Glioblastoma adaptation traced through decline of an IDH1 ...€¦ · 11Centre for Evolution and Cancer, The Institute of Cancer Research, London, United Kingdom 12Children's Hospital

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Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome

Favero, Francesco; McGranahan, Nicholas; Salm, Maximilian P.; Birkbak, Nicolai Juul; Sanborn, J.Zachary ; Benz, Stephen C. ; Becq, Jennifer; Peden, John F. ; Kingsbury, Zoya ; Grocok, Russell J.Total number of authors:11

Published in:Annals of Oncology

Link to article, DOI:10.1093/annonc/mdv127

Publication date:2015

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Favero, F., McGranahan, N., Salm, M. P., Birkbak, N. J., Sanborn, J. Z., Benz, S. C., Becq, J., Peden, J. F.,Kingsbury, Z., Grocok, R. J., & Eklund, A. C. (2015). Glioblastoma adaptation traced through decline of an IDH1clonal driver and macro-evolution of a double-minute chromosome. Annals of Oncology, 26(5), 880-887.https://doi.org/10.1093/annonc/mdv127

https://doi.org/10.1093/annonc/mdv127

https://orbit.dtu.dk/en/publications/7a8e169e-625c-4fca-b499-923c7f8dd6b4

https://doi.org/10.1093/annonc/mdv127

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©TheAuthor2015.PublishedbyOxfordUniversityPressonbehalfoftheEuropeanSocietyforMedicalOncology.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon‐CommercialLicense(http://creativecommons.org/licenses/by‐nc/4.0/),whichpermitsnon‐commercialre‐use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre‐use,[email protected]

GlioblastomaadaptationtracedthroughdeclineofanIDH1clonaldriver

andmacroevolutionofadoubleminutechromosome

FrancescoFavero1,2,*,NicholasMcGranahan1,3,*,MaximilianSalm1,*,Nicolai

J.Birkbak1,4,*,J.ZacharySanborn5,StephenC.Benz5,JenniferBecq6,JohnF.

Peden6,ZoyaKingsbury6,RussellJ.Grocok6,SeanHumphray6,David

Bentley6,BradleySpencer‐Dene1,AliceGutteridge4,MichaelBrada7,8,Stupp

Roger9,Pierre‐YvesDietrich10,TimForshew4,MarcoGerlinger1,11,Andrew

Rowan1,GordonStamp1,AronC.Eklund2,ZoltanSzallasi2,12,13,Charles

Swanton1,4

1CancerResearchUKLondonResearchInstitute,London,UnitedKingdom

2CenterforBiologicalSequenceAnalysis,DepartmentofSystemsBiology,

TechnicalUniversityofDenmark,Lyngby,Denmark

3CentreforMathematics&PhysicsintheLifeSciences&ExperimentalBiology

(CoMPLEX),UniversityCollegeLondon,London,UnitedKingdom

4UniversityCollegeLondonCancerInstitute,London,UnitedKingdom

5NantOmics,LLC,SantaCruz,CA,USA

6IlluminaLtd,Cambridge,UnitedKingdom

7DepartmentofMolecularandClinicalCancerMedicine,UniversityofLiverpool,

Liverpool,UnitedKingdom

8DepartmentofRadiationOncology,ClatterbridgeCancerCentreNHS

FoundationTrust,Bebington,UnitedKingdom

9DepartmentofOncology,UniversityHospitalZurich,Zürich,Switzerland

10CentreofOncology,UniversityHospitalsofGeneva,Switzerland

11CentreforEvolutionandCancer,TheInstituteofCancerResearch,London,

UnitedKingdom

12Children'sHospitalInformaticsProgramattheHarvard‐MITDivisionofHealth

SciencesandTechnology(CHIP@HST),HarvardMedicalSchool,Boston,MA,USA

13MTA‐SENAP,BrainMetastasisResearchGroup,HungarianAcademyof

Sciences,2ndDepartmentofPathology,SemmelweisUniversity,Budapest1091

Annals of Oncology Advance Access published March 2, 2015 by guest on M

arch 11, 2015http://annonc.oxfordjournals.org/

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Correspondenceto:Prof.CharlesSwanton,CancerResearchUKLondon

ResearchInstitute,44Lincoln'sInnFields,London,WC2A3LY,UnitedKingdom.

Phone:(+44)2072693463;Fax:(+44)2072693094;

[email protected]

*ContributedequallyAbstract:

Background

Glioblastoma(GBM)isthemostcommonmalignantbraincanceroccurringin

adults,andisassociatedwithdismaloutcomeandfewtherapeuticoptions.GBM

hasbeenshowntopredominantlydisruptthreecorepathwaysthroughsomatic

aberrations,renderingitidealforprecisionmedicineapproaches.

Methods

Wedescribea35year‐oldfemalepatientwithrecurrentGBMfollowingsurgical

removaloftheprimarytumor,adjuvanttreatmentwithtemozolomide,anda3‐

yeardisease‐freeperiod.Rapidwholegenomesequencing(WGS)ofthree

separatetumourregionsatrecurrencewasperformedandinterpretedrelative

toWGSoftworegionsoftheprimarytumour.

Results

Wefoundextensivemutationalandcopynumberheterogeneitywithinthe

primarytumour.WeidentifiedaTP53mutationandtwofocalamplifications

involvingPDGFRA,KITandCDK4,onchromosomes4and12.AclonalIDH1

R132Hmutationintheprimary,aknownGBMdriverevent,wasdetectableat

onlyverylowfrequencyintherecurrenttumour.Aftersubclonaldiversification,

evidencewasfoundforawholegenome‐doublingeventandatranslocation

betweentheamplifiedregionsofPDGFRA,KITandCDK4,encodedwithina

doubleminutechromosomealsoincorporatingmiR26a‐2.TheWGSanalysis

uncoveredprogressiveevolutionofthedoubleminutechromosomeconverging

ontheKIT/PDGFRA/PI3K/mTORaxis,supersedingtheIDH1mutationin

dominanceinamutuallyexclusivemanneratrecurrence,consequentlythe

patientwastreatedwithimatinib.Despiterapidsequencingandcancer‐genome

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guidedtherapyagainstamplifiedoncogenes,thediseaseprogressed,andthe

patientdiedshortlyafter.

Conclusions

ThiscaseshedslightonthedynamicevolutionofaGBMtumor,definingthe

originsofthelethalsubclone,themacroevolutionarygenomiceventsdominating

thediseaseatrecurrenceandthelossofaclonaldriver.Evenintheeraofrapid

WGSanalysis,casessuchasthisillustratethesignificanthurdlesforprecision

medicinesuccess.

Keywords:

Glioblastoma,multiregionsequencing,intratumourheterogeneity,double

minute

Key Message: "In a glioblastoma tumour with multiregion sequencing before and

after recurrence, we find an IDH1 mutation that is clonal in the primary but lost at

recurrence. We also describe the evolution of a double minute chromosome

encoding regulators of the PI3K signaling axis that dominates at recurrence,

highlighting the challenges of an evolving and dynamic oncogenic landscape for

precision medicine."



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Introduction

Glioblastoma(GBM)isthemostcommonmalignantbraincanceroccurringin

adultsandisassociatedwithpoorprognosisandamedianoverallsurvivalof

only15months[1].NearlyallGBMtumoursrecuraftersurgery,radiotherapy

andchemotherapy,withamediantimetorecurrenceof7months[1].

Accumulatingevidencesuggeststhattreatmentfailureincancermaybedriven

byintratumourheterogeneity(ITH)andbranchedtumourevolutioninvolving

geneticallydistinctsubclones[2].Recentstudieshavedocumentedwidespread

ITHinGBM.Sottorivaetal[3]foundthateachindividualtumourcanharbour

multipledistinctcopy‐numberprofilesandtranscriptomicsubtypes

simultaneously.Johnsonetal[2]revealedspatialandtemporalheterogeneityin

GBM,confirmedtheimportanceofTP53andIDH1asearlydrivermutations[4,

5],anddemonstratedtheimpactoftemozolomide(TMZ)treatmentontumour

evolution,with6of10tumoursshowingevidenceofTMZ‐induced

hypermutationatrecurrence.

InordertofullyassessITHwithinthelife‐historyofasingletumourandattempt

toofferthepatientacancergenome‐guidedtherapy,weimplementedrapid

multi‐regionwhole‐genomesequencing(WGS)inapatientwithrecurrentGBM.

ThisanalysisrevealsthetemporalandspatialevolutionofaGBMtumour,

definingtheoriginsofthelethalsubclonefromasubcloneintheprimarytumour

andtheassociatedmacroevolutionarygenomiceventsdominatingthediseaseat

recurrence,confoundingtreatmentsuccess.

Methods

Ethics

WritteninformedconsentwasobtainedfromthepatientintheHospitaux

UniversitairesdeGeneve“Analysedelareponseimmunologiquecontreles

tumeurscerebrales”translationalapprovedprotocolIRB03‐126.Tumour

materialwasanalysedundertheUCL‐CancerInstituteandPathologybiobank

(UCLHRTB10/H1306/42).Thepatientprovidedwritteninformedconsentto

tumoursequencinganalysiswithinacompassionatesetting.Thestudywas



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conductedaccordingtotheprovisionsoftheDeclarationofHelsinkiandthe

GoodClinicalPracticeGuidelinesoftheInternationalConferenceon

Harmonization.

WGSdataprocessingandanalysis

WGSwasperformedbyIllumina,UK.Mutationcallingandfilteringwas

performedusingVarScan2asdescribed[6],annotationofcodingmutationswere

performedusingANNOVAR[7].Structuralvariant(SV)breakpointmechanism

classificationwasperformedaccordingtothecriteriadefinedinYangetal[8].

Reconstructionoftheputativedoubleminutechromosomeswasperformedas

describedinSanbornetal[9]andbreakpointsmappingtothefocal

amplificationswerevalidatedbyPCRandSangerSequencing.Copynumber

variation(CNV)analysiswasperformedontheWGSdata.Purity,ploidyand

allele‐specificcopynumberestimateswereobtainedwithSequenza[10].Clonal

analysiswasperformedasdescribedinBollietal[11],estimatingthecancer

cellfraction(CCF)byintegratingvariantallelefrequencyestimateswithcopy

number,purityandploidyestimates.Singlesampleandmulti‐sampleDirichlet

processclusteringwasperformedusingtheDPpackageRpackage[12].Inthis

work,mutationsarereferredtoas“subclonal”iftheirCCFindicatestheyare

presentinonlyasubsetofcancercellswithinagivensample(CCF<1).

Mutationspresentinallcancercellsofagivensample(CCF=1)arereferredto

as“clonal”.Genomedoublingwasdeterminedfromthecomparisonofthe

sequencingofthegradeIIandthegradeIVregionsandbyconsideringthe

mutationslocatedintheportionofthegenomewhereacleardoublingofthe

numberofalleleswasdetected,seeSupplementaryInformationfordetails.All

dataanalysiswasperformedinRversion3.0.2,allp‐valuesaretwo‐sided.

Results

Clinicalcasereport

A35‐year‐oldfemalepresentedwithpartialcomplexseizuresinJanuary2007,

increasinginfrequencyafterthedeliveryofhersecondchildinSeptember2007

(Figure1A).MRIcarriedoutinJune2008,revealedarighttemporalmass

(6x6.7x4.5cm)withslightcontrastenhancement.Sheunderwentcomplete



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removalofthetumouronJuly3rd,2008andthediagnosisofWHOgradeIV

astrocytoma(GBM)wasestablished(thetumourconsistedofagradeIIand

gradeIVhistologicalcomponents).Shereceivedconcomitanttreatmentwith

irradiation(60Gyin30fractions)andTMZ,followedby6monthlycyclesofTMZ

(200mg/m2D1‐D5)untilMarch2009.Shewasfreeofsymptomsfortwoyears

butpartialseizuresreappearedinearly2011.AfurtherMRIshowedamultifocal

recurrenceintherighttemporalareaextendingtothethalamusandthecorpus

callosum.Consideringthelongdiseasefreeinterval(3years)betweenfirst

treatmentandrecurrence,TMZatasimilardoseandschedulewasprescribed

again.

InMarch2012,shepresentedwithacuteheadacheandintracranial

hypertension.MRIshowedmassiveprogressionmainlyintherightfrontalarea

withriskofherniation.SheunderwentpartialremovalofthetumouronMarch

29th,2012.ThehistologyconfirmedgradeIVastrocytomawithMGMTgene

promotermethylation.ShereceivedbevacizumabandTMZ;aftertransient

clinicalimprovement,herclinicalconditiondeterioratedandTMZwasreplaced

by800mgimatinibdaily,guidedbytheWGSdata,sequencedandreported

within7daysbyIllumina,indicatingamplificationofKITandPDGFRA.The

tumourprogressedrapidlyontherapyandshedied3monthsafterthe2nd

surgicaldebulkingprocedure.

Wholegenomesequencing

Archivalformalinfixedparaffinembedded(FFPE)specimensofthegradeIIand

IVprimarysamplesalongwiththreefresh‐frozensamplesfromtherecurrence

andagermlinereferencewereWGStoadepthof30X(~1.3x106pairedreads

persample;TableS1inSupplementaryInformation).Thethreerecurrence

regionswerehomogeneousattheSNVlevelindicatinglimitedclonalvariationin

therecurrence,howevertheSNVcallingwashamperedbystromal

contamination(TableS1).The3recurrentregions,referredtoasA1,A2andA3,

respectively,weremergedinsilicoinasinglealignmentfile,termedAs,to

increasetheresolutionandimprovethecapacitytodefinetheevolutionary

trajectoryoftherecurrencespecimen.1271and1935high‐confidencesomatic



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silentandnon‐silentSNVswereidentifiedinthegradeIIandIVregions

respectively,and1435intherecurrencespecimen.WhencomparingthegradeII

andgradeIVtotherecurrence,thegradeIVshared338mutationswiththe

recurrencenotfoundinthegradeIIregion,whilethegradeIIonlyshared1

mutationwiththerecurrencenotfoundinthegradeIVregion(Figure1B,coding

mutationsonlyinFigureS1,detailedmutationinformationinSupplementary

File).GiventhatthegradeIIregionexhibitedfewerprivatemutations(69/1271,

Figure1B,1C),thisindicatesitmostcloselyresemblesthemostrecentcommon

ancestor(MRCA),andthattherecurrencespecimenevolvedfromthegradeIV

region.

ExtensivemutationalvariationfoundbetweengradeIIandgradeIV

regions

Severalclonalmutationswerefoundinbothprimarylesionssuggestingashared

clonalorigin.TheseincludeaTP53Y220Cmutation,aframeshiftmutationin

ATRX(K1871fs),andanIDH1mutation,R132H.Thesegeneshavepreviously

beendescribedasdrivereventsforGBM[4,13],andATRXmutationshasbeen

showntoco‐occurwithTP53andIDH1mutations[2],andtobeadriverof

alternativetelomerelengthening[14].ClusteringthemutationCCFsinthegrade

IIgradeIVregionsrevealedsixdistinctclusters(Figure1D).Mostmutations

wereidentifiedasclonalinbothprimarylesionsorasclonalinonebutmissing

fromtheother(clusters1,3and6).However,wealsofound202mutationsthat

wereclonalinthegradeIVbutsubclonalinthegradeII(cluster2),andtwo

clustersofmutations(4and5)thatweresubclonalinthegradeIVandabsentin

thegradeII.Thecluster2mutationslikelyrepresentapersistentsubclone

withinthegradeIIregionthatgaverisetothegradeIVregion,whileclusters4

and5mayhavearisenindependentlyinthegradeIV,consistentwithfurther

subclonalevolutionoccuringduringdiseaseprogression.

Primarytumourshowsheterogeneousacquisitionofcopynumberchanges

CNVandSVanalysesidentifiedanumberofsharedeventsintheprimaryregions

(SupplementaryTableS2),includingcopy‐neutrallossofheterozygosity(LOH)

onchromosome17pfollowingTP53mutation(FigureS2A‐B);CDK6andMET



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amplificationviagainof7q;andtwohighlevelfocalamplifications(FigureS3)of

4q12(encodingPDGFRAandKIT)and12q13.3‐q14.1(encodingCDK4,AVILand

miR‐26a‐2).

ITHwasalsodetectedbyCNVandSVanalyses(FigureS4):CDKN2A/Blossand

otherCNVs(gainof6p,19pand20p;lossof10q,12q,13,16q,17qand22)were

detectedonlyinthegradeIVsample.Furthermore,the4q12and12q13.3‐14.1

focalamplificationswerelinkedbynumeroustranslocationsinthegradeIVbut

notinthegradeIIsample(FigureS5,FigureS6).

Allele‐specificCNVanalysisrevealedthatthegradeIVregionwaspredominantly

tetraploid,whilethegradeIIregionwaslargelydiploid.Mutationsinthegrade

IVregionalsoexhibitedabimodalvariantallelefrequencydistribution

consistentwithagenome‐doubling(GD)event,exclusivelyinthegradeIVregion

(FigureS7).Aspreviouslyreported[15],GDispermissiveforchromosome

instability(CIN)andacceleratedcancergenomeevolution.ConsistentwithaGD

eventinthegradeIVregionfollowedbychromosomelossesduetoincreasing

CIN[15],flowcytometryonthefreshtissueofthethreerecurrencesamples,

revealedaDNA‐indexof1.60,1.58and1.55foreachrecurrenceregion(Figure

S8).

Chromosome4rearrangementsandevolutionofadoubleminute

chromosome

Copy‐numberanalysisoftherecurrencetumourrevealedfocalamplificationsin

4q12and12q13.3‐q14.1detectedinthegradeIVregion,withcomparablyhigh

copynumberandseeminglyidenticalbreakpoints(FigureS2C‐D,FigureS6).

AlthoughthegradeIIsamplealsosharedthe12q13.3‐q14.1amplification,the

entire4q‐armwasamplifiedinthissample.Structuralvariantanalysisrevealed

complexchromosomalre‐arrangementslinkingthe4q12and12q13.3‐q14.1

focalamplificationsinthehigher‐gradesamplesonly.Takentogether,these

featuresarereminiscentofadoubleminutechromosome,arelativelyfrequent

cytogeneticeventinGBM[9,16].



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Toinvestigatethisfurther,weemployedapreciseampliconreconstruction

method[9].Thisinvolvedtheidentificationofbreakpointsrelatedtothehighly

amplifiedregions,followedbytheconstructionofabreakpointgraphthatlinks

theamplifiedsegmentsandtheirassociatedbreakpoints,andafinalsearchfor

anoptimalpaththatcompletelytraversesthegraph(FigureS9).Inthefinal

solution,segmenttraversalnumbercorrelateswithobservedrelativecopy‐

numberofthesegment,andcircularpathsareindicativeofadoubleminute

chromosome.

Consistentwithadoubleminutechromosome,the4q12and12q13.3‐q14.1

amplificationsrevealedcircularpaths,indicatingdoubleminutechromosomesin

thegradeIVandrecurrencesamples(Figure2A‐B&S9A).Twochromosomal

intervals(Figure2A)repletewithputativedrivergenes(PDGFRA,KIT,CDK4,

AVILandmiR‐26a‐2)arere‐configuredintocircularassemblies.Figure2B

illustratestheoptimalpathsthataccountfortheobservedbreakpointsandhigh

copy‐numberamplifications.TheabsenceofthisstructureinthegradeIIsample

suggeststhatthe12q13.3‐q14.1focalamplificationprecededDMformation,

consistentwithabreakage‐bridgefusioncycle[17].However,L1elementsflank

the12q13.3‐q14.1amplification(datanotshown),precludingfurtherlocalSV

resolution.Therearenumerouspreciselysharedbreakpointsbetweenthe

doubleminutemodels,andallbreakpointstestedvalidated(Figure2C&S10),

whichsuggestsacommonoriginoftheextra‐chromosomalstructures.

Moreover,thebreakpointsexhibitfeaturesofnon‐homologousend‐joining[8]

whichmaybeindicativeofasinglechromothripticevent[18].

ToinvestigatetheoriginoftheDMs,weperformedhaplotypeanalysisinthe

gradeIVandrecurrenceDMs.Thiswasachievedusingallelefrequenciesof

heterozygousSNPslocatedintheDMlocus(SupplementaryInformation).

ConsistentwithasharedoriginoftheDMs,thealleliccompositionoftheDM

haplotypesappearstobeidenticalatthe4q12and12q13.3‐q14.1loci(Figure

S11).Moreover,theDMderivedfrombothhighercopy‐numberhaplotypes;

conversely,thehighercopy‐numberhaplotypeson4q12werepredominantly

lostinthesegmentsflankingtheDM.Suchapatternisconsistentwithshattering



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ofa“gained”chromosome4qfollowedby“rescue”ofoncogenicfragments

withintheDMandlossoftheremainderofthechromosome.

Double‐minutechromosomeisassociatedwithprogression

Wecomputedadistancematrixfromallthemutationsdetectedintheprimary

regionsandinthethreerecurrenceregions,establishingaphylogenetic

relationshipbetweenthesequencedregions.Thisconfirmedthattherecurrence

specimenwasmostsimilartothegradeIVregion,withtheDMlikelyarising

betweenthegradeIIandgradeIVregions(Figure3A).TheDMcarriesthe

putativeGBMdrivergeneAVIL[19],withamutationrestrictedtothegrade

IV/recurrencelineageandlinkedtothefocalamplificationviadiscordantpaired‐

endreadsaswellasexhibitingahighvariantallelefrequency(chr12:58204830;

FigureS2F).Additionally,theDMunifiesmultipleoncogeniccomponentsofthe

PI3Kpathway:PDGFRA,KIT,andaregulatorofPTEN(miR‐26a‐2)aswellas

CDK4(Figure2B).ToassessifthegradeIVtumourshowsincreasedactivationof

thePI3Kpathway,weperformedimmunohistochemistryagainstPDGFRA,PTEN

andcKITinthegradeIIandgradeIVtumours(Figure3B).Wefoundincreased

levelsofPDGFRA(215/300versus93/300,gradeIVversusgradeII)andcKIT

(222/300versus31/300)inthegradeIVtumour,butnodifferenceinthePTEN

levels(34/300versus23/300).ThissuggeststhatPTENisdeactivatedinboth

thegradeIIandgradeIVtumours,butthatthePI3Kpathwayisfurtheractivated

inthegradeIVtumour,likelyduetoamplificationofgenesencodedwithinthe

doubleminutechromosome.

IDH1drivermutationislostatrecurrence

TrunkeventsincludingtheTP53andATRXmutationsandPDGFRA/KIT,

CDK4/miR‐26a‐2focalamplificationsalongwiththegradeIVprivatemutation

detectedinAVILwereidentifiedathighfrequencyintherecurrencesamples.

Surprisingly,theIDH1R132Hmutationwasnotdetectedintherecurrence

samplesdespitebeingclonalinthegradeIIandIVlesions.Tovalidatethis

observation,weperformeddigitalPCR(dPCR)onIDH1andTP53(usingTP53as

acontrol).WeconfirmedthattheR132Hmutationwasindeedclonalinthe

gradeIIandgradeIVspecimens(foundinapproximately42‐44%ofDNA

molecules,SupplementaryFile),butessentiallyundetectedintherecurrence



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samples,withbetween0.01%and0.1%ofDNAmoleculesshowingthemutation

bydPCR.Conversely,TP53wasfoundinbetween8%and12%oftheDNA

molecules(SupplementaryFile),consistentwithclonalpresenceinthelow‐

purityrecurrencebiopsies(estimatedat10‐15%purity,Supplementary

Information).Itislikelythereforethattherecurrencehasexperiencedlossofthe

mutatedIDH1alleleandretentionofthewild‐typeallele.

ArecentreportindicatesmutualexclusivitybetweenactivationofthePI3K

pathwayandIDH1activity[20].Astherecurrencedemonstratedincreasedlevels

ofPDGFRAandKITamplificationencodedontheDM,activatorsofthePI3K

pathway,weaddressedwhethertheGBMdatafromTheCancerGenomeAtlas

(TCGA)[16]supportsmutualexclusivitybetweenIDH1R132Hmutationand

PDGFRAand/orKITamplification.IDH1andTP53mutationsareenrichedinthe

proneuralsubtype[21].UsingthecBIOportal[22],weidentified137TCGAGBM

casesclassifiedasproneuralinBrennanetal[16],withbothsequenceandcopy

numberdata.Ofthese,12showedtheR132Hmutation,25showeddualKITand

PDGFRAamplification,and10showedPDGFRAamplificationonly.Nooverlap

betweencaseswiththeIDH1R132HmutationandKITand/orPDGFRA

amplificationwasdetected,indicatingmutualexclusivitybetweenthese

oncogenicevents(P=0.036,Fisher’sexacttest).ThisalsosuggeststhattheIDH1

R132Hmutationwasindeedanearlydrivereventthatwassubsequentlylost

duringrecurrence,asamorepotentpolyoncogene‐oncomirclusterwasselected

forintheDM.

AnanalysisofTCGAGBMdatafrom264tumourswithwholeexomesequencing

processedbySanbornetal[9]toinferDMstructures,identifiedfoursamples,of

thesethreeproneural,withamplificationsofbothPDGFRAandCDK4,possibly

encodedindoubleminutechromosomes.Thesedatasuggestthatatleast1.5%

(4/264)ofGBMtumoursoverall,and8%(22/264)oftheproneuralsubtype,are

drivenbytheacquisitionofsuchamacro‐evolutionaryeventtypifiedby

PDGRA/CDK4/miR‐26a‐2DMs.



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Discussion

Thisstudyisthefirstreportofamulti‐regionlongitudinalWGSofaglioblastoma

fromdiagnosistodeath,performedspecificallywiththeintentiontoimprove

patientoutcomebytheapplicationoftailoredtherapy.Unfortunately,despite

identifyingmultipleamplifiedtargetableoncogenesandapplyingtargeted

therapy,diseasecontrolwasnotachieved,andthepatientdiedfollowingdisease

progression.

Toourknowledge,thisisthefirstdescriptionofalossofatier1clonaldriver

event(IDH1,R132H)duringdiseaseprogression,andmayreflectcomplex

epistaticrelationshipsbetweentumoursomaticeventsandtheselection

pressureoftherapy.Whileitisformallypossiblethattherecurrenceevolved

fromasubclonelackingtheIDH1mutation,thiswouldrequireextensiveparallel

evolutionsincethemajorityofsomaticaberrationsweresharedbetweenrelapse

andthegradeIVregion.Rather,theprogressiveenrichmentoftheDMsfromthe

gradeIVtorecurrencesuggestsincreasedoncogenicpotentialbasedonthePI3K

pathway.WiththewaningofthedriverIDH1eventthisindicatesamacro‐

evolutionaryswitchfromthedominanceoftheIDH1‐mutatedtumourtoaDM‐

driventumourinamutuallyexclusivecontext.AnanalysisoftheTCGAdataalso

revealednooverlapbetweenIDH1R132HmutationsandPDGFRA/KIT

amplification,suggestingthathighlevelPDGFRA/KITamplificationwouldnotbe

favourablewithanexistentIDH1R132Hmutation.

Thesefindingshaveimportantimplicationsforprecisionmedicine,suggestinga

targetableclonaldrivereventcanbeselectivelylostduringthediseasecourse,

andreplacedinitsentiretybyaninitiallylowfrequencyeventintheprimary

tumour.TheclonaldominanceofIDH1drivereventsmightneedtobe

consideredwithinthecontextoflowfrequencyoncogenicdriverswhen

examiningtheefficacyoftherapeuticstargetingIDH1inthisdisease[23].

Furthermore,despiterapidWGSatrecurrenceandcancergenome‐directed

therapy,imatinibwasunabletocontrolthedisease.Followingradiotherapyand

twosurgicaldebulkingprocedures,itisunlikelythattheblood‐brainbarrierwas

intact,preventingdrugpenetrationintothecentralnervoussystem.Itismore



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likelythattreatmentfailurewasaconsequenceoftheevolutionofthepoly‐

oncogene/oncomirDMtargetingthePI3Kaxisatmultiplenodalpoints.

Moreover,ourresultshighlighttheprofoundeffectsofcancercellgenome

doubling,resultinginacceleratedcancergenomeevolution,characterizedbya

toleranceofCINandpropagationofaneuploidprogeny[15].TheacceleratedCIN

permittedfollowingthegenomedoublingeventinthegradeIVregionofthe

primarytumour,andpossiblyachromothripsiseventonchromosome4,

resultedintheformationandsubsequentselectionofahighlypolyoncogene‐

oncomirDMencodingmiR‐26a‐2,PDGFRA,KITandCDK4.

MicroRNAmiR‐26a‐2effectivelytargetsPTEN[24].Immunohistochemistry

demonstratedthatPTENexpressionwasweakorabsentrelativetostromalcells

inboththegradeIIandgradeIVregions,althoughnogenomicaberrationswere

detectedatthePTENlocus.Itislikelytherefore,thatamplificationofCDK4/miR‐

26a‐2region,eitherencodedwithintheDMinthegradeIVregionandrecurrent

tumourorsimplyduetoamplificationasobservedinthegradeIIspecimen,

directlycontributedtolossofPTENproteinexpression.

Takentogether,theseobservationsemphasisethecomplexityofsignal

transductioncascadesactivatedwithinindividualtumours.However,itis

apparentthattheoncogenicdriversinvolvedinGBMpathogenesisarehighly

constrainedandthecombinationoftheseeventsinvolvedinDMsmaybefinite.

Thereisanunmetneedtoenrollpatientswithinlongitudinalcohortstudiesto

definethesegeneticconstraintsinordertoaccelerateourunderstandingofGBM

evolutionthroughoutthediseasecourseandoptimisetherapeuticopportunities

inthisdisease.

Acknowledgments:

ResultspublishedherearepartiallybasedupondatageneratedbytheCancer

GenomeAtlaspilotprojectestablishedbytheNCIandNHGRI.Informationabout

TCGAandtheinvestigatorsandinstitutionswhoconstitutetheTCGAresearch



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networkcanbefoundathttp://cancergenome.nih.gov/.Thedatawereretrieved

throughdbGaPauthorization(AccessionNo.phs000178.v8.p7).

Funding:

ThisworkwassupportedbytheEuropeanCommission7thFramework

Programme[HEALTH‐2010‐F2‐259303];Z.SwasfundedbyTheBreastCancer

ResearchFoundation,theHungarianAcademyofSciences(KTIA_NAP_13‐2014‐

0021).

Disclosure:JB,JFP,ZK,RJG,SHandDBareemployeesofIlluminaInc,apubliccompanythatdevelopsandmarketssystemsforgeneticanalysis.Theremainingauthorsdeclarenocompetingfinancialinterests.Reference:

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FIGURESLEGENDS

Figure1:Timelineandclonalstructure.

Timelineofthepatient’sdiseasefromdiagnosistodeath(A)timingforthe

temozolomide(TMZ),bevacizumab(BEV)andimatinibtreatments.Timelineis

notdrawntoscaleintermsoflengthoftime.

AEuler‐Venndiagram(B)displayingtheoverlapsofnon‐silentandsilent

mutationsinthejointrecurrencecohortandthegradeIIandgradeIVsamples.

Amutationspectrumofnon‐synonymousmutationsisillustratedasanheatmap

ofthedetectedmutationsinthe2primarytumoursectorsandinthejoint3

recurrencesamples(C).Squarescolouredinyellowrepresentmutations

detectedinsub‐clonalpopulationsinthespecificsectorwhileredsquares

representthepresenceofthemutationintheclonalpopulationoftherespective

sector.

(D)Two‐dimensionalclusteringofmutationsinthegradeIIandgradeIV

specimens.Theaxiscorrespondthecancercellfraction(CCF),describingthe

fractionoftumourcellscarryingthemutation.Theordinatecorrespondsto

gradeIVspecimenandtheabscissacorrespondstogradeIIspecimen.Clusters

presentontheupperrightoftheplotcorrespondtoclonalmutationspresentin

bothspecimen;clusterslocatedintheupperleftrepresentclonalmutations

uniqueofthegradeIVandthebottomrightcorrespondtoclonalmutations

uniqueofthegradeIIspecimen.

Thenumbersclosetoeachclusterrepresentthenumberofnon‐silentand

mutationspresentintherespectivecluster,genesymbolsrepresentnon‐

synonymousmutationpresentinthecluster.



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Figure2:Evolutionofthedoubleminute

(A)TheupperpanelrepresentsthegenomicsegmentspriortotheDM

formation,withgenesannotatedbyhorizontallines.Thelowerpanelcontains

circularchromosomeplotsrepresentingthedouble‐minutemodelsforthegrade

IV(G4)andrecurrencesamples(A1/A3),withvalidated(andshared)

breakpointsdenotedbyredlinksbetweensegments.Lightgreylinksrepresent

un‐validatedbreakpointsforwhichdenovocontigscouldbeassembled.

ValidatedbreakpointsareillustratedinpanelsBandC

Figure3EvolutionaGBMtumortorecurrence

(A)Phylogenetictreedescribingtheevolutionofthetumour.Thelengthofthe

branchesiscalculatedusingthemutationrateasdescribedinthemethod

section.TherecurrencespecimensarecharacterisedbylossoftheIDH1

mutationandbythefurtherevolutionofthedoubleminute.Bluedotrepresents

branchingofGradeIIandGradeIVspeciments,orangedotrepresentsthe

genome‐doublingevent,reddotrepresentsbranchingofrecurrencetumour

fromtheGradeIVspecimen.Blackdotsrepresenttumoursampling.ForIDH1,

mutantallelefrequencydetectedbydPCRisindicatedinparenthesis(B)

ImmunohistochemistryshowingincreasedexpressionofcKITandPDGFRAinthe

gradeIVcomponentoftheprimarytumourrelativetogradeII.PTENishighly

expressedintheproliferatingvesselsofboththegradeIIandIVprimarytumour

sectorsbuttheneoplasticastrocytesarelargelynegative.



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Documents

Glioblastoma adaptation traced through decline of an IDH1 ...€¦ · 11Centre for Evolution and Cancer, The Institute of Cancer Research, London, United Kingdom 12Children's Hospital