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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
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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
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Correspondenceto:Prof.CharlesSwanton,CancerResearchUKLondon
ResearchInstitute,44Lincoln'sInnFields,London,WC2A3LY,UnitedKingdom.
Phone:(+44)2072693463;Fax:(+44)2072693094;
*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:
1. WenPY,KesariS.Malignantgliomasinadults.N.Engl.J.Med.2008;359(5):492–507.
2. JohnsonBE,MazorT,HongCetal.Mutationalanalysisrevealstheoriginandtherapy‐drivenevolutionofrecurrentglioma.Science2014;343(6167):189–93.
3. SottorivaA,SpiteriI,PiccirilloSGMetal.Intratumorheterogeneityinhumanglioblastomareflectscancerevolutionarydynamics.Proc.Natl.Acad.Sci.U.S.A.2013;110(10):4009–14.
4. WatanabeT,NobusawaS,KleihuesP,OhgakiH.IDH1mutationsareearlyeventsinthedevelopmentofastrocytomasandoligodendrogliomas.Am.J.Pathol.2009;174(4):1149–53.
5. TurcanS,RohleD,GoenkaAetal.IDH1mutationissufficienttoestablishthegliomahypermethylatorphenotype.Nature2012;483(7390):479–83.
6. DeBruinEC,McGranahanN,MitterRetal.Spatialandtemporaldiversityingenomicinstabilityprocessesdefineslungcancerevolution.Science(80‐.).2014;346(6206):251–256.
7. WangK,LiM,HakonarsonH.ANNOVAR:functionalannotationofgeneticvariantsfromhigh‐throughputsequencingdata.NucleicAcidsRes.2010;38(16):e164.
8. YangL,LuquetteLJ,GehlenborgNetal.Diversemechanismsofsomaticstructuralvariationsinhumancancergenomes.Cell2013;153(4):919–29.
by guest on March 11, 2015
http://annonc.oxfordjournals.org/D
ownloaded from
15
9. SanbornJZ,SalamaSR,GriffordMetal.Doubleminutechromosomesinglioblastomamultiformearerevealedbyprecisereconstructionofoncogenicamplicons.CancerRes.2013;73(19):6036–45.
10. FaveroF,JoshiT,MarquardAMetal.Sequenza:allele‐specificcopynumberandmutationprofilesfromtumorsequencingdata.Ann.Oncol.2014:mdu479–.
11. BolliN,Avet‐LoiseauH,WedgeDCetal.Heterogeneityofgenomicevolutionandmutationalprofilesinmultiplemyeloma.Nat.Commun.2014;5:2997.
12. JaraA,HansonTE,QuintanaFAetal.DPpackage:BayesianNon‐andSemi‐parametricModellinginR.J.Stat.Softw.2011;40(5):1–30.
13. LiuX‐Y,GergesN,KorshunovAetal.FrequentATRXmutationsandlossofexpressioninadultdiffuseastrocytictumorscarryingIDH1/IDH2andTP53mutations.ActaNeuropathol.2012;124(5):615–25.
14. LovejoyCA,LiW,ReisenweberSetal.LossofATRX,genomeinstability,andanalteredDNAdamageresponsearehallmarksofthealternativelengtheningoftelomerespathway.PLoSGenet.2012;8(7):e1002772.
15. DewhurstSM,McGranahanN,BurrellRAetal.Toleranceofwhole‐genomedoublingpropagateschromosomalinstabilityandacceleratescancergenomeevolution.CancerDiscov.2014;4(2):175–85.
16. BrennanCW,VerhaakRGW,McKennaAetal.Thesomaticgenomiclandscapeofglioblastoma.Cell2013;155(2):462–77.
17. BuntingSF,NussenzweigA.End‐joining,translocationsandcancer.Nat.Rev.Cancer2013;13(7):443–54.
18. FormentJV,KaidiA,JacksonSP.Chromothripsisandcancer:causesandconsequencesofchromosomeshattering.Nat.Rev.Cancer2012;12(10):663–70.
19. CrespoI,TãoH,NietoABetal.Amplifiedandhomozygouslydeletedgenesinglioblastoma:impactongeneexpressionlevels.PLoSOne2012;7(9):e46088.
20. CharitouP,Rodriguez‐colmanM,GerritsJetal.FOXOssupportthemetabolicrequirementsofnormalandtumorcellsbypromotingIDH1expression.EMBORep.2015;Epubahead(Feb3):1–11.
21. VerhaakRGW,HoadleyKA,PurdomEetal.IntegratedgenomicanalysisidentifiesclinicallyrelevantsubtypesofglioblastomacharacterizedbyabnormalitiesinPDGFRA,IDH1,EGFR,andNF1.CancerCell2010;17(1):98–110.
22. cBioPortalforCancerGenomics.[http://www.cbioportal.org/public‐portal/].
by guest on March 11, 2015
http://annonc.oxfordjournals.org/D
ownloaded from
16
23. YapTA,GerlingerM,FutrealPAetal.Intratumorheterogeneity:seeingthewoodforthetrees.Sci.Transl.Med.2012;4(127):127ps10.
24. LeeDH,AmanatS,GoffCetal.OverexpressionofmiR‐26a‐2inhumanliposarcomaiscorrelatedwithpoorpatientsurvival.Oncogenesis2013;2:e47.
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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