ClonalevolutioninhematologicalmalignanciesandtherapeuticDALandau1,2,3, - PDF document

ClonalevolutioninhematologicalmalignanciesandtherapeuticDALandau1,2,3,4,SLCarter,GGetzandCJWuTheabilityofcancertoevolveandadaptisaprincipalchallengetotherapyingeneralandtotheparadigmoftargetedtherapyinparticular.Thisabilityisfueledbytheco-existenceofmultiple,geneticallyheterogeneoussubpopulationswithinthecancercell CancerVaccineCenter,Dana-FarberCancerInstitute,Boston,MA,USA;BroadInstitute,Cambridge,MA,USA;DepartmentofHematology,YaleCancerCenter,NewHaven,CT,ParisDiderot,Paris,France;MassachusettsGeneralHospitalCancerCenterandDepartmentofPathology,Boston,MA,USA;DepartmentofMedicalOncology,Dana-FarberCancerInstitute,Boston,MA,USAandDepartmentofMedicine,BrighamandWomensHospital,HarvardMedicalSchool,Boston,MA,USA.Correspondence:DrCJWu, Leukemia(2014),34…43 2014MacmillanPublishersLimitedAllrightsreserved0887-6924/14www.nature.com/leu evolutionaryoptimizationprocess,assubclonescompeteoverresourcesandadapttoexternalpressures,suchascancertherapy.Cancerprogression,therefore,isfundamentallyaprocessofmutationaldiversi“cationandclonalselection.The“rstexperimentalevidencesupportingtheideathattumorsarecomposedofheterogeneoussubpopulationswasobtainedfrommousemodelsofsolidmalignancies.Theseexperimentsshowedthatindividualsubclonespossesseddifferentphenotypiccharacteristics,includingvaryingmetastaticpotential.Importantly,thelinkbetweenheterogeneityandresistancetotherapywasapparenteveninotherearlyexperiments.Forexample,celllinesthatexhibitedahigherdegreeofphenotypicheterogeneityalsoacquiredresistancetochemotherapy(methotrexate)atahigherratecomparedwithcelllineswithlowerphenotypicvariability.Ascancerisadiseasethatresultsfromtheaccumulationofgeneticalterations,anaturalcorollaryoftheabovestudiesisthatphenotypicevolutionmuststemfromunderlyinggenotypicevolution.Thisconcepthasbeenindeedcon“rmedoverthepastseveraldecadeswithincreasingtechnologicalsophistication,usingapproachesbasedoncytogeneticsandSanger(“rst-generationsequencing).Mullighanetal.forexample,inanelegantsingle-nucleotidepolymorphismarrayanalysesofpediatricpre-BcellALL,demonstratedcomplexbranchedevolutionarygrowthassociatedwithdiseaserelapse.Thislandmarkstudyfurthershowedhowrelapseddiseaseisgeneticallyalteredcomparedwithdiseaseatdiagnosis.Inchroniclymphocyticleukemia(CLL),clonalevolutionwasidenti“edinupto43%ofpatientsusing”uorescentinsituhybridizationorcytogenetictechniques,withfrequentacquisitionofthepoorprognosticmarkersdel(11q)anddel(17p),andoccurringatahigherrateinthepoorprognosisgroupofTogether,theseexperimentalobservationshavedemon-stratedthatthegeneticmakeupofhematologicalmalignanciesisconstantlyreshapedduringdiseaseprogression.Overall,theysupporttheprescientideastheorizedbyNowell,whopostulatedthatgeneticinstabilitywouldbeexpectedtoleadtoenhancedheterogeneitywithcancerprogression,resultingindiverse,geneticallydistinct,subpopulationswithinaneoplasm.Thus,theselectionprocesswouldbeexpectedtopromotetheoutgrowthofincreasingly“tsubclones,therebycontinuouslyremodelingthe“tnessoftheoverallIfcancerplasticityisdrivenbyclonalheterogeneity,itisimportanttoconsiderthefeaturesthatfuelthegenerationofclonalheterogeneity.Geneticinstabilityundoubtedlyhasakeyroleinthisprocess.Therateofacquisitionofnovelsomaticmutationsisprobablycloselytiedtothediversi“cationofthecancerpopulationandthereforeforenhancingitsevolutionarypotential,togetherwithotherfeatures,suchasthepopulation(comprehensivelyreviewedelsewhere).ApermissivegeneticcontextthateitherinhibitsDNArepair(forexample,mutations)orincreasestolerancetonovelmutationsbyremovingcriticalcheckpoints(forexample,TP53ATMmutations,enablingtolerancetowardsmassivegenomicdamageislikelytoincreasetheoveralldiversityofthetumorpopulation.Addingtothecomplexity,differentareasofthegenomemayhavedifferentratesofmutationsacquisition,29,30whichwouldneedtobetakenintoaccountwheninferringpastratesofmutationsfromgenomicinformation.Apotentiallyprovocativenotionthatarisesfromthesedataiswhethergeneticinstabilitymaybetargetedasameasuretoinhibitcancerevolution.Forexample,forhematologicalmalignancies,thedocumentedongoingmutagenicactivityofenzymesresponsibleforB-andT-cellreceptorgeneticmodi“cationsmaybeofparticularinterest.UNRAVELINGCLONALCOMPLEXITYWITHMASSIVELYPARALLELSEQUENCING(MPS)Althoughcon“rmingthebasictenetofcancerasanevolutionarydisease,theabovedescribedstudiesareinherentlylimitedintheirabilitytodecipherthetrueextentofgenomichetero-geneity,giventhelimitedamountofgeneticlesionsstudiedatanyexperimentandthelimitedsensitivityofexperimentaltechniquesthatwerethenavailabletodetectsmallersubclones.BothoftheselimitationshavebeenlargelyovercomewiththeadventofMPS.MPSoftumorshasaffordedanexponentialincreaseintheabilitytocharacterizethegeneticlandscapeofcancer.Ithasrevealedaveryhighdegreeofintertumoralheterogeneity(thatis,differentgeneticlesionaffectingdifferenttumors),withhundredsofdifferentmutationsaffectingdifferenttumorswithaprobableeffecton“tness.34,35Moreover,thistechnologyhasalsorevealedahighlevelofintratumoralgeneticheterogeneity(thatis,differentgeneticlesionsaffectingdifferentsubcloneswithinanindividualtumor),whichalsoaffectsputativedriverevents.Inparticular,theadventofMPShasallowedresearcherstoidentifybothsubclonalsomaticcopynumberalterations(SCNA)andsubclonalsomaticsingle-nucleotidevariations(SSNV),37,38whichcanbetrackedovertimetostudytumorevolution.39,40ThisabilitytoreconstructtheclonalstructureisderivedfromaninherentpropertyofMPS.Itinvolvesgeneratingbillionsofindependentsequencingreads,eachderivedfromasingleDNAmolecule.Thus,MPSdatarepresentaninformativerandomsampleofindividualDNAmoleculescontainedwithinatumor.AtSSNVsites,thenumberofsequencingreadssupportingthealternateandreferencebasescanbeusedtocalculateaquantitativemeasurementofthevariantallelicfraction(VAF).Insamplesderivedfromdiploidcancerssuchasacutemyeloidleukemia(AML)thatessentiallylackSCNAsandarenotcontaminatedwithnon-malignantcells,allelicfractionscanbeusedtoestimateSSNVclonalitydirectly(inwhichanyclonalSSNVshouldhaveaVAFof0.5;whileSSNVsinsubcloneswillhavelowerVAFs).However,thevastmajorityofhumancancerscontainfrequentSCNAs,withmanyofthemhavingundergonewholegenomedoublingduringtheirevolution.Inaddition,mosttumorspecimenscontainasubstantialfractionofnormalcells.Thus,inordertoaccuratelyinferthefractionoftumorcellsthatcontainanSSNVfromMPSdata,itisnecessarytoaccountforboththecopynumberattheSSNVsiteandtheoveralltumorpurityinthesample.Recently,inferencemethodshavebeendevelopedwhichattempttoaccountforthesefactorsinordertoestimatetheactualcancercellfractionharboringaspeci“cmutation(Figure1).Althoughmoderatesequencingdepthmayresultinconsiderableuncertaintyinthecancercellfractionestimatesofindividualmutations,thefactthatsubclonalmutationsareexpectedtoco-occurindiscretesubclonalcellpopulationshasformedthebasisofusingclusteringtechniquestobetterresolvethesubclonalstructureofbulktumorsamples.BecauseDNAfromallcellspresentinthebulksampleismixedtogetherbeforesequencing,informationregardingwhichmuta-tionsco-occurinspeci“csubclonesisnotreadilyaccessiblefromanalysisofasinglesample.Ingeneral,analysisofbulkDNAfromasinglecancersamplecannotruleoutthepossibilitythatallsubclonesarenestedinsideoneanotherinalinearphylogeny.Oneexceptiontothiswasreportedinastudythatapplieddeepwholegenomesequencing(188X)ofasingleprimarybreastcancersampleandcouldinferbranchedevolutionbasedonco-occurrenceofinformativeallelesonsequencingreads.thisapproachrepresentsanelegantsolutiontothephylogeneticinferenceproblem,thedeepcoverageneededoverthewholegenomerendersitimpracticalforlargestudiesusingexistingtechnology.Wenotethatsequencingplatformscapableofreliablyproducinglongerreadswillmakethisapproachfarmorepowerful. ClonalevolutionandtherapeuticstrategiesDALandauetal 2014MacmillanPublishersLimitedLeukemia(2014)34…43 AnattractiveapproachtoinferringphylogeneticstructuresfromanalysisofbulktumorDNAistosequencemultiplespecimensfromthesameindividualscancer.Branchedevolutionaryrelation-shipscanbedetectedasclustersinwhichonesubclonemayincreaseinfrequencywhileanothersiblingsubclonemayexhibitaconcomitantdecreaseinfrequency.Thisapproachhasbeenusedtoidentifybranchedevolutioninleukemiasamplestakenbeforeandaftertreatmentaswellasinsolidtumorssampledatmultipleanatomicallocations.THESURPRISINGASPECTSOFCLONALCOMPLEXITYOFHEMATOLOGICALMALIGNANCIESOneofthe“rstkeylessonsgleanedfromgenome-widestudiesofhematologicalcancersisthatclonalevolutionwhichfollowsacomplexbranchedpath(wheremultiplesubpopulationco-existinthesametumorandcompeteforascendency)isatleastascommonasamorelineartrajectory(inwhichprogenyclonesreplaceparentclonesinfullselectivesweeps).Atraditionallinearmodelofsuccessiveclonalexpansionscouldhavebeenexpectedofhematologicalmalignancies,byvirtueofthemobilenatureoftheircellularnormalcounterparts(comparedwithsolidtissuesthatareoftenembeddedin“xedtissuearchitecture).Intheory,thisfeaturecouldtheoreticallyhaveledtoadecreasedlevelofclonalcomplexityascancercellscanreadilymoveacrosstissuesandhenceundergomorehomogenouscellularmixing.Thisscenarioisunlikesolidtumormalignancies,inwhichthespatialcompartmentsareformed.Tothecontrary,however,wholegenome/exomeinvestigationofclonalevolutioninAML,myelodysplasticsyndrome,multiplemyelomaandCLLhaveallconsistentlydemonstratednotonlyahighdegreeofclonalheterogeneityandmarkedchangesinthegeneticmakeupofthediseaseuponrelapsebutalsobranchingratherthanlinearasthepredominantpatternofevolution(Table1).Amajorimplicationofthese“ndingsisthattheevolutionaryprocessisexpectedtoresultfromcomplexinteractionsamongmultiplehighlydiversepopulationsratherthanaclearsuccessionofselectivesweeps.Clonalcompetitionamongco-existingsub-populationsthatharbordriverlesionsthusshapestheeventualcompositionofthetumorsuchthatmultipleclonalvariantsarepresentatthesametime.Itisimportanttonotethatthepublishedanalysestodatehavebeenlimitedtothedetectionofmacroscopicclonalheterogeneity(clonesize1…10%oftheentirecellpopulation).Thisisbecauseonlyclonesthateitherrepresentasubstantialproportionofthecancercellmassorclonesthatbecomedominantatsomepointduringthestudiedperiodaretrackableusingcurrentmethodologies.EmergingtechnologiescapableofachievingdeepersequencingdepthofbulkDNAorsingle-cellgenomicsequencingmethodsmayenablethestudyofsmallersubpopulations.Delineatingthefullextentofcancerheterogeneitydowntothesingle-celllevelwillenableustounderstandhowtheseeminglystochasticprocessoftrialanderroratthesingle-celllevelisintegratedthroughselectiontoshapethegeneticmakeupofthetumor.Itcarriesthepotentialtore“nethedichotomyofdrivervspassengermutations,byquantifyingthe“tnesscontributionofeachindividualmutationtoselection(manifestedinvaryingclonesizes).EPIGENETICCLONALHETEROGENEITYAlthoughgeneticalterationhasbeenthemainfocusofevolutionarydynamicsincancerthusfar,epigeneticmodi“cationsareprobablyresponsibleforalargepartofphenotypicthatultimatelyaffect“tness.Similartogeneticalterations,epigeneticmodi“cationsareheritableandthereforesubjecttonaturalselection.Thecontributionofepigeneticmodi“cationtoselectionincancerisprobablysubstantial,asepigeneticalterationsaccumulateasthecellpopulationevolvesanddiversi“esatratesestimatedtobeordersofmagnitudehighercomparedwithsomaticgeneticalterations.Indeed,alargedegreeofintratumorepigeneticheterogeneitywasrecentlydescribedinlymphomausingDNAmethylationarrays.Geneticandepigeneticchangesprobablyhavecomplexbidirectionalinteractionsandco-operatetomoldtheevolutionarylandscape.Thiscomplexandbidirectionalinterplaybetweengeneticandepigeneticfeaturesincancerhasbeenperhapsmostdeeplyexploredintheareaofcancerstemcells.Speci“cally,earlyxenograftstudiesofALLrevealedleukemicrepopulationthatrecapitulatedthegeneticheterogeneityofthepatientsoriginalSimilar“ndingswerealsodemonstratedinsolidtumormalignancies.etal.concludedthatcancerstemcells„anepigeneticallyuniformpopulation„aregeneticallydiverse.Ontheotherhand,evengeneticallyuniformcellsubpopulationshavebeenreportedtorevealprofoundepigeneticdifferencesleadingtodifferencesinthephenotypesofsurvivalcapacityandpluripotencypotential. Figure1.Inferringthesizeofasubpopulationaffectedbysomaticmutationsfromgenomicdata.MPSprovidesanestimateofVAF,whichiscalculatedbycountingthenumberofreadswiththevariantallelesanddividingitbythetotalnumberofreadingfromthespeciÞclocation.Thecertaintyoftheestimateisafunctionofthedepthofcoverage,usingtheBetadistribution().Subsequently,theVAFestimatesareintegratedwiththepurityandlocalcopynumberinformation()toyieldcancercellfractions().Intheexampleprovided,asomaticmutationwithaVAFof0.125,alocalcopynumberof3andapurityof67%yieldscancercellfractionestimatesof0.5. Clonalevolutionandtherapeuticstrategies DALandauetalLeukemia(2014)34…432014MacmillanPublishersLimited Together,theseobservationspromptamodelofcancerevolutioninwhichepigeneticandgeneticheterogeneityareintegrated,therebyaccountingforepigeneticheterogeneityofgeneticallyuniformpopulationsandgeneticheterogeneityofepigeneticallyuniformpopulations.Suchamodelwoulddesig-natecellswithhighself-renewalcapacity(anattributeencodedintheepigeneticstate)asthecrucialunitssubjectedtoselectionforcesingeneticevolution.Hence,theappearanceofanewsomaticmutationwithinthesespecializedcellscouldleadtotheirclonalpropagation.Atthesametime,suchamodelwouldalsoacknowledgetheexistenceofafarlesshomogenouscancerstemcellpopulationthenpreviouslyconsideredwithregardtovariousfeatures,includingdrugsensitivity.Itisimportanttoconsiderthatincancerthemovementbetweendifferentepigeneticstates(alongthespectrumofpluripotencytodifferentiation,forexample)maybealteredaswell.Inmulticellularorganisms,epigenetictransitionsaretightlycontrolledthroughnumerousregulatorymechanisms.Neoplastictransformationcanunhingethosemechanisms,revertingtoastatemorecloselyresemblingunicellularinwhichthe”uidmovementacrossdiversestatescanachievehighadaptivitybytrialanderror.Epigeneticheterogeneity,thus,canbeahedgingstrategyforenhancedCancerprogression,therefore,maybeviewedasascenarioinwhichbothgeneticandepigeneticpopulationstructuresbecomeincreasinglymalleable,suchthatthelinesbetweenpopulationswithdifferentstemnesspotentialbecomemoreblurred.Withinthisframework,stemnessmayexistasafunctionalphenotype,whichcanbemanifestedbyanymemberofamalignantpopulationgiventheappropriateendogenousandexogenousfactors.Thus,ahighdegreeofinterclonalcompetitionwouldprobablyselectforcellswiththehighestself-renewingcapacityattheexpenseofmoredifferentiatedcells,ashasbeendemonstratedinCML.Therapymayalsoacceleratethisprocessbyprovidingastrongselectionforcancerstemcellsurvivalandproliferation.Acquiredgeneticalterationsprobablyhaveanimportantroleinthisscenarioaswell.Forexample,thelossof,oftenseenwithdiseaseprogression,provokesstem-cell-liketranscriptionalprograms.oncogenesmayalsoaffordleukemogenicpotentialtocommittedmyeloidprogenitors,againdemonstratingthatgeneticlesionsmayenlargetheavailablepoolofcellswithstem-likefeatures.Insummary,integratingthestemcellhierarchyandthegeneticphylogenetictreeyieldsacomplexevolutionarypicturethathasonlybeguntobeunraveled.Inconcertwithgeneticdiversi“cationand“tnessoptimization,asimilarprocessverylikelyoccursattheepigeneticlevel.Cancerstemcellsconstituteagrowingproportionwithinthecancercellmassandhaveagreaterplasticityintermsofbidirectionalconversionfromstemcellstomoredifferentiatedcells.Together,thisleadstoenhancementofthecellularsubstrateavailableforselection,withlarge,treatment-resistantandgeneticallyheterogeneouscancerstemcellHOWDOESCLONALEVOLUTIONCONTRIBUTETORESISTANCETOTHERAPY?Relapsedmalignancyshowsanalmostuniversalphenotypicevolution,resultinginamoreaggressiveandtreatment-refractoryWeandtheothershaveshownthatfrequentgeneticevolutionunderliesthephenotypicevolution.Therefore,acentralquestionincancertreatmentiswhatistheprecisenatureoftheinteractionofclonalevolutionwithcancertherapy.Initialstudieshighlightedthepotentialroleofchemotherapytoinducenovelmutagenesisandtherebytoenhancetheprocessofgeneticdiversi“cation(Figure2a).AlthoughstudiesofWGS(wholegemonesequencing)areinherentlylimitedbythepowertodetectminutesubcloneswithinasample,studiesinacutemyeloidmalignancieshavenonethelesssuggestedthatthenovelmutagenesismayresultfromthegenotoxiceffectsofchemotherapy,supportedalsobyachangingspectrumofsomaticsingle-nucleotidealterations.contrast,incaseofindolentbloodmalignanciessuchasCLL,evidenceforthecontributionofthechemotherapysmutagenizingeffectislimited.Previouspurineanalog-basedtherapywasnotassociatedwithanincreasedtotalnumberofmutationsinCLLandalsowasnotassociatedwithanalteredmutationalpattern.Therefore,althoughchemotherapy-inducedmutagenesishasthepotentialtocontributetofurtherclonaldiversi“cation,othersourcesforgeneratingevolutionaryshiftsappeartobeatplay,andprobablyinvolvepre-existinggeneticvariantsorsubclones.Howthendoestherapyinduceevolutionfrompre-treatmentgeneticvariation?Twoexplanationsareconsidered,dependingonthetumorkinetics,theef“cacyofcellkillwithtreatmentandotherfactorsrelatedtoboththetumortypeandthespeci“ctreatmentstrategy.The“rstisthatresistantclonesmaybeactivelyselectedbytherapy(Figure2b).Examplesforthismodelarenumerous,79…81mismatchrepairgenemutationsinrecurrentglioblastomamultiformeaftertreatmentwithtemozolomideandtheBCR-ABLT315ImutationsinCML.Indeed,thismodelofclonalevolutioninducedbytheselectivepressureoftherapymaybeparticularlyrelevantinthecontextoftargetedtherapy,as Table1.Next-generationsequencingstudiesofclonalevolutioninhematologicalmalignanciesDiseaseMethodologyNumberofcasesInsightsWGS,followedbytargeteddeepsequencing8RelapseafterchemotherapyisassociatedwithclonalevolutionandacquisitionofnewmutationsSecondaryAMLWGS,followedbytargeteddeepsequencing7SecondaryAMLclonesareoftenevolvedprogenyofMDSclonesMultiplemyelomaWES1ClonalshiftsoccuralongthehistoryofthediseaseMultiplemyelomaWES1ClonalshiftsoccuralongthehistoryofthediseaseWGS,followedbytargeteddeepsequencing3DifferentpatternsofevolutionevidentthroughcyclesoftherapyWES149(18longitudinalSubclonaldriverscananticipateclonalevolutionandimpactoutcomeEssentialSingle-cellWES1ETismonoclonalinoriginFollicularWES8EarlyandlatedriversidentiÞedAbbreviations:AML,acutemyeloidleukemia;CLL,chroniclymphocyticleukemia;ET,essentialthrombocytosis;WES,wholeexomesequencing;WGS,wgemonesequencing. ClonalevolutionandtherapeuticstrategiesDALandauetal 2014MacmillanPublishersLimitedLeukemia(2014)34…43 thetherapyisoftendirectedataparticulargeneticcontextwhichmaynotbesharedbyallsubclones.Thisrelationshipbetweentherapyandgeneticadaptationislikelytoresultinconvergentevolution,inwhichamutationthatconfersresistancewillbecomehighlyprevalentinrelapseddisease.Indeed,thisprocesshasbeenreportedinrelapsedT-cellALLaftertreatmentwithnucleoside-analogchemotherapydrugs.Analternativeprocesscontributingtotheemergenceofcontinuouslymoreaggressiveclonesmaybeentirelyindependentofdifferentialsensitivitytotherapy(Figure2c).Werecentlyobservedahighernumberoflargesubclones(10%ofcancercells)in149CLLcasesthatwereexposedtotreatmentbeforesamplingcomparedwithpatientswhoreceivedtherapyafterthesamplewasobtained.This“ndingofincreasedclonaldiversitywithtreatmentheldtrueevenafteraccountingforpotentialconfounders,suchaslongerfollow-uptime.Weinterpretthisobservationtoresult,atleastinpart,fromtheoutgrowthofmanydiversepre-existingminorbut“tsubclones.Thislatterinterpretationisfurthersupportedbyourobservationofanincreasedfrequencyofsubclonal-driverevents(presumably“tter)intreatedrelativetountreatedpatients.Overall,ourdatasupporttheideathatCLLtherapy,bymarkedlyreducingdiseasebulk,mayactasaclassicevolutionaryrestrictionpointandresetinterclonalWithinthisconceptualframework,whensubcloneswithhigh“tnessalreadyexistwithinatumorpopulation,treatmentcouldfavorthedevelopmentofmoreaggressiveclones,potentiallyreducingpost-relapsesurvival.Inthiscontext,cytotoxictherapywouldeffectivelyremovetheincumbentclone„actinglikeamassextinctionevent„andtherebyshifttheevolutionaryinfavorofoneormoreaggressivesubclones.Thus,highly“tsubclonesprobablybene“tfromtreatmentandexhibitrapidoutgrowth.Thesedataprovidemechanisticsupporttotheobservationthatthewatchandwaitstrategyfor Figure2.Threemodelsofhowcancertherapymayaccelerateclonalevolution.First,cancertherapy,particularlycontaininggenotoxicagents,caninducenovelmutagenesis().Second,therapycanaccelerateclonalevolutionbyselectingaclone(hereillustratedinred)containingamutationthatconfersresistancetothetherapeuticagentused().Theresistanceoftheselectedcloneisreßectedinthedepictionofthecellpopulationaftercytoreduction,composedalmostentirelyoftheresistantclone(inred).Athirdmodelpostulatessimilarsensitivitytotreatmentofthedifferentsubpopulations,reßectedinsimilarproportionsbeforeandaftercytoreduction().TheclearingnichealtersthedynamicevolutionarylandscapeallowingafasterriseofaÞtterclone. Clonalevolutionandtherapeuticstrategies DALandauetalLeukemia(2014)34…432014MacmillanPublishersLimited CLLleadstosuperiorclinicalresults,astheearlieradministrationofchemotherapymayaccelerateclonalevolutionandtheemergenceof“ttercloneswithmoreaggressivediseasephenotypes.ThisformofrelationshipbetweentherapyandevolutionmaybeparticularlyimportanttoCLL,asthiscancertypeishighlydependentongrowthandsurvivalsignalsprovidedbythelocalmicroenvironment.ThisdependencymayaugmenttheimportanceoftheroleofinterclonalcompetitionintheevolutionarydynamicsofCLL.Futurein-depthstudieswouldassistincon“rmingthismodelaswellaswhetheritisgeneralizabletomalignanciesotherthanCLL,andinparticularinothermoreindolentcancers.TRANSLATINGCLONALEVOLUTIONTOTHECLINICAmajorpriorityofprecisioncancergenomicsistouseinformationongeneticlesionstode“nepatientprognosis.Inanillustrativeexample,Pateletal.coulddemonstratethatlesionssuchasinternaltandemduplicationof-ITD),partialtandemduplicationin-PTD),aswellasmutationsinassociatedwithreducedoverallsurvivalinAML,whilemutationsassociatedwithimprovedoverallsurvival.Theseassociationswereindependentofestablishedriskfactors.Similareffortshavebeencarriedoutinotherhematologicalmalignancies,includingCLL,andmultiplemyeloma.Acrossthebloodmalignancies,patientswithapparentlypoorprognosticmarkerscannonethelessexhibitgoodsurvival,andviceversa.Theseveralstudiesreviewedabove(Table1)suggestthatintratumoralclonalheterogeneitymaybeanimportantcontributortothiscomplexpicture.Inaggregate,studiesofclonalevolutionhaverevealedcancerstobegeneticallyheterogeneousinspaceandtime.Hence,simplylabelinganindividualcancerasharboringageneticlesionornotisnotfullyprecise.Fromapracticalstandpoint,forasolidtumormass,orevenleukemiacellsthatarepresentindifferenttissuecompartments(thatis,bloodvsmarrowvslymphnode),multiplesamplingsmayberequiredtocorrectlyassertthegeneticlandscapeofanindividualcase(Figure3).Finally,clonalheterogeneityinandofitselfmayimpactclinicaloutcome.Ourstudieshaveshownthatthepresenceofastrongsubclonal-driverevent,butnotaclonaldriver,negativelyimpactsclinicaloutcomeinCLL.Thelinkbetweenclonalheterogeneityandspeci“callythepresenceofasubclonaldrivertoadverseclinicaloutcomeaddsanadditionaldimensiontothecurrenteffortsoflinkingdiscretesomaticmutationstooutcome.Inotherwords,itisnotonlythepresenceorabsenceofamutationthatshouldbeconsideredbutalsothesizeofthesubpopulationitFromthetherapeuticstandpoint,studiesofcancergenomicshighlighttheconceptthatcancerisnotasinglediseaseentitybutratheracollectionofrelateddisorders;hence,treatmentshouldbetargetedtothemolecularsubtypeofdisease.Forexample,high-dosedaunorubicin,ascomparedwithstandard-dosedau-norubicin,improvestherateofsurvivalamongpatientswithmutationsortranslocationsinAMLbutnotamongpatientswithwild-type,andpotentialtointegratetheavailablehigh-throughputsequencingtechnologies(forexample,DNA-seq,RNA-seqandChIP-seq)toprovideapatient-speci“cgenomic-epigenomicmapmayprovidecrucialprognosticperspectiveandinformtherapeuticchoices.Furthermore,targetedtreatmentsthatarebasedonthepresenceofspeci“cmolecularlesionsmaygreatlyimprovetherapeuticresponse,asseenin,forexample,associatedmyeloproliferativedisorders.Theseactionablemutationswhereaclinicianmatchesatumormutationtoacancerdrugmayeitherbemissedgivengeneticheterogeneityintimeandspace,oralternativelymightinvolveonlyasmallsubclone,whichbegsthequestionoftheclinicalef“cacywereittobesolelytargeted.Forinstance,syntheticlethalapproacheswerefoundtobehighlyeffectiveinsituationsinwhichallcancercellscontainthetargetedvariation,aswitnessedbythepotentef“cacyofPARP(polyADP-ribosepolymerase)inhibitionintumorsgermlinecarriers.Theseobservationstogetherraisestheprovocativequestionofwhetheritispreferabletotargetgeneticvariationsthatarefoundinthetrunkcomparedwiththosefoundinbranchesoftheevolutionaryphylogenetictree.Intuitively,theformermaybeconsideredthesuperiorapproach.Trunkevents,byde“nition,aremutationspresentinallthecellsofthemalignantprocess.Targetingofthiseventintheorycarriesthepotentialofacompleteextinctionoftheentirepopulationofmalignantcells.Conversely,itisunclearwhetherthecellremainsdependentonthespeci“ctrunktargetafteracquiringadditionaloncogenicevents(abranchtarget),andtherefore,howwelltheywillbeimpactedbytherapydirectedagainstthesefoundertargets.Insolidmalignancies,suchasnon-smallcelllungcancer,mutationsarerarelydetectedtogether;however,whentheyco-occur,targetingthetrunk-typemutation(thatis,)isnolongereffective.canonicalmutationsarediscoveredinbenigncolonicpolypsandarethereforeprobablytobeearlier,trunk-typeevents.However,theresponsetotargetinghasbeendisappointing.Onemayhypothesizethatatleastinmoreindolentmalignancies,targetingbranchmutations(pruning)maybeaneffectivestrategy,whichcouldpromoteclonalequilibriumandhindertheselectionofmoreaggressiveThedifferentialeffectsoftargetingbranchvstrunklesionsmaybedetermined,inpart,bythecomplexepistaticrelationshipbetweendifferentgeneticlesionswithinthesameclonalpopulation.Asnewmutationsdonotoccurinisolationbutratherenterintoanestablishedgenomiclandscape,theexistinggenenetworkmayhaveaprofoundeffectonthefateofthecellanddeterminewhetherthenovelmutationwillresultincelldeathorclonalexpansion.Forexample,activationofmanyoncogenestogether,including,canleadtoastateofoncogene-inducedAsimilarrelationshiphasbeendemonstrated-inducedapoptosisthatisrelievedinthecontext Figure3.Translatingclonalheterogeneityinsightstotheclinic.Possibleprognosticandtherapeuticimplicationsofclonalheterogeneityareoutlined.ImagecourtesyBroadInstitute/LaurenSolomon;hourglassphotoiStockphoto/DominikPabis. ClonalevolutionandtherapeuticstrategiesDALandauetal 2014MacmillanPublishersLimitedLeukemia(2014)34…43 Hence,furtherstudyoftheepistaticrelationshipsinmodelsystemsaswellasinclinicaltrialswillhelpclarifyinwhatcontextoptimaleffectswillresultfromtargetingthetrunkeventandwhenitispreferabletotargetthebranches.Thebroaderevolutionaryperspectiveallowsustoviewcancerasanecologyofdifferentsubpopulationsinthecontextoftheirenvironment.Intriguingdatasuggestthat,atleastinsomecases,complexco-dependencyrelationshipsbetweensubpopulationsmayexist,inadditiontocompetition.Theunderstandingthatdiseaseiscomposedofdiversesubpopulationsisachallengetoourtraditionalschemesofclinicaltrials.Afutureinwhichbothtrunkandbrancheventsarecharacterized,andinwhichnotwocancerssharethesamegenomicfeatures,maybeenvisaged.Inthissetting,performinglarge-scaleclinicaltrialsusingpresent-daymethodologies,inparticularutilizingcombinationsoftargetedagents,mayprovehighlychallenging.Thediseasecannolongerbede“nedasasingleentitycontainingauniformsetofgeneticabnormalities.Furthermore,thedegreeofgeneticheterogeneityofatumorislikelytobeanimportantdeterminantoftherapeuticAbetterunderstandingisneededoftheimpactoftherapyontheevolutionarylandscape,possiblythroughtheuseofthemoreapplicablewhole-exomesequencingtechnologiestostudylargecohortsonpatients.ResearchersmayconsiderincorporatingapproachessuchasWES(wholeexomesequencing)thatidentifyatleastlargersubpopulations(1…10%ofcancermass)andcharacterizetheirevolutioninancillarystudiesofprospectiveclinicaltrials.Suchinformationmayinformusregardingtheadaptiveprocessesresponsiblefortreatmentfailureaswellaseventuallysparkthedevelopmentofnoveltherapeuticparadigms.Forone,ithasbeenproposedthatalternativeapproachescouldpotentiallymaintaininterclonalequilibriumattheexpenseoftryingtomaximizecellkill.Thisapproachsupportspreventingtheeliminationoftherapy-sensitiveclones,asthey(theoretically)couldcontinuetosuppressthegrowthoftherapy-resistantclonesinacompetitivemannerandtherebymaintainanequilibriumstate.Asecondapproachthatrequiresfurtherconsiderationistheideaoflimitingtheunderlyingdiversi“cationthatservesasthesubstrateforclonalevolutionbeforethefullexpressionofthegeneticortheepigeneticheterogeneityincancerisevident.Finally,thetherapeuticchallengeposedbyacontinuouslyadaptingandreshapingmalignantprocessprovidesstrongrationaletosupportthepursuitofimmunity-basedtherapies,asthisapproachmayeffectivelypitonecomplexadaptiveprocessagainstanother.Thereisalreadylimitedevidencethatallogeneichematopoieticstemcelltransplant(anon-speci“cexampleofaimmunity-basedtherapy)imposesevolutionarypressuresonthetumorthataredistinctfromothertherapeuticmodalities(leading,forexample,tolossofdonor…recipientmismatchedormultiplecytogeneticabnormalities).Thesealterationsdemonstratethattheleukemiccellpopulationisbeingmoldedbyapowerfulimmuneresponseandhencetotheef“cacyoftheimmunity-basedtherapy.Theprocessofco-evolutionofthecancercellsandtheimmuneresponseinthesettingofeffectiveimmunotherapyisanareaofgreatinterestforfuturestudy.CONCLUSIONSANDFUTUREDIRECTIONSUnderstandingtheevolutionarycapacityofcancerisemergingasakeyelementindevelopingimprovedtherapeuticstrategiesintheeraofprecisionmedicine,asitpresentsoneofthemostformidableobstaclestothesuccessfulapplicationoftargetedAsaforementioned,theintensiveapplicationofhigh-throughputgenomicplatformshasenabledrapidprogressinourunderstandingoftheprocessofclonalevolutioninhematologicalmalignancies.Collectively,thesestudieshaveprovidedseveralcoreinsights,including:“rst,thatclonalheterogeneityiscommoninmalignancybothatthegeneticandtheepigeneticlevel;second,thatclonalevolutionisfrequentlyobservedinrelationtotherapy,leadingtoemer-genceofmoreaggressiveandresistantdisease;and“nally,thattheprocessofclonalevolutionislinkedtoadverseclinicalAlthoughtheserecentstudiespointtothekeyroleplayedbyclonalevolutionincancerprogression,currentperspectivesofcancerasanevolutionaryproblemarefairlylimited.Evenasknowledgeaboutgermlineandacquiredgeneticlesionsasso-ciatedwithcancershasgrownexponentially,westillknowonlylittleaboutthebackgroundrateofheterogeneity„whichisthesubstrateofevolution„andpossessonlyarudimentaryunder-standingofhowtheepigeneticprogramaffectsthissubstrate.Inthisrespect,developingmethodologiestointegratedatafromcomplementary„geneticandepigenetic„high-throughputplat-formsiskey,bothatthecellpopulationandatthesingle-celllevel.Moreover,thedynamicsofinteractionsbetweenclones„whethertheycompeteorco-dependoneachother„hasnotbeenelucidated.Additionally,theexaminationofkeymechanisticquestionrelatingtoclonalevolutionusinggenomictools(forexample,differenttypesofselectivepressure,interactionwithmicroenvironmentnichesandinteractionsbetweenmultiplegeneticlesionswithinthesamecell)hasyettobeaccomplished.Thus,theabilitytoforeseetheevolutionarytrajectoryofanyindividualcancerispresentlystillinitsinfancy.Improvingthiscapacitytopredicthowcancerwillevolvewithtreatmentcarriesasigni“cantpotentialtoallowustoanticipateandtailortreatmenttotheprobablefuturetrajectory(so-calledanticipation-basedOngoingtechnologicaldevelopmentsarenowgeneratingtoolsideallysuitedforthestudyofthesequestions.Recently,proof-of-principlestudiesofsingle-cellsequencinghavebeenconductedthathavecataloguedthepointmutationsinprotein-codingregions.115,116Inthenottoodistantfuture,single-cellsequencingwillallowthedetailedstudyofgeneticheterogeneitythatprovidethebackdropagainstwhichevolutionatthesubpopulationleveloccurs.Theapplicationofsingle-cellRNA-seq117tothestudyofhematologicalmalignancieswouldenablethestudyoftheheterogeneoustranscriptionalchangesandsignalingnetworksthatstemfromheterogeneoussomaticgeneticalterations.Inadditiontosingle-cellexamination,novelmethodologiesarecapableofdeconvolutingsubpopulationsfrombulkmaterialandmaybehelpfulindelineatingthebasicunderlyingprinciplesofevolutionininvivoinvitromodels,withdeepersequencingprovidingbothhighersensitivityforsmallersubclonesaswellasmorepreciseestimatesoftheirsize.TheabilitytouseWESasanalternativeapproachtoWGS,aswellastheprojecteddowntrendofsequencingcosts,willenablemulti-samplingintimeandspaceofhematologicalmalignancies,clarifyingthenatureofspatialheterogeneityinbloodmalignanciesaswellasquestionsregardingthenaturerepopulationoftheecologicalnicheuponrelapse.Itmayalsoallowthestudyofthesequestionsinlargeclinicaltrials.Theseeffortscanpotentiallyanswerquestionsoffundamentalimportancetotheclinicalapplicationoftheseinsights,namelywhatistheprognosticsigni“canceofthesizeofthesubclonethatharborsageneticmarker,whenshouldwetargetbranchortrunklesionsandhowtointegratethisknowledgeincombinatorialtherapies.CONFLICTOFINTERESTTheauthorsdeclarenocon”ictofinterest. Clonalevolutionandtherapeuticstrategies DALandauetalLeukemia(2014)34…432014MacmillanPublishersLimited 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