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ORIGINAL ARTICLE
Interest of next-generation sequencing in BCG-treated high-risk bladder cancer
Intérêt du séquenc ¸age de nouvelle génération dans le cancer de la vessie à haut risque traité par BCG
C. Jungels
a, N. Martinez Chanza
b, S. Albisinni
a, M. Mercier
c, N. d’Haene
c, S. Rorive
c,
T. Roumeguère
a,∗aDepartmentofurology,Erasmehospital,universitéLibre-de-Bruxelles,Brussels,Belgium
bDepartmentofoncology,Erasmehospital,universitéLibre-de-Bruxelles,Brussels,Belgium
cDepartmentofpathology,Erasmehospital,universitéLibre-de-Bruxelles,Brussels,Belgium
Received30December2017;accepted19March2018 Availableonline17April2018
KEYWORDS Nextgeneration sequencing;
Prognostic;
NMIBC;
Highrisk;
BCG
Summary
Objectives.—Thereareonlyfewpredictivefactorsforresponseofnon-musculo-invasiveblad- dercancer(NMIBC)toBacillusCalmette-Guérin(BCG)therapy.Ourstudyanalyzedtheresultsof thesequencingofnewgeneration(NGS)targetedon50genesofoncologicalinterestobtained onbladderresectionpartsinhigh-riskNMIBCpatientstreatedwithBCG,todescribethispopu- lationfromamolecularpointofviewandtrytocorrelatetheseresultsinpatientswhopresent ornotrecurrenceafterBCG.
Methods.—Wereviewed63patients withhighgradeNMIBCtreatedbetween2014and2016 withBCGafter endoscopicresection.EachonehadNGSanalysis.Associationtestsbetween mutationsdetectedbyNGSandrecurrenceorprogressionwererealized.
Results.—The 45 remainingpatients were fully analysed. For 73%of casesa mutationhas beenfound,mostfrequentone’sbeingFGFR3,TP53andPIK3CA.Withamedianfollow-upof 24months(4—40),recurrencewaspresentin15patients(33.3%),with10NMIBC(22.2%)and 5progressionstomuscular-invasivecancer(11.1%).Ifsomemutationsweremorefrequentin differentprognosticgroupsnosignificantassociationhasbeenfound.NopatientpresentingCIS hadFGFR3mutation(P<0.0001).
∗Correspondingauthorat:UniversityclinicsofBrussels,urologydepartment,Erasmehospital-ULB,routedeLennik,808,1070Brussels, Belgium.
E-mailaddress:Thierry.roumeguere@erasme.ulb.ac.be(T.Roumeguère).
https://doi.org/10.1016/j.purol.2018.03.008
1166-7087/©2018ElsevierMassonSAS.Allrightsreserved.
Conclusion.—NextgenerationsequencinginNMIBCcouldbeasupplementaryaidintreatment decisionmakinginthefuture. Inanareawherepersonalizedmedicineisrapidlygrowingin importancewe needlargerstudies todefinemolecularcharacteristicsintumours todetect genomicassociationsbetweenclinicalphenotypesandrecurrenceorprogressionofthedisease.
Levelofevidence.—3.
©2018ElsevierMassonSAS.Allrightsreserved.
MOTSCLÉS Séquenc¸agede nouvellegénération; Pronostic;
TVNIM; Hautrisque; BCG
Résumé
Introduction.—Ilexistepeudefacteursprédictifspourlaréponsedescancersdevessiesnon- musculo-invasifs(TVNIM)auBacilledeCalmetteetGuérin(BCG). Notreétudeaanalyséles résultatsduséquenc¸agedenouvellegénération(NGS)ciblant50gènesd’intérêtoncologique obtenussurpiècesderésectiondevessiepourdesTVNIMàhautrisquetraitéesparBCGafin decorréleruneanalysemoléculairepourchaquepatientaveclerisquederécidivetumorale.
Matérielsetméthodes.—Entre 2014 et 2016, nous avons revu les caractéristiques de 63 patientstraitésparBCGpourTVNIMdehautgrade.UneanalyseNGSaétéréaliséepourchaque patient.Destestsd’associationsontétéréalisésentrelesdifférentesmutationsdétectéeset lerisquederécidiveetprogression.
Résultats.—Autotal,45patientsétaientdisponiblespourl’analysefinale.Dans73%descas unemutationaététrouvée.LesplusfréquentesétaientFGFR3,TP53etPIK3CA.Avecunsuivi médiande24mois(4—40),unerécidiveaététrouvéechez15patients(33,3%),avec10TVNIM (22,2%)et5progressionsversuneinvasionmusculaire(11,1%).Sidesmutationsétaientplus fréquentesdanscertainsgroupespronostiquesaucuneassociationstatistiquementsignificative n’aétéretrouvée.AucuncasprésentantduCISn’avaitdemutationdeFGFR3(p<0,0001).
Conclusion.—LeNGSdanslesTVNIMpourraitapporteruneaidesupplémentairedanslaprisede décisionthérapeutique.Desétudespluslargessontnécessairespourdéfinirlescaractéristiques moléculairesdestumeursafindedétecterlesassociationsgénomiquesentrelesphénotypes cliniquesetlarécidiveoulaprogressiondelamaladie.
Niveaudepreuve.— 3.
©2018ElsevierMassonSAS.Tousdroitsr´eserv´es.
Introduction
Bladder cancer is the 9th most diagnosed cancer in the world and even takes the 7th place in men [1]. At diag- nosis,almost80%of urothelialcarcinomasarestadifiedas non-muscular-invasive.Intotal,30%ofnon-muscleinvasive bladder cancers (NMIBC) are high-risk tumours. European guidelinesdefinehigh-risk urothelialcarcinomaasT1infil- tration stage,high grade (Grade3), withthe presenceof carcinoma insitu (CIS), or for lowergrades andstages as concomitantpresenceoflarge(>3cm),recurrentandmulti- pletumours[2].High-risktumoursassociatehighrecurrence (70% at 5 years) with mortality rates (10% at 5 years) [3,4]. First line conservative treatment consists of endo- scopicresectionwithadjuvantimmunotherapy(intravesical instillationswithBacillus Calmette-Guérin(BCG)or direct cystectomy.
These NMIBC represent a heterogeneous group with widevariationsinprognosisandnaturalhistoryevaluation.
Nonetheless, patient care has not been much evaluated latelyandwehavegotonlyfewprognosticfactorstoadapt treatmentandsurveillancetoourpatients.
Severalfactors(numberandtimetorecurrence,multi- plicityandtumoursize,presenceofCIS)havebeenproposed
topredictrecurrenceandprogressionofthesetumoursusing risk calculators like the one presented by the club uro- logicoEspagnol detratamiento-oncologico (CUETO)or the Europeanorganisationforresearchandtreatmentofcancer (EORTC)[5,6].
Extensive catalogues of geneticaberrations in cancers havebeengeneratedbyhighthroughputtechnologiessuch asnext-generationsequencing(NGS).
NGSisaDNAsequencingtechnologyforcancergenomes research.NGSplatformsperformsequencingofmillionsof smallfragmentsofDNAinparallel.Bioinformaticsanalyses areusedtopiecetogetherthesefragmentsbymappingthe individualreadstothehumanreferencegenome.
Itmayprovideamoreprecisediagnosisandclassification ofthediseaseswithamoreaccurateprognosis.Therefore, usingNGS,mainlyaimingtoidentifymutationsintumours, couldbethebasisofpersonalisedcancermanagement.
Ourstudyanalyzedtheresultsofthesequencingofnew generation(NGS)targetedon50genesofoncologicalinter- estobtainedonbladder resectionpartsinhigh-risk NMIBC patientstreatedwithBCG.Thepurposewastodescribethis populationfromamolecularpointofviewandtotrytocor- relatetheseresultsinpatientswithorwithoutrecurrence afterBCG.
Methods
Patient identification
Patient medicalfiles with high-risk NMIBC [2], which had BCGinstillationsbetweenJanuary2014andDecember2016 inErasme-ULBhospital,havebeenretrospectivelyreviewed after ethics committee approbation (n◦P2017/355). BCG instillationsscheme included6 inductioninstillations with control cystoscopies and biopsies and afterwards every 3 months, 1 weekly instillation for 3 weeks to complete a minimumof18instillationsinoneyear.
Clinical and histologicalcharacteristics have been ret- rospectively reviewed: age, gender, tumour grade and stage, multiplicity, tumour size, number of BCG instilla- tions,recurrencerate(definedbyrecurrenceconfirmedon anatomopathological analysis after endoscopic resection) andprogressionrate(definedasanatomopathologicalcon- firmedmuscular-invasive(MI)tumourinfollow-up).
Anatomopathological analysis
Anatomopathologicalanalysishasbeencarriedoutbasedon 1973and2004WHOandInternationalurologicpathological societyclassifications,andthesampleshavebeenreviewed bytwoseniorpathologists.
NGS analysis
Onerepresentativeblockofthetumourhasbeenselected by the pathologist: a tumour zone surrounded by a haematoxylin-eosin (HE) section and the percentage of tumourcellsestimatedinthiszone.10-mparaffinsections havebeen realizedandthetumourzonehasbeen macro- dissectedtoextractDNAwithaKitQiAampDNAFFPETissue kit(Qiagen)followingthemanufacturer’sinstructions.
TennanogramsofDNAhavebeenusedforsequencingby NGS.ThetechniqueofsequencingbyNGShasbeenrealized accordingtothemanufacturer’sinstructionsintheanatomy- pathologylaboratoryofErasme-ULBhospitalonaIontorrent platform(LifeTechnologies)withthepanel‘‘IonAmpliSeqTM Cancer Hotspot Panel’’ permitting sequencing in several regionsof interest (n=207) of 50 genesoften mutated in cancers:ABL1,AKT1,ALK,APC,ATM,BRAF,CDH1,CDKN2A, CSF1R,CTNNB1,EGFR,ERBB2,ERBB4,EZH2,FBXW7,FGFR1, FGFR2,FGFR3,FLT3, GNA11,GNAQ,GNAS, HNF1A,HRAS, IDH1,IDH2,JAK2,JAK3,KDR,KIT,KRAS,MET,MLH1,MPL, NMP1, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1,RET,SMAD4,SMARCB1,SMO,SRC,STK11,TP53,VHL.
This technique is carried out in 4 steps: preparation of libraries by ‘‘Multiplex polymerase chain reaction’’, then one step of emulsion PCR permitting clonal ampli- fication of DNA fragments of interest purified which are then loadedandsequenced by an Ion torrentchip. Inter- pretationoftheresultsconsistsindetectingmutationsthat are manually checked in order to keep only the exonic andnon-silentoneswhicharereportedinCOSMICdatabase (http://cancer.sanger.ac.uk/cosmic).
Statistics
Statistic tests fromPearson khi carré association for val- ues without evaluation in time and Kaplan-Meier method afterlog-rankverificationinthedifferentprognosticgroups have been used. We used STATA 12.0 statistical soft- ware. Value for statistical significance was defined for P<0.05.
Results
Atotalof63patientswithhigh-riskNMIBCandadjuvantBCG treatment have been identified. Five patients have been excludedforupperurinarytractinvolvement(whichpoten- tially presents different molecular pathways), 2 patients for incomplete data,3 lostin follow-up andtwo patients died froma concurrent pathology. NGS was successful in 45 outof the51 remainingpatients (88%). 3out ofthe 6 unsuccessful NGS presented CIS without papillarytumour.
These45remainingpatientsformthebasisofouranalysis population.
Medianage was 72 years (ranging from39 to 85) and median follow-up was24 months (ranging from 4 to 40).
Men/womenratiowas4/1.82.2%oftumourswereT1high grades withor withoutassociated CIS,andat the timeof analysis97.8%ofthepatientshavehadatleastBCGinduc- tion instillations. Table 1 shows the patients and tumour characteristics.
In33cases(73%ofpatients),amutationhasbeenfound.
FGFR3, TP53 and PIK3CA were the most frequent. In 12 patients, more than onemutation wasfound, 3 had dou- bleFGFR3/TP53mutationand3haddoubleFGFR3/PIK3CA mutation.Table2showsthefrequenciesofthevariousmuta- tionsfound.
Recurrencehasoccurredin15patients(33.3%).Median time torecurrence was11 months (4—37) and 5 patients out of 15 (33.3%) were high-risk NMIBC. Ten patients had NMIBC recurrence (22.2%) and 5 progressed to muscular- invasive(MI) tumours(11.1%).A totalof10 patientswere treated by cystectomy (5 NMIBC, 5 MIBC). 5 recurrences occurred after BCG induction instillations and a total of 9 within the first year after endoscopic resection. Fig. 1 shows the distribution of the most frequent mutations detectedbyNGSindifferentgroups:patientswithoutrecur- rence,withrecurrence,withprogression, withrecurrence after BCG induction and recurrence within the first year after resection respectively. One patient with a mutated FGFR3 has progressed and presented nowmetastatic dis- ease after cystectomy. Nine cases of PIK3CA mutation have been noted,3 of them at the same time as FGFR3 mutation.
In16patientspresenting alsoCIS,44% (n=7) hadTP53 mutationcomparedto24%(n=7/29)intumourswithoutCIS.
NopatientswithFGFR3orPIK3CAmutationspresentedCIS concurrently.
No statistically significant association was found between molecular alteration for FGFR3 (P=0.094) and TP53 (P=0.139) and the risk of recurrence after BCG (Figs.2and3).
Table1 Patientsandtumourcharacteristics.
Medianage 72years (39—85years)
Gender 36men(80%) 9women(20%)
Medianfollow-up 24months (4—40months)
Previousurothelialcarcinoma Patients
Yes 12 27%
No 33 73%
Stade+grade
TaLG: 1(hist.pT1HG) 2.2%
TaHG: 4 8.9%
T1HG: 24 53.3%
T1LG+CIS: 1 2.2%
T1HG+CIS: 12 26.7%
CIS: 3 6.7%
NumberofBCGinstillations (5—30)
<6 1 2.2%
6 10 22.2%
7—12 12 26.7%
>12 22 48.9%
Numberofrecurrence 15 33.3%
Numberofprogression 5 11.1%
HG:highgrade;LG:lowgrade;CIS:carcinomainsitu.
Table2 NGScharacteristics.
Patients %
0mutationdetected 12 26.7
FGFR3 15 33.3
TP53 14 31.1
PI3KCA 9 20.0
RAS(HRAS,NRAS,KRAS) 4 8.9
CTNNB1 2 4.4
BRAF 1 2.2
NOTCH 1 2.2
CDKN2A 1 2.2
FBXW7 1 2.2
KIT 1 2.2
MET 1 2.2
≥2mutations 12 26.7
FGFR3+TP53 3 6.7
FGFR3+PI3KCA 3 6.7
Discussion
PatientcareinhighriskNMIBCisamajorchallengeregarding limitationstoclassifyprognosticrisksstillgenerallybased onhistopathologicaldatasubjecttointerpretationvariabil- ity[3,5—9]andalackof biomarkerscapableofpredicting responsetoBCGtreatment[6,10].
WefoundhighfrequenciesofTP53andFGFR3mutations similartotheresultsofthecancergenomeatlas(TCGA).Asa reminder,TCGAprojectprovidebroadinsightintotheunder- lying genetic aberrations existing across multiple cancer
Figure1. Distributionof mostfrequentmutations indifferent groups.
typesandthepotentialfordeterminingtheclinicalsignif- icance of critical genetic aberrations.Mutation of FGFR3 genewasthemostfrequentfoundinthisstudy(33%)andwas morecommoninpatientswithoutrecurrenceorprogression butwithoutreachingstatisticalsignificance,incomparison to recurring or progressing patients. Literature describes FGFR3mutations in mostly50% of bladder cancers,being morefrequent in low grade and stagetumours [9,11—17]
withfavourableprogressionrisk [11,13,16,18].Implication ofFGFR3inrecurrenceriskisstillcontroversialwithdiver- gentresultsinliterature[16,18—21].Onlyonestudyshowed betterspecificsurvivalinNMIBCpresentingFGFR3mutation [14]. The study of van Rhijn and al. [9] reached statisti- callybetterprognosticvaluescombiningmolecularfactors (FGFR3 andMIB-1) withEORTCscore, whichis one ofthe
Figure2. Kapan-Meiersurvivalcurveinmonthswithout recur- renceinfunctionofFGFR3status(0=wildtype;1=mutation).
Figure3. Kapan-Meiersurvivalcurveinmonthswithout recur- renceinfunctionofTP53status(0=wildtype;1=mutation).
recommendedstandardfeatures[4].DeregulationofFGFR3 bymutation,over-expression or both at thesame timeis frequentinbladdercancer,namelyin81%ofNMIBCand54%
ofMIBC[13].Thisexplainstheinterestindevelopingtreat- mentstargeting FGFR3 by differentpossible mechanisms:
Anti-FGFRtreatmentsliketyrosinekinaseinhibitorsoranti- bodiesblocking FGFR3 [12]. Several trials with anti-FGFR agentsaloneorincombinationtherapiesinNMIBCandMIBC areinprogress.
Beside FGFR3, alterations of TP53 are frequently described in urothelial tumours. These are more often linked to high grade and stage tumours [12,15,22,23], withworseprognosticoutcomesandhigherprogressionrisk [12,15,23—25]. TP53 alterations are often present at an early stage in tumours presenting CIS [12,26,27]. Predic- tive character of TP53 in response to BCG treatment or chemotherapyisstillcontroversial[22].Recenttrialsstudy thepossibilitytoreintroduceTP53activityintumourswith the mutation to promote apoptosis in cancer cells and
diminish abnormalcellproliferation, botheffects of TP53 [22].TP53mutationwasthe2ndmostfoundinthepresent study with14/45 cases (31%). Even if the frequency was higher inearly recurrent patients (afterBCGinduction or within12months),nostatisticallinkhasbeenreached.
FGFR3 andTP53 mutations arerarely associatedwith- outbeingexclusive[12,15].Theabsenceofthisexclusivity is confirmed in this study with three patients presenting bothmutations.HighgradeNMIBCpresentaheterogeneous groupwhere mutationalpathways maycross.Theassocia- tionofFGFR3mutationtoTP53seemstohaveaprotective effect [16]. None of the patients presenting FGFR3/TP53 hadoccurred recurrenceor progressionatthe momentof analysis.
PIK3CAmutationsarefrequentlylinkedtoFGFR3muta- tionsandhavebeendescribedmoreofteninlowgradeand stage tumours, evenif this association is less established [17,27].SpecificPIK3CAinhibitorsoccurclinicalevaluation [28].
MutationsinRASgenes(HRAS,KRAS,NRAS)arepresent in13%ofbladdercancerswithouthavingclearassociationto specificgrade,stageorestablishedprognosticvalue[17,27].
ThesemutationsaremutuallyexclusivetoFGFR3mutations [17,29],afactwehavefoundoutinthecurrentstudywith none of the 4 RASmutations found in presence of FGFR3 mutation.Thissuggestsdifferentpathways[29].
SeveralgenemutationsfoundinourstudylikeCTNNB1, CDKN2A, BRAF, NOTCH, FBXW7, MET or KIT have been described in urothelial carcinoma with unknown clinical impact.
Carcinoma in situ is a less explored disease withfew available prognostic factors. In our study, NGS has had results inonly3 outof 6caseswithsolelyCIS.A possible explanationisthatcollectingenoughDNAmaterialfromthe tumoursitecanbedifficultinthesecases.Ifmutationsof TP53arefrequentlyseenintumourswithCIS,FGFR3muta- tions are rare in these tumours [16,27]. In our study, no mutationofFGFR3wasseenwhenCISwaspresent.
ArecentstudyfailedtoconfirmTP53orFGFR3asprog- nosticfactorsforrecurrenceafterBCGinstillationsbutwas able to statistically bind ARID1A, a chromatin-modifying gene torecurrence[30]. This mutation wasnot tested in thecurrentstudybutispoorlydescribedintheliteratureso far.
ThisstudyisoneofthefirsttofocusonNGSinthespe- cificgroupofpatientswithhigh-riskNMIBCtreatedbyBCG instillations.Ourstudyhaslimitationsduetoitsmonocentric characterwithasmallnumberof patients,ashortperiod of follow-up and haslimited power in detecting genomic significant associations between the mutations found and recurrenceorprogressionofthedisease.In73%ofthecases, atleastonemutationofthe50genessearchedinourpanel has been found which arouses our interest in performing molecularanalysisinthispopulationtofindprognosticand predictivebiomarkerswithpersonalizedtreatments,which maybedeveloped.
Conclusion
Nextgenerationsequencinginnon-muscularinvasiveblad- der cancer could be an additionalaid in our therapeutic
decision making in the near future. In an area where personalized medicine is rapidly growing, we need larger studiestodefinemolecularcharacteristicsintumours,and try to find predictive biomarkers and specific targeted treatments.
Disclosure of interest
Theauthorsdeclarethattheyhavenocompetinginterest.
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