Cancer

"Deregulated circular RNAs circRNAs are associated with the development of cancer and therapyresistance However functional research of circRNAs mostly focus on potential miRNA or protein binding and morepotential regulation of circRNA on host gene DNA in cancers are yet to be inspectedMethod We performed total RNA sequencing on clinical breast cancer samples and identified the expressionpatterns of circRNAs and corresponding host genes in patient blood tumor and adjacent normal tissues qPCRnorthern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5 A series ofprocedures including Rloop dotblotting DNARNA immunoprecipitation and mass spectrum etc were conductedto explore the regulation of circSMARCA5 on the transcription of exon of SMARCA5 Moreoverimmunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5with drug sensitivitiesResults We found that circRNAs has average higher expression over its host linear genes in peripheral bloodCompared to adjacent normal tissues circSMARCA5 is decreased in breast cancer tissues contrary to host geneSMARCA5 The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro andin vivo Furthermore we demonstrated that circSMARCA5 can bind to its parent gene locus forming an Rloopwhich results in transcriptional pausing at exon of SMARCA5 CircSMARCA5 expression resulted in thedownregulation of SMARCA5 and the production of a truncated nonfunctional protein and the overexpression ofcircSMARCA5 was sufficient to improve sensitivity to cytotoxic drugsConclusion Our results revealed a new regulatory mechanism for circRNA on its host gene and provided evidencethat circSMARCA5 may serve as a therapeutic target for drugresistant breast cancer patientsKeywords Breast cancer circRNA DNA damage repair Rloop Host gene Correspondence kechenhusteducn chewhueducnleiweifrhotmailcom Xiaolong Xu Jingwei Zhang Yihao Tian and Yang Gao contributed equallyto this work3Department of Urology Tongji Hospital Tongji Medical College HuazhongUniversity of Science and Technology Wuhan China1School of Basic Medical Sciences Wuhan University Wuhan HubeiChinaFull 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 0cXu Molecular Cancer Page of IntroductionCircular RNAs circRNAs are novel RNAs that havebeen ubiquitously discovered in many species by highthroughput sequencing in recent years [ ] CircRNAsare generated by the backsplicing of intronic exonic orintergenic regions circRNAs are resistant to RNase Rand the stability of their structures makes these molecules ideal candidates for disease [] Extensive studieshave revealed that dysregulated circRNAs are involvedin the development of various cancers In gastric cancercircRNAssuch as circPVT1 circLARP4 has_circ_ and circ_100269 have been shown to play arole in promoting tumor growth and their expression iscorrelated with high TNM stage and poor prognosis [“]In colon and hepatic carcinoma ciRS7 promoted tumordevelopment and progression by activating the EGFR andPI3KAkt pathway [ ] CircRNAs such as circKIF4Ahsa_circ_0001944 hsa_circ_0001481 and circRNA_0025202have been implicated in molecular typing brain metastasisand drug resistance in breast cancer [“] Although greatprogress has been made the roles of circRNA and relevantmolecular mechanisms remain largely unknownPrevious studies have shown that circRNAs exert theirfunctions in different ways As noncoding RNAs circRNAs regulate the expression of other genes by servingas sponges for microRNA and RNAbinding proteins[ ] In addition some circRNAs have been shownto be translated into functional proteins [ ] Inaddition circRNAs have also been shown to directlyinteract with the genomic DNA of the host gene inplant which results in altered parent gene expression[] However the interaction of circRNAs and hostgene DNA were less studied in human cancersSMARCA5 is a member of the SWISNF complex withATPdependent chromatin remodeling activity [“]In the process of DNA damage repair SMARCA5 isinvolved in chromatin remodeling in DNA damageregions providing a structural basis for the recruitmentof DNA damage repair factors [ ] In tumorsSMARCA5 is highly expressed in hepatic carcinoma andprostate cancer and its expression levelis inverselyrelated to tumor radiosensitivity [ ]In this study we established circRNAs have averagehigher expression than their host genes in peripheralblood comparing to tissues Then we identified acircRNA derived from SMARCA5 circSMARCA5 issignificantly decreased in breast cancer cell lines andbreast cancer samples Differentto previous worksrevealing circSMARCA5 can also function as a competing endogenous RNAs by binding with miRNAmoleculesour mechanism explorationdisplayed circSMARCA5 is involved in regulating DNArepair capacity by binding exon DNA directly Andfurther functionalinvestigation of this circRNA may[“]contribute to the therapeutic implications for cytotoxicdrugresistant breast cancer patientsResultsIdentification of expression of circRNAs in breast cancerWe performed high throughput sequencing on tumorT and adjacent normal tissue AN and peripheralblood B of six breast cancer patients Total RNA withrRNAdepleted library wereconstructed and thencircRNAs expressed in those samples were identifiedCompared to tumor and adjacent normal tissue we observed average higher CIRCscore expression of circRNA linear host genes in blood than both tumor andadjacent normal tissue In all circRNAs which wereexpressed across all six patients we observed averageCIRCscores from to in blood which is higherthan tumor to and adjacent normal tissue to in six patients Fig 1a This result indicated average higher expression of circRNAs than theirhost genes in peripheral blood comparing to tissueswhich might contribute to the exploration of diagnosticbiomarker for breast cancer We then selected sixcircRNAs with high CIRCscores average to in patients and performed further experimental validation in patients Realtime PCR results establishedtwo circRNAs circHIPK3 and circSMARCA5 weresignificantly differentially expressed between tumor andadjacent normal tissue Fig 1b and Figure S1A Especially circSMARCA5 was lower expressed in tumorsamples and less studied in previous work Furthermorethe ratio of circtolinear expression of circRNA linearhost genes of circSMARCA5 in blood sample of healthvolunteers were significantly higher than those of breast cancer patients P Fig 1c and Figure S1BWe nextcirctolinear ofcircSMARCA5 and clinical relevance in patients withbreast cancer and observed significant difference in thedistribution of the patients according to pathologic T P Table S1 Together these results indicating thepotential function and candidate biomarker attributes ofcircSMARCA5 in breast cancerexamined theratio ofand functionallyinvestigatefound thatTo characterizecircSMARCA5 we firstly detected the expression of circSMARCA5 in cell lines circSMARCA5 is derived fromthe backsplicing of exon and exon of SMARCA5Fig 1d As expected endogenous circSMARCA5 butto RNase R digestionnot premRNA was resistantFig 1dthe ntcircSMARCA5 was further confirmed by Northern blotassaycircSMARCA5 was mainly present in the nucleus whereasits parent mRNA was present exclusively in the cytoplasm as evidenced by qPCR Northern blotting andRNA in situ hybridization Fig 1fh and Figure S2Fig 1e Furthermore wethe existence ofIn addition 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig Identification of circRNAs in breast cancer a Heatmap of CIRCscore FBPcircFBPlinear in tumor T adjacent normal tissue AN and bloodsample B from six breast cancer patients b Expression of six circRNAs with high CIRCscore were validated by RTqPCR assay in breast tumor andadjacent normal tissue represents P CircRNAs IDs are according to circBase through their genomic coordinates c The ratio of circtolinear ofcircSMARCA5 in blood sample of breast cancer patients and health volunteers Total RNA from blood sample of breast cancer patients and healthvolunteers was extracted and detected by RTqPCR The expression level was normalized with βactin as reference P was consideredstatistically significant d Schematic illustration showing the genomic region of circSMARCA5 derived from exons and of the SMARCA5 geneConvergent gray and divergent black primers were designed to amplify the linear or backsplicing products upper Total RNA from MCF7 cellswith or without RNase R treatment was subjected to RTPCR lower and further validated by Sanger sequencing Right e Northern blot using ajunctionspecific probe or an exon probe showing the endogenous existence of circSMARCA5 and SMARCA5 mRNA from MCF7 cells with orwithout RNaseR treatment R or R The bp marker indicates the SMARCA5 fulllength transcript transcribed in vitro The bp markerindicates exon and exon of SMARCA5 transcribed in vitro f The nucleus and cytoplasm mRNA of MCF7 were extracted and SMARCA5 andcircSMARCA5 expression levels were quantitated by RTPCR GAPDH and hU6 serve as internal references of the cytoplasm and nucleus respectivelyœ indicates P g The nucleus and cytoplasm mRNA of MCF7 were extracted SMARCA5 and circSMARCA5 were examined by Northernblotting and the SMARCA5 exon probe was applied in this experiment h Subcellular localization of circSMARCA5 and SMARCA5 in MCF7 cellsThe signals were examined by indirect RNA FISH and confocal microscopy The nucleus was counterstained with DAPI The circSMARCA5 probe waslabled by biotin while the SMARCA5 probe was labled by DIG They were stained with red and green fluorescent secondary antibodies respectively IThe expression of circSMARCA5 detected by northern blot MDAMB231 BT474 MCF7 SKBR3 are breast cancer cell lines MCF10A are normal breastcell line N1N2N3N4N5 are adjacent normal tissues T1T2T3T4 are breast cancer tissues œ indicates P Next we examined the expression of circSMARCA5 invarious breast cancer cell lines MCF7 SKBR3 BT474MDAMB231 and immortalized but nontransformedbreast epithelial cells MCF10A as well as in adjacentnormal tissues and breast cancer tissues Northern blotresultsthe expression levels of circSMARCA5 in MCF10A and normal adjacent tissuesare higher than breast cancer cell lines and cancer tissues Fig 1i These results indicated that circSMARCA5is downregulated in breast cancer tissues and cellsrevealed thatsequencecircSMARCA5 decreases the expression of SMARCA5 incancer cellsTo clarify the mechanisms of circSMARCA5 we investigated its effects on the expression of its parent geneSMARCA5 The expression levels of circSMARCA5 andSMARCA5 were detected by the primers of junctionsequence and “ exonsrespectivelyKnockdown of circSMARCA5 increased both mRNAand protein levels of SMARCA5 while converselycircSMARCA5 overexpression decreased SMARCA5levels Fig 2ac and Figure S3 Consistently the proteinof SMARCA5 was high expressed in breasttumorsamples as compared with the corresponding controlsFigure S4 Moreoverthe ratio of circtolinear ofcircSMARCA5 was significantly lower in breast andrenaltumor tissue than the corresponding adjacenttissue specimens Fig 2de and Figure S1C Besides asignificant negative correlation was also found betweencircSMARCA5 and SMARCA5 expression in various celllines and primary cancer tissues Fig 2df and FigureS5 which corroborates our observation that circSMARCA5 decreased the expression of SMARCA5 incancer cellscirSMARCA5 terminates the transcription of SMARCA5 atexon We further investigated the mechanism of circSMARCA5in regulating the expression of SMARCA5 Interestinglywe found that the overexpression of circSMARCA5 indeed decreased the expression of SMARCA5 exons “but had minimal effects on the expression of exons “Fig 3a Next we designed a primer location in exon for the amplification of ² cDNA ends by rapid amplification of cDNA ends RACE PCR Fig 3b left As shownin Fig 3b SMARCA5 can give rise to multiple isoformsImportantly we found a decrease in a band of bpupon circSMARCA5 overexpression while an bpband displayed the opposite phenomenon Fig 3b rightSanger sequencing showed that the bp band andthe bp band are derived from fulllength and truncated mRNA exons to respectively of the SMARCA5 gene Fig 3c Consistent with the RACE resultsNorthern blot assay further demonstrated that ectopiccircSMARCA5 expression decreased SMARCA5 levelsand promoted truncated mRNA levels Fig 3d The observations gathered thus far have led us to hypothesizethat circSMARCA5 prevents transcription from exon of SMARCA5 Indeed ChIP analysis indicated that thebinding of pol II to exons “ of SMARCA5 was higherthan that to exons “ Fig 3e left and the ectopic expression of circSMARCA5 decreased the binding of Pol IIto exons “ of SMARCA5 Fig 3e right To furtheraddress whether circSMARCA5 could terminate the transcriptional elongation of SMARCA5 we cloned a series ofexons of SMARCA5 in a luciferase plasmid reporterFig 4a upper The transient transfection of these luciferase reporters containing the “ exon sequence revealed that luciferase activity was significantly decreasedwhen circSMARCA5 was overexpressed Fig 4a lower 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases the expression of SMARCA5 in cells a Generation of circSMARCA5knockdown and circSMARCA5overexpressingcells MCF7 cells were infected with lentiviruses expressing shRNA against circSMARCA5 shcircSMARCA5 three different oligonucleotides orcircSMARCA5 pLCDHcircSMARCA5 RTqPCR was performed to evaluate the expression of circSMARCA5 GAPDH was used as an internal controlb RTqPCR showing the levels of circSMARCA5 and SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciR control orpLCDHcircSMARCA5 c Western blot showing the levels of SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciRcontrol pLCDHcircSMARCA5 pLCDHcircSMARCA5Δ without splicinginducing sequence GAPDH was used as an internal control DF Theratio of circtolinear of circSMARCA5 in tumor tissue were significantly lower than normal tissue in breast cancer samples d and RCC samplese P A negative correlation between circSMARCA5 and SMARCA5 expression was observed in breast cancer samples d RCC samplese and various cell lines fTo further confirm the effect of circSMARCA5 on thetranscriptional elongation of SMARCA5 we insertedexons of SMARCA5 between DsRED and EGFP as indicated Fig 4b upper The EGFP level was significantly decreased by circSMARCA5 when exons “were present Fig 4b lower We further investigatedthe role of circSMARCA5 in the regulation of SMARCA5 at the protein level As expected circSMARCA5overexpression downregulated the protein levels ofSMARCA5 and upregulated truncated SMARCA5ΔSMARCA5 protein levels Fig 4c and Figure S6which was confirmed by mass spectrometry Fig 4dMoreover we found that ΔSMARCA5 is more susceptible to proteolysis by the proteasome than SMARCA5 Fig 4e Together these results show the roleof circSMARCA5 in the termination of transcriptionalelongation at exon of SMARCA5circSMARCA5 can form Rloops with its parent geneTo further dissect the mechanism of SMARCA5 transcriptional termination mediated by circSMARCA5 weinvestigated whether circSMARCA5 can bind genomic 0cXu Molecular Cancer Page of Fig cirSMARCA5 terminates the transcription of SMARCA5 at exon a RTqPCR analysis of the expression of SMARCA5 in MCF7 cells using aseries of paired primers œ indicates P b Rapid amplification of cDNA ends RACE PCR analysis of SMARCAC5 transcripts The PCRproducts were readily identified by agarose gel electrophoresis Each set of samples was repeated three times c Sanger sequencing of twotranscripts of SMARCAC5 that are regulated by circSMARCA5 overexpression d Northern blotting using the junctionspecific probes for exons and to show the expression levels of the transcripts of SMARCAC5 mRNA from MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 circOE e CircSMARCA5 prevents transcription from exon of SMARCA5 ChIPseq analysis showing that the binding of pol II toexons of SMARCA5 was higher than that to exons ChIPqPCR showed that the ectopic expression of circSMARCA5 decreased thebinding of Pol II to exons of SMARCA5SMARCA5 DNA to form an Rloop Dotblotting withRloopspecific S96 antibody supported our hypothesisthat circSMARCA5 can bind exons “ of SMARCA5genomic DNA Fig 5a Additionally we performedDNARNA immunoprecipitation DRIP qPCR and confirmed the interaction between circSMARCA5 andexons “ pretreatment with RNase H ablated thisinteraction confirming that the interaction is Rloopspecific Fig b and Figure S7 The interaction of circSMARCA5 with the DNA of SMARCA5 was directlyverified by fluorescence in situ hybridization Fig 5cConsistent with previous findings [] dotblotting ofthe genome without RNA digest revealed that the binding of circRNA to genomic DNA may be widely presentin cancer cells Fig 5d We next determined the specificsequence of exons “ required for Rloop formationA series of fragments from exons “ were hybridizedwith circSMARCA5 for the dotblotting assay As shownin Fig 5e the bp fragment of the ² end of exon plays important role in interacting with circSMARCA5Moreoverthe secondary structure of circSMARCA5was determined by the software MFOLD [] whichrevealed the sequence ²AACAAAAUUGGGAAAGAUGAAAUGCUUCAAAU3² from the ² end of exon located in the loop region of circSMARCA5 Fig 6aWe thus hypothesized that this sequence might play akey role in mediating the circSMARCA5DNA interaction To this end we synthesized the wildtype andmutant phosphorylated DNA fragments ANT andANTmut respectively corresponding to this sequenceFig 6b Dotblotting demonstrated that wildtypeoligonucleotides ANT can bind to circSMARCA5 but 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig cirSMARCA5 blocks the transcription of SMARCA5 and promotes the generation of a truncated SMARCA5 protein ΔSMARCA5 aSchematics of luciferase reporter constructs containing the SMARCA5 exon sequence as indicated upper The SMARCA5 exon sequenceplays an important negative role in mediating the effect of circSMARCA5 overexpression on luciferase activity lower b Schematics offluorescence reporter constructs containing the SMARCA5 exon sequence as indicated upper MCF7 cells were transiently transfected withthese fluorescence reporters along with or without circSMARCA5 cooverexpression After transfection for hours the reporter transcriptionactivities were measured by flow cytometry assay c circSMARCA5 overexpression downregulated the protein levels of SMARCA5 whileupregulating truncated SMARCA5 ΔSMARCA5 protein levels MCF7 cells stably overexpressing circSMARCA5 or control cells were treated withDMSO or MG132 Western blot analysis was performed using an antibody targeting the Nterminus of SMARCA5 to evaluate the expression ofSMARCA5 and ΔSMARCA5 GAPDH was used as an internal control d The ΔSMARCA5 protein was identified by mass spectrometry and detectedSMARCA5 peptides were showed in the map The redlabeled portion is the amino acid sequence of the translated defective transcript e MCF7cells expressing FlagSMARCA5 and FlagΔSMARCA5 were treated with cycloheximide CHX μgml The cell lysates were subsequentlyharvested at sequential time points or h after treatment and then the cell lysates were immunoblotted with antiFlag or antiActin antibodymutant oligonucleotides ANTmut cannot bind to circSMARCA5 Fig 6c As expected DRIPqPCR showedthat ANT inhibited circSMARCA5 binding to the DNAat exons “ whereas ANTmut had no effect on thisinteraction Fig 6d Furthermore the transfection ofANT prevented the decrease in SMARCA5 proteinlevels in MCF7 cells stably expressing circSMARCA5whereas ANTmut had no effect on SMARCA5 proteinlevels Fig 6e Importantly the mutation of the keysequence in circSMARCA5 impaired the interactionwith its parent gene which was confirmed by dotblotting and DRIPqPCR assays Fig 6fh and FigureS8 Unlike circSMARCA5 circSMARCA5mut had littleeffect on SMARCA5 protein levels Fig 6i Theseresults suggested that circSMARCA5 formed Rloopswith its parent gene to inhibit the expression of SMARCA5 in cancer cellscircSMARCA5 inhibits DNA damage repair functionTo explore the roles of circSMARCA5 in cancer progression we overexpressed and depleted circSMARCA5in MCF7 cells by lentiviral vectors and then examinedthe effect of circSMARCA5 on cell proliferation migration and apoptosis However the results showed thatboth overexpressed and depleted circSMARCA5 had noeffect on these three activities Figure S9 Previousstudies have indicated that SMARCA5 plays an important role in regulating the DNA repair process and main[ “]taining theConsistent with previous reports SMARCA5 overexpression improved DNA repair capacity and reducedthe expression of Chk1 and Chk2 after DNA damagerepair Figure S10A Given that circSMARCA5 canpromote the production of the truncated ΔSMARCA5protein we tested whether the truncated protein is alsofunctional The overexpression of FlagΔSMARCA5had a minimal effect on the expression of Chk1 andChk2 after DNA damage repair Figure S10B suggesting that ΔSMARCA5 isa nonfunctional proteinproduct We next assessed whether circSMARCA5 canthe genomestability ofshowedlowerthan thatsignificantlyformation assaysaffect the function of DNA damage repair capacityCCK8 and clonerevealed thatcircSMARCA5 overexpression increased sensitivity tocisplatin or bleomycin in MCF7 cells Fig 7a b NextMCF7 cells were treated with the indicated concentration of cisplatin or bleomycin for h and then theDNA damage was evaluated by single cell gel electrophoresis SCGE at and h MCF7 cells expressingcircSMARCA5repaircapacity than did control cells Fig 7c In parallelDNA damage was examined after h of treatmentwith cisplatin or bleomycin by using an antiγH2AXantibody Consistent with the SCGE results the γH2AXin MCF7 cells expressing circSMARCA5 wassignalsignificantly higherin MCF7 cells asevidenced by immunostaining Fig 7d Consistentlycisplatin significantly enhanced the levels of DNAdamage response proteins Chk1 and Chk2 in MCF7cellsexpressing circSMARCA5 Fig 7e whereasseveral key cellcycle genes were reduced specificallyupon circSMARCA5 overexpression Fig 7f To testwhether circSMARCA5 Rloop formation is necessaryfor its DNA repair function we transfected ANT orANTmutinto circSMARCA5expressing cells TheSCGE assay and γH2AX measurement showed thatANT significantly enhanced the DNA repair capacitywhile ANTmut had no effect on this activity Fig 7gand Figure S11 Furthermore ANT significantlydecreased the degree of colocalization between circSMARCA5 and its cognate DNA locus Figure S12In addition unlike circSMARCA5 the overexpressionof circSMARCA5mut had little effect on the DNArepair rate Fig 7h and Figure S13A B Next we determined whether SMARCA5 could mediate the effects ofcircSMARCA5 in preventing DNA damage repair Asshown in Fig 7i the γH2AX signal was much lower incircSMARCA5expressing cellscomplemented withSMARCA5 than that in cells expressing circSMARCA5alone As expected ΔSMARCA5 could not rescue theinhibition of DNA damage repair function induced by 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig circSMARCA5 interacts with its site of transcription a circSMARCA5 interacts with the exon sequence of the SMARCA5 locus A seriesof exon DNA fragments were hybridized with circSMARCA in vitro The DNARNA hybridization strength was quantified by dotblot with Rloopspecific S96 antibody Hybridization stringency was altered by decreasing ionic strength mM NaCl b DRIPqPCR analysis of the exon sequence of SMARCA5 to detect the association of circSMARCA5 in MCF7 cells RNase Htreated andor DRIPqPCR analysis of the exon sequence as a control c CircSMARCA5 partially localized at its site of transcription Double FISH of circSMARCA5 red and its parent DNAregion green The nucleus was stained by DAPI d Dotblot of Rloops in MCF7 cell genomic DNA preparations treated with DNase I RNase Hor RNase R The DNARNA hybrids in genome DNA were analyzed by S96 antibody e Mapping of the Rloop formation region of circSMARCA5A series of exon deletion mutants were hybridized with circSMARCA5 for the dotblotting assay The DNARNA hybridization intensity wasanalyzed by dotblot with an S96 antibody targeting the DNARNA hybrid strand Hybridization stringency was altered by decreasing ionicstrength mM NaClcircSMARCA5 Figure S13C D Moreover the overexpression of SMARCA5 could significantly rescue thegrowth defects of cells expressing circSMARCA5 asdemonstrated by a colony formation assay Fig jTogetherthese results demonstrated the roles ofcircSMARCA5 in regulating the DNA repair process inMCF7 cellstheevaluatecircSMARCA5 overexpression enhances the cisplatinresponse in breast cancerTo furthertherapeutic potential ofcircSMARCA5 in breast cancer in vivo we establishedcircSMARCA5 overexpression clones in MCF7 cells Asshown in Fig 8a the overexpression of circSMARCA5efficiently enhanced the sensitivity of MCF7 xenograftsto concurrent cisplatin treatment Fig 8a b The overexpression of circSMARCA5 was confirmed by in situhybridization and qPCR analysis Fig 8c along with decreased SMARCA5 protein levels and increased γH2AXlevels Fig 8d In addition qPCR analysis demonstratedthat circSMARCA5 can be detected in the bloodsuggesting that circSMARCA5 is a secretory moleculeCollectively these data demonstrate that circSMARCA5could serve as a potential therapeutic target to restoresensitivity to cisplatin therapy in breast cancerDiscussionPrevious studies have indicated that circRNAs have multiple functions in cancer development and progression[“] In this study we identified multiple expressedcircRNAs in breast cancer samples and observed averagehigher abundance of circRNAs over their host genes inperipheral blood than tissues which might contribute tothe exploration of diagnostic biomarkerfor breastcancer We then identified that circSMARCA5 is significantly decreased in breast cancer tissues using RNAseqMore importantly we define a critical role for circSMARCA5 in the regulation of DNA damage repaircapacity and the drug sensitivity of breast cancer cellsin vitro and in vivo through the negative regulation ofits parent gene SMARCA5 These findings are of highclinical relevance because chemotherapy with cisplatinand bleomycin remains the standard of care in breastcancer [“] Hence the restoration of circSMARCA5levels provides an approach to overcome treatmentresistance in breast cancer patientsSMARCA5 also known as SNF2H is a member of theSWISNF chromatinremodeling complex During DNAdamage repair processes SMARCA5 is recruited toDNA damage sites where it induces the ubiquitinationand phosphorylation of histone H2A which facilitateschromatin remodeling and DNA damage repair [ ]In this study we show that circSMARCA5 expressionresulted in the downregulation of SMARCA5 and theeffect of circSMARCA5 overexpression on DNA repaircapacity was reversed by concomitant SMARCA5 overexpression suggesting that the effect of circSMARCA5on DNA repair capacity is mediated through SMARCA5circRNAs exert functions in various ways such as forming an Rloop with DNA to regulate splicing and transcriptional pausing [] For example circSEPALLATA3regulates the splicing of its parent mRNA through Rloop formation [] In addition circRNAs are a novelclass of ceRNAs that sponge miRNAs thus positivelyregulating gene expression [ ] Additionally circRNAs such as exonintron circRNAs regulate geneexpression through specific RNARNA interactions withU1 snRNA [] Furthermore circRNAs also exertfunctions by binding to proteins and regulating theiractivities [] We identified one mechanism by whichcircSMARCA5 regulates the drug sensitivity of breastcancer cells to cisplatin and bleomycin through thedownregulation of SMARC5 circSMARCA5 is recruitedto its parent gene locus leading to Rloop formationtranscriptiontruncatedΔSMARCA5 protein upregulation and decreased SMARCA5 expression This regulatory mechanism has alsobeen verified in cervical cancer Hela cells Figure S14However our evidence demonstrates that circSMARCA5has no significant effect on the proliferation migrationand apoptosis of breast cancer cells suggesting that thismolecule functions in a celltype and contextdependentmanner Notablythatnonfunctionalterminationweprovideevidence 0cXu Molecular Cancer Page of Fig circSMARCA5 can form an Rloop with its parent gene a Secondary structure prediction for circSMARCA5 using the Mfold program Thesequence KEY shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red b Thethiophosphorus nucleic acid analog ANT complementary to KEY and its mutant ANTmut were synthesized in vitro c Dotblot verifying theinteraction between circSMARCA and ANT or ANTmut d DRIPqPCR analysis on exon or exon sequences of SMARCA5 to detect theassociation of circSMARCA5 in MCF7 cells overexpressing ANT or ANTmut RNase Htreated andor DRIPqPCR analysis of the exonsequence as a control œ indicates P e Western blot analysis shows that transfection of ANT into circSMARCA5overexpressing cells canrestore SMARCA5 protein levels but ANTmut cannot f g Dotblot analysis quantifying Rloop strength between the SMARCA5 locus andcircSMARCA5 or circSMARCA5mut guanine converted to cytosine of the KEY sequence h DRIPqPCR in MCF7 cells transfected withcircSMARCA5 or circSMARCA5mut RNase Htreated genomic DNA and qPCR of exon1314 were treated as controls œ indicates P iWestern blot analysis shows that overexpression of circSMARCA5 to MCF7 cells can decrease SMARCA5 protein levels but circSMARCA5mutcannot 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases DNA repair capacity a circSMARCA5 increases sensitivity to cisplatin or bleomycin in MCF7 cells MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 were treated with cisplatin or bleomycin for h and CCK8 was used to measure cellviability b Relative colony formation units of MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After hours the drugs were replaced by fresh medium The number of colonies was quantified c d e Single cell gelelectrophoresis SCGE assay indicating that circSMARCA5 overexpression inhibits cell recovery from DNA damage MCF7 cells stably expressingcontrol vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After incubation for h the cells were recoveredwith fresh medium for or hours and then collected for SCGE analysis c immunofluorescence assay using an antiγH2AX antibody d andwestern blot assay with the indicated antibodies e f RTqPCR assay showing the relative levels of several key cell cycle genes in MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 treated with DMSO or μM cisplatin for h and replaced with fresh medium for hœ indicates P g SCGE assay showing that the cotransfection of ANT in circSMARCA5overexpressing cells can restore the DNA repaircapacity

Thyroid_Cancer

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

Thyroid_Cancer

"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [“] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [“] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[“] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ‰¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure„¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening„¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009“ of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU “ Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE “ Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at “ A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at ×g for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturer™s instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturer™s instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope × objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ΔΔCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncan™s multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncan™s multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncan™s multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining × Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup“ BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Fir“micutesand Proteobacteria “ There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella“ Helicobacter “ and Clostridium “ were the most abundant followed byOscillospira “ Bacteroides “ andLactobacillus “Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncan™s multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c

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

"Sintilimab blocks the interaction between programmed death1 PD1 and its ligands The safety andefficacy of sintilimab combined with oxaliplatincapecitabine CapeOx as firstline treatment were evaluated inpatients with gastric Ggastroesophageal junction GEJ adenocarcinoma in a phase Ib clinical trialMethods Patients with locally advanced or metastatic GGEJ adenocarcinoma without previous systemic treatmentwere enrolled as one cohort of a multicohort study Sintilimab was administered at a dose of mg intravenously IV incombination with CapeOx mgm2 capecitabine orally bid D1“ and mgm2 oxaliplatin IV D1 every daysfor up to cycles After combination treatment patients continued to receive sintilimab mg at weekly intervals asmaintenance therapy until progressive disease PD unacceptable toxicity withdrawal of informed consent or for up to months Adverse events AEs were monitored to assess safety in terms of their frequency intensity and causality Theefficacy endpoints included the objective response rate ORR disease control rate DCR progressionfree survival PFSand overall survival OS Tumor mutation burden TMB was evaluated for its association with clinical responseResults A total of patients were enrolled and received sintilimab plus CapeOx All patients reported treatmentrelatedAEs TRAEs Grade “ TRAEs were found in patients Seventeen patients obtained partial response and theORR was CI “ Three had stable disease and DCR was CI “ Asdata cutoff of May the median followup was months The median PFS was months CI “ andmedian OS had not been reached The OS rates at months and months were and No association wasobserved between TMB and efficacyContinued on next page Correspondence Nongxuzhjphoutlookcom1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cJiang BMC Cancer Page of Continued from previous pageConclusions Sintilimab combined with CapeOx as firstline treatment demonstrated acceptable safety andpromising efficacyTrial registration ClinicalTrialsgov NCT02937116 Registered October Keywords Sintilimab Capecitabine Oxaliplatin Gastricgastroesophageal junction adenocarcinoma Tumormutation burdenBackgroundThe fifth most commonly diagnosed cancer worldwide isgastric cancerGC accounting for about ofcancerrelated deaths globally and the third most common cancer in China with almost half of worldwide newGC cases occurring in China annually [ ] The standard treatments exhibit regional differences among western countriesJapanKorea and China which areconsidered to be associated with different screening andearly detection methods as well as different biologicalbehaviors disease characteristics and ethnicity [“]treatment mainlySurgical resection is the only radical therapy for gastricgastroesophageal junction GGEJ cancer Howeversystemic chemotherapy is an alternative main therapyfor GGEJ cancer because of the high relapse rate afterpostresection surgery and for the many patients diagnosed at an advancedstage For advanced GGEJ cancerinvolves platinumbasedfirstlinechemotherapy using a combination oftwo or threedrugs trastuzumab is given to patients whose tumor ishuman epidermal growth factorreceptor2 HER2positive but the overall survival OS is disappointingsince the maximum OS time has been reported to be months [“] Any potential novel drug that willincrease patient survival times is urgently neededinparticular for firstline treatmentinhibitor treatmentImmune checkpointis a newapproach for tumor immunotherapy [ ] The treatment diminishes the immune system tolerance to tumorcells and improves the effective identification and eradication of tumor cells by blocking T cell inhibition [] Theprogrammed death1 PD1 antibody specifically binds toPD1 thereby inhibiting apoptosis of antigenspecific Tcells and thus reducing regulatory T cell Treg apoptosisby inhibiting the activation of PDL1 [ ]patients with recurrent or advanced GC In the ATTRACTION2 study nivolumab monotherapy improvedOS from to months hazard ratio CI“ P compared with a placebo in advanced GC that was refractory or intolerant to previoustreatment regimens []However between and of patients exhibit noresponse to PD1 blockade which is considered to beassociated with T cell exclusion or exhaustion or inadequate T cell trafficking and many immunosuppressivefactors accumulate in the tumor microenvironment []New therapy regimens that improve the response andlongterm efficacy are desperately needed The efficacy ofantiPD1 therapy in combination with chemotherapy hasbeen confirmed in nonsmallcell lung cancer [ ] Inaddition to direct tumor killing conventional cytotoxicchemotherapy has demonstrated immunoregulatory properties by enhancing tumor antigenicity disturbing immunesuppressive pathways inducing immunogenic cell deathand increasing effector Tcell reactions [] It is safe tohypothesize that antiPD1 antibodies in combination withchemotherapy may further improve the clinical outcomesof patients with advanced GC Sintilimab is a highly selective monoclonal IgG4 antibody that inhibits interactionsbetween PD1 and its ligands with strong antitumorresponse [] A phase 1a study for dose escalation hasdemonstrated the tolerance and pharmacological activity ofsintilimab in patients with advancedstage solid tumors butthere is limited evidence for the efficacy of antibodiesagainst PD1 plus chemotherapy in Chinese GGEJ adenocarcinoma patients Thus the present trial was conductedto investigate the safety and efficacy of sintilimab combinedwith CapeOx as firstline therapy for a cohort of patientswith GGEJ adenocarcinomaThe efficacy of antiPD1 antibodies monotherapy inpatients who had prior chemotherapy for advanced GChas been demonstrated and supported by several trialsIn the KEYNOTE012 and KEYNOTE059 trials pembrolizumab monotherapy showed objective responserates ORR of n [] and n []respectively in PDL1 positive advanced GCpatients after at least two prior systemic therapies Basedon such results the Food and Drug Administration approved pembrolizumab forthirdline treatment ofMethodsStudy design and patientsThe present study was an label multicenter phase Ibstudy to evaluate the safety and efficacy of sintilimab in cohorts of patients with solid tumors Patients age range“ years with cytologically or histologically confirmedunresectable GGEJ adenocarcinoma were enrolled in theGGEJ cohort Tumor nodes and metastases TNM staging has been evaluated according to the Union for International Cancer Control UICC TNM classification 8th 0cJiang BMC Cancer Page of edition [] The patients had received no previoussystemic treatment for advanced disease or had disease progression PD more than months after systemic adjuvant therapy Other major inclusion criteriawere at least one measurable lesion as defined by theResponse Evaluation Criteria in Solid Tumor RECISTversion criteria score or for Eastern TumorCollaborative Group Performance Status ECOGPSadequate an and bone marrow functions and lifeexpectancy ‰¥ weeks Patients with amplification oroverexpression of the HER2 gene were excluded fromthe trial Appendix contains a complete list of allinclusion and exclusion criteriaThe institutional review boards of all centers approvedthe protocols and the study was carried out in strictaccordance with the declaration of Helsinki principlesall participating patients signed consent forms beforetaking partProceduresAccording to NCCN guideline the preferred firstlinechemotherapy regimens for advanced gastric cancer arefluorouracil or capecitabine combined with cisplationor oxaliplatin [] Howeverthe results from theREAL2 study [] revealed significant clinical benefitof the oxaliplatincapecitabine CapeOx regimen whichled to the longest OS time of months comparedwith other regimens Oxaliplatin produces less renaltoxicity there is no requirement for hydration and ithas a lower emetic potential compared to cisplatinwhile capecitabine has no requirement for continuousintravenous IV infusion and is administered orallywhich should ensure an improved quality of life for patients in their homes Therefore a CapeOx regimen hasbeen chosen During the combination phase enrolledpatients were given sintilimab in combination withCapeOx for up to cycles every weeks Each cycleconsisted of intravenous sintilimab mg plus oxaliplatin mgm2 on day and capecitabine mgm2 twice daily orally from day to day Aftercombination treatment patients without PD continuedto receive sintilimab mg at weekly intervals asmaintenance therapy until PD unacceptable toxiceffects withdrawal of informed consent or for up to monthsStudy assessmentsAdverse events AEs were monitored for days after thelast administration of a treatment dose Responses wereassessed by computed tomography CT or magnetic resonance imaging MRI every weeks until PD new treatment initiation withdrawal of informed consent or deathEndpointsSafety was assessed as collected AEs according to theirtype frequency causality and severity grading defined bythe National Cancer Institute Common TerminologyCriteria CTCAE ver The efficacy endpoints werethe ORR disease control rate DCR time to responseTTR duration of response DOR progression freesurvival PFS and OS Efficacy was determined by an investigator according to RECIST v11 guidelinesExploratory endpoints were to evaluate the correlationof tumor mutation burden TMB with clinical efficacyTumor mutation burden analysisThe tumor biopsies and blood samples were collected atbaseline DNA sequences were extracted from biopsiesof tumors with matched blood samples and submittedfor next generation sequencing using a designed gene panel Genecast Beijing China TMB was determined by analysis of the quantity of somatic mutationsper megabase Mb Median TMB was used as a cutoffto define a tumor as highTMB HTMB and lowTMBLTMBStatistical analysisAll patients who received at least one study treatmentwere included in the safety and efficacy analyses AEswere coded following the Medical Dictionary for Regulatory Activity and tabulated by system an class andpreferred terms Causality between AEs and the studytreatment was assessed by the investigator ORR was calculated as the proportion of patients who had achieved acomplete response CR or partial response PR and the CIs were evaluated by the binomial distributionDCR was calculated as the proportion of patients whoobtained PR CR and stable disease SD and data arepresented with the CIs Median DOR TTR PFSOS and the PFS and OS rates at and months weredetermined using the KaplanMeier methodology Fisher™s test was used to compare the ORRs between patientswith HTMB and LTMBResultsFrom Dec to Oct patients werescreened and were enrolled in the GGEJ adenocarcinoma cohort Fig The median interval between initial diagnosis and screening was days range “Most patients had metastatic disease status and had ECOG scores of Table The TNMstage summary is shown in Table and the staging ofeach patient in Supplementary Table At data cutoff on May the median followuptime was months range “ The median treatment duration was months range “ Allpatients received more than cycles of treatment with 0cJiang BMC Cancer Page of Fig Flowchart of the studythe median doses of received sintilimab being range“SafetyAll of the patients reported at least one treatmentrelated adverse event TRAE and the most commonTRAE was platelet count decreased n Grade or treatmentrelated AEs TRAEs occurredin patients the most common also being aplatelet count decreased n Table NoTRAE was fatal and patient discontinued the treatment due to treatmentrelated Grade hepatic functionabnormal Sintilimabrelated AEs occurred in patients Grade “ sintilimabrelated AEs occurred in patients the most common being platelet countdecreased n Supplementary Table Chemotherapyrelated AEs were found in all patientsGrade “ chemotherapyinduced AEs were found in patients the most common being platelet countdecreased n Supplementary Table Fivepatientsadverseevents platelet count decreased n abnormal hepatic function n hypothyroidism n pneumonitis n and autoimmune colitis n reported treatmentrelated seriousEfficacyAll patients experienced a decrease in the sum oftheir target lesions Fig 2a and in the majority the lesions kept smaller than at baseline Fig 2b The medianTTR was months CI “ and the medianDOR was months CI “ According tothe best tumor response following RECIST guidelines patients reached a PR CI “ and patients obtained a confirmedobjective response ie by two continuous PRs at intervalsof weeks In addition patients had SD and DCR was CI “ Table One patient achieved a CR after the primary analysisby May This patient began the study treatmenton October and completed cycles of treatment before CRThe median PFS time was months CI “ and the month PFS rate was Median OSwas not reached and the 6month and 12month OSrates were and respectively Fig 2c dTumor mutation burdenValid results were obtained from patients The median TMB value was Mb The ORR was CI “ in patients with HTMBand CI “ in patients with LTMB No significant difference in clinical responseswere found between HTMB and LTMB patientsP Fig 2eDiscussionIn the present study the results from the GGEJ adenocarcinoma cohort in a Phase Ib study demonstratedmanageable safety and favorable antitumor activity ofsintilimab combined with a CapeOx regimen as firstlinetreatment for unresectable advanced metastatic GGEJadenocarcinoma 0cJiang BMC Cancer Page of Table Demographics and disease characteristicsTable Treatmentrelated adverse events TRAEsAge median range in yearsAll patients N to Gender n MaleFemaleECOG PS n Time since initial diagnosismedian range in daysDisease status n Locally advancedMetastaticLocation of the primary tumor n UpperMiddleLowerTNM staging n T3T4TxM0M1N1N2N3NxHistology n Poorly differentiated adenocarcinomaModerately differentiated adenocarcinomaUnknown differentiated adenocarcinoma “ ECOG Eastern Cooperative Oncology Group T tumor N node M metastasisIn terms of safety the incidence and severity of TRAEswith sintilimab and CapeOx were generally consistentwith those of known toxic effects of conventional chemotherapy [“] and previously reported side effects ofother antiPD1 antibody combined with chemotherapyregimens [ ] Platelet count white blood cell countand neutrophil count decreases were most commonly andmostly grade to reported TRAEs and are expected AEsassociated with CapeOx [“] Only patient reporteddiscontinuation of investigational drug application due toa TRAE abnormal hepatic function No treatmentrelated death occurred in this study and in general theaddition of sintilimab to CapeOx showed a manageablesafety profile and did not bear extra safety risksAll TRAEs nPlatelet count decreasedWhite blood cell count decreasedNeutrophil count decreasedHypothyroidismRashAlanine aminotransferase increasedAspartate aminotransferase increasedAnemiaHepatic function abnormalVomitingNauseaHyperchlorhydriaThyroid function test abnormalHypokalemiaHypesthesiaPyrexiaProteinuriaγglutamyl transferase increasedDiarrheaAutoimmune colitisAll graden Grade “n Pneumonitis Listed are any grade TRAE found in ‰¥ patients and all grade “ TRAEs In the present study after treatment with sintilimabplus CapeOx patients with unresectable GGEJ adenocarcinoma obtained an ORR of CI “ which is higher than that of conventional firstline chemotherapy For GGEJ adenocarcinoma firstline treatment mainly involves platinumbased chemotherapy and fluoropyrimidine[] The ORR ofcapecitabinebased or oxaliplatinbased therapies fGEJ adenocarcinoma was about “ [ ]The ORR for antiPD1 antibodies with a chemotherapy regimen were variableIn the KEYNOTE059study the ORR was CI “ forpembrolizumab plus cisplatin5fluorouracilm 5FUas firstline treatmentIn the KEYNOTE062study ORRs were and in patients with a ‰¥ and ‰¥ combined positive score CPS respectivelyafter they received pembrolizumab plus cisplatin5FUor capecitabine regimen as firstline therapy [] InATTRACTION04 the ORR for nivolumab with S1oxaliplatin was CI “ and theORR for nivolumab with CapeOx was CI“the ORR wasIn another study[][] 0cJiang BMC Cancer Page of Fig Evaluation of efficacy and tumor responses a Maximum change in tumor size from baseline Seventeen of patients obtained PR basedon the percentage changes of the sum of the maximum diameter of the tumor lesion range ˆ’ to ˆ’ b the change of lesiondiameters over time from baseline each line represents the changes in one patient c PFS KaplanMeier curve d OS KaplanMeier curve e theobjective response rate in low and high TMB groups OS overall survival PFS progressionfree survival PR partial response SD stable diseaseTMB tumor mutation burdenreported to be for an antiPD1 antibody toripalimab plus CapeOx treatment []Sintilimab plus CapeOx also showed favorable longterm efficacy Median PFS was months CI “ and the 6month PFS rate was Median OS wasnot reached and the 6month and month OS rateswere and respectively which was higherthan for conventional treatments with a median PFS of months CI “ for capecitabinecisplatinregimen [] and a median OS of months CI“epirubicinoxaliplatincapecitabineregimen [] The median PFS times for antiPD1foran 0cJiang BMC Cancer Page of Table Efficacy evaluation of sintilimabEfficacy evaluationCRnSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1288502007251zAdditional file Table S1 TNM stages of each patient Table S2Sintilimab related adverse events Table S3 Chemotherapyrelated adverse events Appendix Inclusion and exclusion criteriaPRSDPDORR CI CI “ CI “DCR CICI confidence interval CR complete response DCR disease control rate ORRoverall response rate PD progressive disease PR partial response SDstable diseaseAbbreviationsAEs Adverse events CapeOx Oxaliplatincapecitabine CPS Combinedpositive score CR Complete response CT Computed tomographyDCR Disease control rate DOR Duration of response ECOGPS EasternTumor Collaborative Group Performance Status G Gastric GC Gastric cancerGEJ Gastroesophageal junction HER2 Human epidermal growth factorreceptor2 HTMB High tumor mutation burden LTMB Low tumormutation burden Mb Megabase MRI Magnetic resonance imagingNGS Nextgeneration sequencing ORR Objective response rate OS Overallsurvival PD Progressive disease PD1 Programmed death1 PDL1 Proteinprogrammed deathligand PFS Progressionfree survival PR Partialresponse SD Stable disease TMB Tumor mutation burden TNM Tumornodes and metastases TRAEs Treatmentrelated AEs TTR Time to responseUICC Union for International Cancer ControlAcknowledgementsWe thank the patients and their families and the participating study teamsfor making this study possible and Yuan Fang Innovent Biologics IncSuzhou China for assistance with drafting the manuscriptAuthors™ contributionsHPJ NX LS and HZ were responsible for the design of the study HPJ YLZJQ CM XX NL CX HW LST and LS were responsible for acquisition of datafurthermore NX LS HPJ SYW and DLZ were in charge of analysis andinterpretation of data HPJ drafted the manuscript NX LS HZ SYW and DLZrevised and commented the draft BP conducted the biomarker analysis Allauthors read and approved the final manuscriptFundingThe study was sponsored by Innovent Biologics Inc and cofunded by EliLilly and company The study was also supported by the National Health andFamily Planning Commission Research Fund Zhejiang Provincial Medicaland Health major Science and Technology Plan Project Grant No KWJZJ and the Public Welfare Technology Application Research Project ofZhejiang Province Grant No LGF20E030004 Innovent Biologics Inc involved in the design of the study and collection analysis and interpretationof data and in writing the manuscript All remaining funding bodies had norole in the design of the study and collection analysis and interpretation ofdata and in writing the manuscriptAvailability of data and materialsThe datasets generated andor analyzed during the current study are notpublicly available since the new drug is being submitted to the NationalMedical Products Administration for approval but are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe trial protocol was approved by the Institutional Review Boards andEthical Committee of The First Hospital Affiliated to Zhejiang UniversitySchool of Medicine and Beijing Cancer Hospital and the study was carriedout strictly following the declaration of Helsinki principles all participatingpatients signed consent forms before taking part in the trial The trialnational registration number is NCT02937116Consent for publicationNot applicableCompeting interestsHui Zhou Shuyan Wang Donglei Zhu Bo Peng are the staff of InnoventBiologics Inc Suzhou China Suzhou China Lin Shen is the associate editorof BMC Cancer All remaining authors declare that they have no competinginterestsantibodies with a chemotherapy regimen were variableranging from to months a finding which mightbe associated with different populations and diseasestatus [“]Nextgeneration sequencing NGS hasresearcherenabled to perform target capture sequencing which hasbeen proposed as a reliable technique to identifymutated driver genes and for the estimation of TMBsIts use has led to the detection of actionable alterationsin various cancer related genes [] Regarding highTMB and the efficacy of PD1 treatments inconsistentresults have been reported in previous studies Wang suggested that TMB might be associatedwith better efficacy for PD1 monotherapy [] whereasMishima [] did not find a significant relationshipbetween TMB and the response of gastric cancers toPD1 therapy [] The latter data is in accordance withour finding that after treatment with sintilimab in combination with CapeOx no significant difference in theclinical responses was found between HTMB and LTMB patients However using the median TMB as acutoff is difficult to extrapolate to the real world clinicand bias due to the small sample size could not beexcluded in the present study In addition it has beennoted that up to now there is no uniform standard forHTMB [] and further investigations are urgentlyrequiredConclusionsOur results strongly indicate that sintilimab combined withCapeOx is an option for the firstline treatment of patientswith advanced or metastatic GGEJ adenocarcinoma However the sample size was small and it was a singlearmstudy without a comparator The large scale doubleblinded and randomized Phase III clinical trial ORIENT16for previously untreated advanced GGEJ adenocarcinomapatients is being conducted to evaluate the efficacy andsafety of sintilimab combined with CapeOx vs CapeOxalone ClinicalTrialsgov Identifier NCT03745170 0cJiang BMC Cancer Page of Author details1Department of Medical Oncology The First Affiliated Hospital School ofMedicine Zhejiang University No Qingchun Road Hangzhou China 2Department of Surgical Oncology The First Affiliated Hospital Schoolof Medicine Zhejiang University Hangzhou China 3Department of MedicalScience and Strategy Oncology Innovent Biologics Inc Suzhou China4Department of Translational Medicine Innovent Biologics Inc SuzhouChina 5Department of Medical Oncology Beijing Cancer Hospital BeijingChinaReceived March Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “Chen W Zheng R Baade PD Zhang S Zeng H Bray F Cancer statisticsin China CA Cancer J Clin “Bickenbach K Strong VE Comparisons of gastric Cancer treatments east vsWest J Gastric Cancer “Lui FH Tuan B Swenson SL Wong RJ Ethnic disparities in gastric cancerincidence and survival in the USA an updated analysis of SEERdata Dig Dis Sci “Ye XS Yu C Aggarwal A Reinhard C Genomic alterations and molecularsubtypes of gastric cancers in Asians Chin J Cancer Van Cutsem E 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phase III noninferiority trial AnnOncol “ Bang YJ Van Cutsem E Feyereislova A Chung HC Shen L Sawaki A et alTrastuzumab in combination with chemotherapy versus chemotherapyalone for treatment of HER2positive advanced gastric or gastrooesophageal junction cancer ToGA a phase label randomisedcontrolled trial Lancet “ Wei SC Duffy CR Allison JP Fundamental mechanisms of immunecheckpoint blockade therapy Cancer Discov “ Park YJ Kuen DS Chung Y Future prospects of immune checkpointblockade in cancer from response prediction to overcoming resistance ExpMol Med MarinAcevedo JA Dholaria B Soyano AE Knutson KL Chumsri S Lou YNext generation of immune checkpoint therapy in cancer newdevelopments and challenges J Hematol Oncol Lowther DE Goods BA Lucca LE Lerner BA Raddassi K van Dijk D et alPD1 marks dysfunctional regulatory T cells in malignant gliomas JCIInsight 201615e85935 Dong H Strome SE Salomao DR Tamura H Hirano F Flies DB Tumorassociated B7H1 promotes Tcell apoptosis a potential mechanism ofimmune evasion Nat Med “ Muro K Chung HC Shankaran V Geva R Catenacci D Gupta S et alPembrolizumab for patients with PDL1positive advanced gastric cancerKEYNOTE012 a multicentre label phase 1b trial Lancet Oncol “Fuchs CS Doi T Jang RW Muro K Satoh T Machado M Safety andefficacy of Pembrolizumab Monotherapy in patients with previously treatedadvanced gastric and Gastroesophageal junction Cancer phase clinicalKEYNOTE059 trial JAMA Oncol 201845e180013Kang YK Boku N Satoh T Ryu MH Chao Y Kato K Nivolumab inpatients with advanced gastric or gastrooesophageal junction cancerrefractory to or intolerant of at least two previous chemotherapy regimensONO453812 ATTRACTION2 a randomised doubleblind placebocontrolled phase trial Lancet “Song M Chen X Wang L Zhang Y Future of antiPD1PDL1 applicationscombinations with other therapeutic regimens Chin J Cancer Res Gandhi L RodriguezAbreu D Gadgeel S Esteban E Felip E De Angelis F Pembrolizumab plus chemotherapy in metastatic nonsmallcell lungCancer N Engl J Med “ PazAres L Luft A Vicente D Tafreshi A Gumus M Mazieres J et alPembrolizumab plus chemotherapy for squamous nonsmallcell lungCancer N Engl J Med “ Yan Y Kumar AB Finnes H Markovic SN Park S Dronca RS Combiningimmune checkpoint inhibitors with conventional Cancer therapy FrontImmunol Wang J Fei K Jing H Wu Z Wu W Zhou S Durable blockade of PD1signaling links preclinical efficacy of sintilimab to its clinical benefit MAbs“ Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th ed Oxford Wiley National Comprehensive Cancer Network Guidelines for Gastric CancerVersion p Cunningham D Starling N Rao S Iveson T Nicolson M Coxon F et alCapecitabine and oxaliplatin for advanced esophagogastric cancer N Engl JMed “Lordick F Kang YK Chung HC Salman P Oh SC Bodoky G et alCapecitabine and cisplatin with or without cetuximab for patients withpreviously untreated advanced gastric cancer EXPAND a randomised label phase trial Lancet Oncol “ Waddell T Chau I Cunningham D Gonzalez D Okines AF Okines C et alEpirubicin oxaliplatin and capecitabine with or without panitumumab forpatients with previously untreated advanced oesophagogastric cancer REAL3a randomised label phase trial Lancet Oncol “ Bang YJ Kang YK Catenacci DV Muro K Fuchs CS Geva R et alPembrolizumab alone or in combination with chemotherapy as firstlinetherapy for patients with advanced gastric or gastroesophageal junctionadenocarcinoma results from the phase II nonrandomized KEYNOTE059study Gastric Cancer “ Boku N Ryu MH Kato K Chung HC Minashi K Lee KW Safety andefficacy of nivolumab in combination with S1capecitabine plus oxaliplatinin patients with previously untreated unresectable advanced or recurrentgastricgastroesophageal junction cancer interim results of a randomizedphase II trial ATTRACTION4 Ann Oncol “Tabernero J Cutsem EV Bang YJ Fuchs CS Wyrwicz L Lee KW et alPembrolizumab with or without chemotherapy versus chemotherapy inadvanced GGEJ adenocarcinoma the phase keynote062 study J ClinOncol 20193718supplLBA4007 Wang F Wei XL Wang FH Xu N Shen L Dai GH Safety efficacy andtumor mutational burden as a biomarker of overall survival benefit inchemorefractory gastric cancer treated with toripalimab a PD1 antibody inphase IbII clinical trial NCT02915432 Ann Oncol “ Cai H Jing C Chang X Ding D Han T Yang J Mutational landscape ofgastric cancer and clinical application of genomic profiling based on targetnextgeneration sequencing J Transl Med Mishima S Kawazoe A Nakamura Y Sasaki A Kotani D Kuboki Y et alClinicopathological and molecular features of responders to nivolumab forpatients with advanced gastric cancer J Immunother Cancer Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

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

Thyroid_Cancer

"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

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