DiscoveryofCancerBiomarkers張敏躍南京大學生命科學院StatisticsMorethan11millionpeoplearediagnosedwithcancereveryyear.Itisestimatedthattherewillbe16millionnewcaseseveryyearby2023.Fromatotalof58milliondeathsworldwidein2023,canceraccountsfor7.6million(or13%)oftheglobalmortality.Deathsfromcancerintheworldareprojectedtocontinuerising,withanestimated9millionpeopledyingfromcancerin2023and11.4milliondyingin2030.IntheUSin2023,over1.4millionnewcasesofcancerwerediagnosed.Overhalfamillionpeoplediedfromthisdisease,accountingforapproximately25%ofalldeathsintheUSeachyearHowtoImprovetheSituation?PreventionDetectionCancerisadiseaseofgeneticprogressionthatisoftenassociatedwithspecificmolecular,geneticandhistologicalchanges.Theabilitytodevelopbiomarkersthatcandetectthecriticalcomponentsofthesehallmarksofcancertogetherprovidesapowerfulbasisfordiagnosing,monitoringandpredictingoutcomeandresponsetotreatment.HowtoImprovetheSituation?Thegoalofcancerbiomarkerfieldistodevelopsimplenon-invasiveteststhatindicatecancerrisk,allowearlycancerdetection,classifytumorssothatthepatientcanreceivethemostappropriatetherapyandmonitordiseaseprogression,regressionandrecurrence.3.TreatmentConceptofCancerBiomarkersDefinitionofbiologicalmarkersBiologicalmarkers(biomarkers)havebeendefinedbyHulkaandcolleagues(1990)as“cellular,biochemicalormolecularalterationsthataremeasurableinbiologicalmediasuchashumantissues,cells,orfluids.”

HulkaBS.Overviewofbiologicalmarkers.In:Biologicalmarkersinepidemiology(HulkaBS,GriffithJD,WilcoskyTC,eds),pp3–15.NewYork:OxfordUniversityPress,1990.Morerecently,thedefinitionhasbeenbroadenedtoinclude“biologicalcharacteristicsthatcanbeobjectivelymeasuredandevaluatedasanindicatorofnormalbiologicalprocesses,pathogenicprocesses,orpharmacologicalresponsestoatherapeuticintervention”

NaylorS.Biomarkers:currentperspectivesandfutureprospects.ExpertRevMolDiagn3:525–529,2023.ConceptofCancerBiomarkers2.FormsofcancermarkersHormones,metabolites，aswellasdifferentfunctionalsubgroupsofproteinssuchasenzymes,glycoproteins,oncofetalantigensandreceptors.Furthermore,otherchangesintumors,suchasgeneticmutations,amplificationsortranslocations,andchangesinmicroarray-generatedprofiles(geneticsignatures),arealsoformsoftumormarkers.Themarkersareproducedeitherbythetumoritselforbyothertissues,inresponsetothepresenceofcancerorotherassociatedconditions,suchasinflammation.Cancerbiomarkerscanalsobeprocessessuchasapoptosis,angiogenesisorproliferation.ConceptofCancerBiomarkers3.FactorsthatareidealforatumormarkerProducedbythetumorcellsandentersthecirculationPresentatlowlevelsintheserumofhealthyindividualsandthosewithbenigndiseasebutincreasessubstantiallyincancer(preferablyinonecancertypeonly)EasilyquantifiablewithaninexpensiveassayPresentindetectable(orhigherthannormal)quantitiesatearlyorpreclinicalstagesQuantitativelevelsofthetumormarkerreflectthetumorburdenHighdiagnosticsensitivity(fewfalsenegatives)andspecificity(fewfalsepositives)ConceptofCancerBiomarkers3.FactorsthatareidealforaserologicaltumormarkerConceptofCancerBiomarkers4.Typesofcancerbiomarkers4.1.Diagnostic(screening)biomarkerAmarkerthatisusedtodetectandidentifyagiventypeofcancerinanindividual.ThesemarkersareexpectedtohavehighspecificityandsensitivityForexample,thepresenceofBence–JonesproteininurineremainsoneofthestrongestdiagnosticindicatorsofmultiplemyelomaConceptofCancerBiomarkers4.2.PrognosticbiomarkerThistypeofmarkerisusedoncethediseasestatushasbeenestablished.Thesebiomarkersareexpectedtopredicttheprobablecourseofthediseaseincludingitsrecurrence,andtheythereforehaveanimportantinfluenceontheaggressivenessoftherapy.Forexample,intesticularteratoma,humanchorionicgonadotropinandalfa-fetoproteinlevelscandiscriminatetwogroupswithdifferentsurvivalrates.ConceptofCancerBiomarkers4.3.Stratification(predictive)biomarkerThistypeofmarkerservestopredicttheresponsetoadrugbeforetreatmentisstarted.Thismarkerclassifiesindividualsaslikelyrespondersornonresponderstoaparticulartreatment.Thesebiomarkersmainlyarisefromarray-typeexperimentsthatmakeitpossibletopredictclinicaloutcomefromthemolecularcharacteristicsofapatient’stumor.CurrentapplicationsoftumormarkersandtheirlimitationsCurrentapplicationsoftumormarkersandtheirlimitationsCancerbiomarkersthatarecurrentlyinclinicaluseCancerbiomarkersthatarecurrentlyinclinicaluseTheclinicalphasesoftestinganewcancerdrugPhase1Determinationsoftoxicity,pharmacokinetics,andoptimaldoselevelsphase2DeterminationsofbiologicefficacyPhase3Definitivecontrolledtrialsofeffectsonclinicalendpoints.Foreachphase,guidelinesexistforsubjectselection,outcomemeasures,relevantcomparisonsforevaluatingstudyresults,andsoforth.Phasesofbiomarkerdevelopment

－MargaretSP;etal.UniversityofWashington(2023)1.PreclinicalexploratorystudiesPrimaryAims1)Toidentifyleadsforpotentiallyusefulbiomarkers.2)Toprioritizeidentifiedleads.Inthisphase,tumorandnon-tumorspecimensarecompared.Strategiessuchasgeneexpressionprofiling,mass-spectrometry-basedmethodsandotherapproachestobiomarkerdiscoverycanbeusedToidentifygenesorclustersofgenes(orproteins)thatappeartobeoverexpressedorunderexpressedintumortissuerelativetocontroltissue.Toidentifycharacteristicsuniquetotumortissuethatmightleadtoideasforclinicaltestsfordetectingcancer.Thedevelopmentofstatisticalalgorithmsforselectingpromisingbiomarkersfromalargepoolofbiomarkersisanactiveareaofresearch.Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)1.PreclinicalexploratorystudiesSpecimenSelectionTumortissuefromcasesubjectsshouldbeobtainedatdiagnosisandbeforetreatmentbecausetreatmentmayinterferewiththebehaviorofthebiomarker.Noncancercontrolsubjectsshouldbeselectedsothatfactorspotentiallyinfluencingthebiomarker,otherthanthecanceritself,aretightlymatchedtothoseofthecancercasesubjects.Thesefactorsmightincludeage,sex,race,andpossiblylifestyle-relatedcharacteristics,suchassmokinghabits.Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)1.PreclinicalexploratorystudiesSpecimenSelectionFactorsshouldbeconsideredwhenselectingtumorSpecimen1，樣品旳一致性：取材部位、腫瘤亞型、年齡、性別、種族、生活習慣等。2，樣品處理方式旳一致性：預處理條件、保存條件（涉及時間）、處理條件、操作等。Factorsshouldbeconsideredwhenselectingnontumor(control)Specimen1，對照樣品和腫瘤樣品旳對等性2，對照樣品和腫瘤樣品旳處理方式旳對等性Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)PreclinicalexploratorystudiesSampleSizesThenumberdependsontheobjectiveofthestudyandtheextentofthevariabilityofthebiomarkerinthestudy.Thefollowingfactorscontributetovariability:thenumberandrelativeprevalenceofthecancersubtypesamongthestudysamplesthecapacitiesofthebiomarkerstodiscriminateamongthedifferentcancersubtypesthenumberofbiomarkersunderstudythenumberofcaseandcontrolsubjectsandthestatisticalalgorithmusedtoselectpromisingbiomarkers.Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)2.AssaydevelopmentandvalidationPrimaryAimToestimatetheTPRandFPRorROCcurvefortheclinicalbiomarkerassay,toassessitsabilitytodistinguishsubjectswithcancerfromsubjectswithoutcancer.Aclinicalassaythatusesaspecimenofchoice(usuallysomethingthatcanbeobtainednoninvasively)isdevelopedinthisphase.Thepatientsassessedinthisphasehaveestablisheddisease.Theutilityoftheassayindetectingdiseaseearlyisnotdemonstratedinthisphase.

Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)3.RetrospectivelongitudinalclinicalrepositorystudiesPrimaryAims1)Toevaluate,asafunctionoftimebeforeclinicaldiagnosis,thecapacityofthebiomarkertodetectpreclinicaldisease.2)Todefinecriteriaforapositivescreeningtestinpreparationforphase4.Repositoriesofclinicalspecimens,collectedandstoredfromacohortofapparentlyhealthysubjectsmonitoredfordevelopmentofcancer,areusedinphase3ofthebiomarkerevaluation.Phasesofbiomarkerdevelopmentforearlydetetion

－MargaretSP;etal.UniversityofWashington(2023)4.ProspectivescreeningstudiesPrimaryAimTodeterminetheoperatingcharacteristicsofthebiomarkerbasedscreeningtestinarelevantpopulationbydeterminingthedetectionrateandthefalsereferralrate.Inthisphase,individualsarescreenedwiththeassayanddiagnosticproceduresareappliedtothosewhoscreenedpositive.Thiscanhelptoestablishthetumorstageorthenatureofthediseaseatthetimeofdetection.Phasesofbiomarkerdevelopmentforearlydetection

－MargaretSP;etal.UniversityofWashington(2023)5.RandomizedcontroltrialsPrimaryAimToestimatethereductionsincancermortalityaffordedbythescreeningtest.Strategiesandtechniquesfordiscovery

ofcancerbiomarkersGenomiclevelcancerbiomarkerdiscovery1.1.GenomicaberrationSequencing:TheCancerGenomeAtlas(TCGA)isapplyinglarge-scalegenomesequencingtechnologytoidentifynovelgenesinvolvedincancerpathogenesis.Comparativegenomichybridization(CGH)array-CGH(aCGH)Spectralkaryotyping(SKY)1.2.SNPSequencingSNParray1.3.EpigeneticalternationsStrategiesandtechniquesfordiscovery

ofcancerbiomarkers2.Transcriptionallevelcancerbiomarkerdiscovery2.1.mRNAexpressionprofilecDNA-micro-array,Oligo-micro-arrayDifferentialdisplay-PCR(DD-PCR)Serialanalysisofgeneexpression(SAGE),cDNALibrarySubtraction,etc.Strategiesandtechniquesfordiscovery

ofcancerbiomarkers2.Transcriptionallevelcancerbiomarkerdiscovery2.2.miRNAPotentialimportanceofmiRNAs

ascancerbiomarkersExpressionofmicroRNAs(miRNAs)invarioustissueshasbeenassociatedwithavarietyofdiseases,includingcancers.

SerummiRNAscontainfingerprintsforvariousdiseases.

RelatedtechniquesSequencingmiRNA-arrayStrategiesandtechniquesfordiscovery

ofcancerbiomarkers3.Translationallevelcancerbiomarkerdiscovery3.1.Protein(orsubtypes:enzymes,antibodies,secretedproteins,etc)2-dimensioalelectrophoresis/massspectrometry(2-DE/MS)Surface-enhancedlaserdesorptionionizationtime-of-flightmassspectrometrytechnology(SELDI-TOP-MS):proteomicpatterndiagnosticsMulti-dimensionalproteinidentificationtechnology(MudPIT)/MSStrategiesandtechniquesfordiscovery

ofcancerbiomarkersPrincipleofSELDI-TOF-MSOnemicrolitreofraw,unfractionatedserumisappliedtothesurfaceofaprotein-bindingchip.Thechipisrinsedtoremoveunboundproteins,treatedwithaMATRIXCOMPOUND,washedanddried.Alaserirradiatesanddesorbstheadherentproteins.Thetime-of-flight(TOF)oftheionisdetectedbyanelectrode.Aproteomicsignatureoftheserumiscreated.Strategiesandtechniquesfordiscovery

ofcancerbiomarkersProteomicpatterndiagnosticsWiththisapproach,theunderlyingidentityoftheindividualcomponentsofthepatternisnotnecessaryforitsuseasapotentialdiagnosticfordisease.Strategiesandtechniquesfordiscovery

ofcancerbiomarkers3.Translationallevelcancerbiomarkerdiscovery3.1.Protein(orsubtypes:enzymes,antibodies,secretedproteins,etc)2-dimensioalelectrophoresis/massspectrometry(2-DE/MS)Surface-enhancedlaserdesorptionionizationtime-of-flightmassspectrometrytechnology(SELDI-TOP-MS):proteomicpatterndiagnosticsMulti-dimensionalproteinidentificationtechnology(MudPIT)/MSStrategiesandtechniquesfordiscovery

ofcancerbiomarkersPrincipleofMudPITStrategiesandtechniquesfordiscovery

ofcancerbiomarkers4.Post-translationalmodificationsofproteins(cleavageproducts,alteredglycosylation,etc)

4.1.Cleavageproductsoftumour-derivedproteinshavebeenproposedaspotentialcancerbiomarkers.4.2.Alteredproteinglycosylationincancerisanothersourceofpotentialcancerbiomarkers.Identificationofglycosylatedproteinsreliesonvariousglyco-capturestrategies,ameansofglycosylated-proteinsub-selectionbyaffinitychromatography.Electron-transferdissociation(電子轉移解離)allowslabilemodificationstoremainintactwhileobtainingpeptidesequenceinformation,enablingthestudyofmodificationssuchasglycosylationandphosphorylation.CurrenttumormarkersunderdevelopmentContributionofoncoproteomicstocancerbiomarkerdiscoveryPublished:2April2023MolecularCancer2023,6:25PotentialimportanceofmiRNAs

ascancerbiomarkers1.HistoryDiscoveredinCaenorhabditiselegansin1993andformallynamedin2023Havebeenidentifiedineveryplantandanimalspeciesexamined2.Features2.1.GeneralfeaturesLength:AclassofnoncodingRNAs,18–25nucleotidesSpeciecs:miRNAshavebeenidentified5withupto1,000predicted.Location:miRNAsareencodedbyDNAthatmaybesituatedintheexonsorintronsofgenesorscatteredamongintergenicDNAPotentialimportanceofmiRNAs

ascancerbiomarkers2.2.Transcriptionandmaturation(i)nuclearprocessingintoaprimarymiRNA(pri-miRNA)andthenaprecursor(pre-miRNA);(ii)exportintothecytoplasm;(iii)furtherprocessingintomaturemiRNA;(iv)incorporationintoanRNA-inducedsilencingcomplex(RISC)withanArgonauteproteincatalystPotentialimportanceofmiRNAs

ascancerbiomarkers2.3.FunctionandtargetsThemiRNA-RISCcomplexhybridizestonucleotidesequencesofvaryingcomplementarityinthe3’untranslatedregion(UTR)ofmRNAandinhibitsproteinsynthesisordegradesthetargetmRNAPlayskeyrolesintheregulationoffundamentalcellularprocessesPotentialimportanceofmiRNAsascancerbiomarkersDysregulatedexpressionofmicroRNAs(miRNAs)invarioustissueshasbeenassociatedwithavarietyofdiseases,includingcancers.miRNAsexpressedincancermayactlikeoncogenesortumor-suppressorgenesbyregulatingproliferationand/orapoptosis.NormalandmalignanttissueshavespecificmiRNAsignaturesandshowdifferentialexpressionacrosstumortypes.OverexpressionorlackofexpressionofspecificmiRNAsappearstocorrelatewithclinicallyaggressiveormetastaticphenotype.miRNAexpressionhastissuespecificityandhasbeenusedfortumorclassification.——CancerbiomarkerprofilingwithmicroRNAsApril2023,NatureBiotechnologyVol26/4.PotentialimportanceofmiRNAincancerHerewedemonstratethatmiRNAsarepresentintheserumandplasmaofhumansandotheranimalssuchasmice,rats,bovinefetuses,calves,andhorses.ThelevelsofmiRNAsinserumarestable,reproducible,andconsistentamongindividualsofthesamespecies.EmployingSolexa,wesequencedallserummiRNAsofhealthyChinesesubjectsandfoundover100and91serummiRNAsinmaleandfemalesubjects,respectively.WealsoidentifiedspecificexpressionpatternsofserummiRNAsforlungcancer,colorectalcancer,anddiabetes,providingevidencethatserummiRNAscontainfingerprintsforvariousdiseases.Throughtheseanalyses,weconcludethatserummiRNAscanserveaspotentialbiomarkersforthedetectionofvariouscancersandotherdiseases.

CharacterizationofmicroRNAsinserum:anovelclassofbiomarkersfordiagnosisofcancerandotherdiseases.CellResearch,2023,18:997-1006.Organizations1.TheInternationalCancerBiomarkersConsortium(ICBC)()UndertheleadershipofDr.LeeHartwell,PresidentandDirectorofFredHutchinsonCancerResearchCentertheICBCispioneeringanewmodelforbiomarkerdiscoveryanddevelopingtechnologiesandmethodologiestomakeitpossible.Organizations1.TheInternationalCancerBiomarkersConsortium(ICBC)()ThegoaloftheInternationalCancerBiomarkerConsortium(ICBC)istoadvancemedicalresearchandimprovepatientoutcomesbydiscoveringbiomarkers(indicators)formultipletypesofcancer.Throughalarge-scaleeffortsimilartotheHumanGenomeProject,theconsortiumaimstofacilitatehighlycoordinatedresearchandbyleveragingresourcesandexpertisefromaroundtheworldtoovercomethecurrentobstaclesinbiomarkerresearch.Organizations1.TheInternationalCancerBiomarkersConsortium(ICBC)()Atthesametime,theICBCwillprovideastructureforinternationalteamstoworktogetheronglobalissuessuchasadoptionofdatastandardsandthesharingdataaswellasonscientificdetailssuchasthelogisticsoftissuesamplesharingandinvestigationofmousemodelsofcancer.Organizations2.NationalInstituteofHealth’s(NIH)NationalCancerInstitute(NCI)()CancerBiomarkersResearchGroupThisgrouppromotesandsupportsresearchtoidentify,develop,andvalidatebiologicalmarkersforearliercancerdetectionandriskassessment.Thegroupintegratesbasicandclinicalsciencestudiesalongwithcomputational,statisticalandepidemiologicapproachesforacomprehensiveunderstandingofbiomarkers.ItcoordinatestheEarlyDetectionResearchNetwork.Organizations3.EarlyDetectionResearchNetwork(EDRN)()AninitiativeoftheNationalCancerInstitute(NCI),bri