Exploring genetic alterations in circulating tumor DNA from ...

Medulloblastoma (MB) is the most common type of brain malignancy in children. Molecular profiling has become an important component to ... Skiptomaincontent Thankyouforvisitingnature.com.YouareusingabrowserversionwithlimitedsupportforCSS.Toobtain thebestexperience,werecommendyouuseamoreuptodatebrowser(orturnoffcompatibilitymodein InternetExplorer).Inthemeantime,toensurecontinuedsupport,wearedisplayingthesitewithoutstyles andJavaScript. Advertisement nature scientificreports articles article ExploringgeneticalterationsincirculatingtumorDNAfromcerebrospinalfluidofpediatricmedulloblastoma DownloadPDF Subjects CancergenomicsCNScancerPaediatriccancer AbstractMedulloblastoma(MB)isthemostcommontypeofbrainmalignancyinchildren.Molecularprofilinghasbecomeanimportantcomponenttoselectpatientsfortherapeuticapproaches,allowingforpersonalizedtherapy.Inthisstudy,wesuccessfullyidentifieddetectablelevelsoftumor-derivedcell-freeDNA(cfDNA)incerebrospinalfluid(CSF)samplesofpatientswithMB.Furthermore,cfDNAfromCSFcaninterrogatefortumor-associatedmolecularclues.MB-associatedalterationsfromCSF,tumor,andpost-chemotherapyplasmawerecomparedbydeepsequencingonnext-generationsequencingplatform.SharedalterationsexistbetweenCSFandmatchedtumortissues.MorealternationsweredetectedincirculatingtumorDNAfromCSFthanthoseingenomicDNAfromprimarytumor.ItwasfeasibletodetectMB-associatedmutationsinplasmaofpatientstreatedwithchemotherapy.Collectively,CSFsupernatantcanbeusedtomonitorgenomicalterations,asasuperiortechniqueaslongastumor-derivedcfDNAcanbeisolatedfromCSFsuccessfully. DownloadPDF IntroductionMedulloblastoma(MB)isthemostcommonprimarymalignanttumorinchildren,accountingfor20%ofallpediatricintracranialtumors1.Currently,MBisrecognizedasanumbrellatermthatencompassesvariousmolecularpathologicalentities.Basedon2016WorldHealthOrganization(WHO)classificationofcentralnervoussystem(CNS)tumors,MBwasdividedintofoursubgroups,i.e.,WNT,SHH,Group3andGroup42.Aboutone-thirdofchildrenwithMBhavecerebrospinalfluid,cranialorspinalmetastasesatdiagnosis3.Patientswhosufferfromrecurrenceortumorprogression,evenwithadvancedtreatmentprotocols,havedismalprognosis.Nowadays,cancergenomelandscapeisessentialfordiagnosis,prognosticevaluationandtreatmentselection.Unfortunately,forpatientswithtumorprogressionorrecurrence,itisdifficulttodetectmolecularalterations,becauseofunobtainabletumortissueandlackofbiopsysample.Onlyadvancedimagingtechnologies,suchasMRI,CTandPET,areappliedtodiagnoseandmonitorprogressivediseaseforMB. Thenext-generationsequencing(NGS)hasbeenappliedtosequencecirculatingtumorDNA(ctDNA)fromblood,especiallyforidentifyingEGFRmutationsinnon-smallcelllungcancer.However,blood–brainbarrierimpedesdetectionofctDNAinbloodofapatientwithbraintumor4.Recently,accumulatingevidencehassupportedthatcirculatingcell-freenucleicacidsincludingDNA(cfDNA)andRNA(cfRNA),andevenproteinsincerebrospinalfluid(CSF)arepotentialtorevolutionizediagnosisandclinicalcareforCNStumors,especiallyforglioblastoma(GBM)andMB5.WeaimtoexplorethefeasibilityofliquidbiopsyusingCSFtofacilitatediagnosisandtopredictprognosisinMB.TodecipherpathogenesisofMBandapplytargetedtherapy,itisnecessarytoexploregeneticbackgroundindifferenttypesofsamples,suchastumortissueandCSF,basedonNGSplatform.Becauseinourpreviousclinicalgenetictestreports,fewMB-associatedalterationsweredetectedintumortissue.Therefore,deepsequencingonctDNAinCSFandformalin-fixedparaffin-embedded(FFPE)tissuewasperformedtoobtaincomprehensivegenomeprofiling.WehypothesizedthatgeneticalterationinCSFmayserveasacomplementaryroletotissueformonitoringdiseaseprogression.Circulatingcell-freeDNA(cfDNA)ispresentinbodyfluidofhealthyindividualsaswellaspatientswithcancer,suchasplasma,urineandCSF.CirculatingtumorDNA(ctDNA)isthoughttobeshedintocirculationbyapoptoticandnecrotictumorcellsinpatientswithcancer6.ThusdetectionofctDNAisofgreatvalueforearlydiagnosisofmalignancy.FordetectingctDNAintheblood,thecontentofctDNAisloweratearlierstage,comparedtorelativelylate-stage4,7.Additionally,thedifferenceoffragmentssizeinctDNAandcfDNAwasutilizedforearlydiagnosisofmalignancies8.Furthermore,high-grade(WHOgradesIIIandIV)braintumorsweremorelikelytoharbordetectablectDNAinCSFthanlow-gradeones9.Therefore,ctDNAdetectioninCSFplaysapredictiveroleinmonitoringdiseaseprogression.Here,wedeterminedthepresenceorabsenceofcfDNAin58CSFsamples.Inordertodeciphermedulloblastoma-associatedalterations,deepsequencingbasedonNGSplatformwasappliedtoexamineCSF,tumortissueandbloodinpatientswithMB.Notably,positivecfDNAinCSFisassociatedwithdiseaseprogressionormetastasis.Itwasmorelikelytodetectmedulloblastoma-associatedalterationsinCSFratherthantumortissue.SharedalterationsbetweenCSFandmatchedtumortissuescouldbedetectedwhensamplecollectedinshorttimeinterval.MethodsPatientsandsamplecollectionTheprotocolwasapprovedbytheinstitutionalreviewboardofResearchEthicsCommitteeatBeijingShijitanHospital.WritteninformedconsentwasobtainedfromtheirparentsofMBpatients.Allexperimentswerecarriedoutinaccordancewithrelevantguidelinesandregulations.Inthisprospectivestudy,58patientswithMBwererecruitedfromBeijingTiantanHospitalofCapitalMedicalUniversityandBeijingShijitanHospitalofCapitalMedicalUniversitybetweenApril2019andDec2019.Foreachpatient,CSFwascollectedbylumbarpunctureoratthetimeofsurgery.Additionally,tumortissuesampleswerecollectedfrom4patientsonprimary-careand7patientswithrelapse.Thematchedplasmawasisolatedfrom5patientswithrelapseandtreatedwithadjuvantradiotherapyorchemotherapy.Furthermore,wholebloodwasobtainedfromeachpatient,servingasgermlineDNAcontrol.IsolationofcfDNAfromCSFandplasmaThemedianvolumeofcollectedCSFis5 mL.FreshCSFwasstoredat4 °C,andcentrifugated(at4 °C,1400 rpm,for5 min)within2–3 haftercollection.Cellularpelletwasdiscarded.Thesterilecentrifugetubewaspre-cooledat − 20 °C.CSFsupernatantwastransferredtoacryotubesandstoredat − 80 °C.Immediatelybeforeuse,CSFwasthawedinathermostatwaterbathat37 °C.Then,cfDNAwasextractedfromCSFsupernatantorplasmaaccordingtothemanufacturer’sprotocolusingQIAampCirculatingNucleicAcidKit(catalog#55114;QIAGEN,Valencia,CA).Isolationoftumor/germlineDNAIsolationofgenomicDNAfromFFPEtumortissueormatchedwholebloodleukocyteswasperformedusingQIAampDNAFFPETissueKit(Qiagen,Hilden,Germany)andblood/cellcultureDNAkit(Qiagen,Valencia,CA,USA),respectively.Concentrationofisolated(cell-freeandgenomic)DNAwasquantifiedwithQubit2.0FluorometerusingQubitdsDNAHSAssaykit(LifeTechnologies,Carlsbad,California,US)accordingtothemanufacture’sprotocol.DNAwasstoredat − 20 °Cuntilitwasused.AnalysisofcfDNAandctDNAThepresenceandabsenceofcfDNAwasinferredaccordingtoisolatedDNAfragmentlengthdistribution,insteadofquantity.ToverifyisolatedcfDNA,thesizeofcfDNAfragmentwasassessedusingAgilent2100Bioanalyzer.Ifpeakappearedat150–200basepair(bp),itisconsideredascfDNA,otherwise(> 1500 bp)asCSFcellulargenomicDNA.AndNGSlibraryconstructionwasnotperformedagainstCSFcellulargenomicDNA.ThedetectionmethodcannotdeterminewhethercfDNAisderivedfromtumortissue.Therefore,weadoptedtheterm“cfDNA”.Theterm“ctDNA”wasusedonlywhentumor-associatedmutationsweredetectedinCSForplasma10.DNAlibraryconstructionandNGSsequencingFirst,tumorgenomicDNAwasfragmentedinto150–200 bpfragmentswithBioruptorPico.LibrariesoffragmentedDNAandcfDNAwereconstructedwithKAPALibraryAmplificationKit(KK2611/KK2612)accordingtostandardprotocols.Next,cfDNAlibrarieswerecapturedwithadesigned500-geneor952-genepanel(Agilent),whichcontainedthemajorityofbraintumorrelatedgenes.ThegenomicDNAlibrarieswereappliedwithwholeexomesequencing(WES).Thecapturedsamplesweresubjectedtopaired-endsequencingontheIlluminaNovaSeq6000platform.Finally,thelowqualityreadswereremovedbasedonbioinformatics;whilethehighqualityreadsweremappedonhumanreferencesequence(hg19)(HumanGenomeversion19)usingtoolBurrows–WheelerAlignersoftware(BWA)11.TheSNVsandIn/DelsweredetectedusingGenomeAnalysisToolkit(GATK)12andVarScan213andfilteredoutbydbSNPand1000Genomedatasets.Weselectedatleast3mutatedreadswithqualityscoresof > 30.Inthisstudy,wefocusedonmutationslocatedinexons,suchasnonsynonymous,synonymous,frameshift,non-frameshiftandsplicingsites.AllthegenestestedwereverifiedwithIntegrativeGenomicsViewer(IGV).StatisticalanalysisAssociationsbetweenCSF-cfDNAandclinicalcharacteristicsincludingageandquantityofDNAinCSFwereevaluatedbyMann–WhitneyUtests.Clinicalfeaturessuchasrecurrence,metastasis,tumorprogressionandquantityofDNAinCSFwerecomparedbetweenpositiveandnegativeCSF-cfDNAgroupswithFisher’sexacttestsorchi-squaretests.Allstatisticaltestsweretwo-sidedwithap ≤ 0.05definedasstatisticalsignificance.ResultsThebasiccharacteristicsofpatientsThepatientpopulation(64%maleand36%female)includedinfants,childrenorteenager,agedat2–15 yearsold(median:7).DemographicandclinicalfeaturesweresummarizedinTable1,includingage,gender,medialhistology,primarytumorlocation,initialdiagnosis(Mstage,molecularsubgroup),recurrence,progression,withorwithoutdetectableCSF-cfDNA,andquantityofDNAinCSF.Tumorswerelocatedinthe4thventricleinthemajorityofpatients(n = 48).7patientshadspinalspread,3hadspinalsolitarymetastasis,2hadcraniospinal(cerebrospinal;cerebellumandspinalaxis)spreadandonehadsubventricularspread.Othertumorswerelocatedinvermis(n = 5),cerebellarhemisphere(n = 4)orcerebellopontineangle(CPA)(n = 1).Totally,15patientsdevelopeddistantmetastasisorlocalspread.Diseaserecurrenceoccurredin7patients.Among58patients,15haddetectablecfDNAinCSF(asanalyzedbyAgilent2100Bioanalyzer).Eightpatientsdevelopedprogressivedisease(PD)ordiedofPD.Onepatientexhibitedpartialremission(PR)afterchemotherapyandsurgery.Therestofpatientsachievedstabledisease(SD)(n = 3)orcompleteremission(CR)(n = 46)aftersurgery,nevertheless,thepatient192D0463diedfromradiation(RT)complication.Table1Clinicalcharacteristicsof58patientswithmedulloblastoma.FullsizetableRelationshipbetweenCSF-cfDNAandclinicalcharacteristicsInourpatientswithMB,CSF-cfDNAwasnotassociatedwithage(p = 0.50,Mann–WhitneyUtest)orgender(p = 0.37,chi-squaretest).However,thepresenceorabsenceofcfDNAinCSFwasassociatedwithseveralclinicalfeatures,includingrecurrence(p = 0.0098,Fisher’sexacttest),metastasis(p = 0.001,Fisher’sexacttest),tumorprogression(p = 0.003,Fisher’sexacttest),aswellasquantityofDNAinCSF(p