Interest of next-generation sequencing in BCG-treated high-risk bladder cancer

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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

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Conclusion.—NextgenerationsequencinginNMIBCcouldbeasupplementaryaidintreatment decisionmakinginthefuture. Inanareawherepersonalizedmedicineisrapidlygrowingin importancewe needlargerstudies todeﬁnemolecularcharacteristicsintumours todetect genomicassociationsbetweenclinicalphenotypesandrecurrenceorprogressionofthedisease.

Levelofevidence.—3.

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.

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.

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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 instillations,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‘‘IonAmpliSeq^TM 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 disease 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).

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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

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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].TP53mutationwasthe2^ndmostfoundinthepresent 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,FGFR3mutations 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

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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.

References

[1]AntoniS,FerlayJ,SoerjomataramI,ZnaorA,JemalA,BrayF.

Bladdercancerincidenceandmortality:aglobaloverviewand recenttrends.EurUrol2017;71(1):96—108.

[2]Babjuk M, Böhle A, Burger M, Capoun O, Cohen D, Com- pérat EM, et al. EAU Guidelines on non-muscle-invasive urothelialcarcinoma of thebladder: update 2016. EurUrol 2017;71(3):447—61.

[3]ChamieK,LitwinMS,BassettJC,DaskivichTJ,LaiJ,Hanley JM,etal.Recurrenceofhigh-riskbladdercancer:apopulation- based analysis: burden of high-risk bladder cancer. Cancer 2013;119(17):3219—27.

[4]Sylvester RJ, van der Meijden AP, Oosterlinck W, Witjes JA, Boufﬁoux C, Denis L, et al. Predicting recurrence and progression in individual patients with stage Ta T1 blad- dercancerusing EORTC risktables: a combined analysisof 2596patientsfrom sevenEORTCtrials.EurUrol2006;49(3):

466—77.

[5]Fernandez-GomezJ,MaderoR,SolsonaE,UndaM,Martinez- Pi˜neiroL, Gonzalez M,et al. Predicting nonmuscleinvasive bladdercancerrecurrenceandprogressioninpatientstreated withBacillusCalmette-Guerin: the CUETOscoring model. J Urol2009;182(5):2195—203.

[6]CambierS, Sylvester RJ, Collette L, Gontero P, Brausi MA, vanAndel G, et al. EORTC nomograms and risk groups for predicting recurrence,progression, and disease-speciﬁcand overallsurvival in non-muscle-invasive stage Ta—T1 urothelial bladder cancer patients treated with 1—3 years of maintenanceBacillus Calmette-Guérin.EurUrol 2016;69(1):

60—9.

[7]MurphyWM,TakezawaK,MaruniakNA.Interobserverdiscrep- ancyusingthe1998WorldHealthOrganization/International Society of Urologic Pathology Classiﬁcation of urothelial neoplasms: practical choices for patient care. J Urol 2002;168(3):968—72.

[8]WitjesJA,MoonenPMJ,vanderHeijdenAG.Reviewpathol- ogyinadiagnosticbladdercancertrial:effectofpatientrisk category.Urology2006;67(4):751—5.

[9]van Rhijn BW, Zuiverloon TC, Vis AN, Radvanyi F, van LeendersGJ, Ooms BC, etal. Molecular grade(FGFR3/MIB- 1) and EORTC risk scores are predictive in primary non-muscle-invasive bladder cancer. Eur Urol 2010;58(3):

433—41.

[10] SaintF, Salomon L, QuintelaR, Cicco A, Hoznek A, Abbou CC, et al. Do prognostic parameters of remission versus relapseafterBacillusCalmette-Guérin(BCG)immunotherapy exist? Analysis of a quarter century of literature. Eur Urol 2003;43(4):351—60.

[11] di Martino E, Tomlinson DC, Knowles MA. A decadeof FGF receptorresearchinbladdercancer:past,present,andfuture challenges.AdvUrol2012;2012:1—10.

[12] NeuzilletY,PaolettiX,OuerhaniS,Mongiat-ArtusP,Soliman H,deTheH,etal.Meta-analysisoftherelationshipbetween

FGFR3 and TP53 mutations in bladder cancer. Koul H, édi- teur.PLoSOne2012;7(12):e48993,http://dx.doi.org/10.1371/

journal.pone.0048993.

[13]Tomlinson D, Baldo O, Harnden P, Knowles M. FGFR3 pro- tein expression and its relationship to mutation status and prognosticvariablesinbladdercancer.JPathol2007;213(1):

91—8.

[14]vanOersJM,ZwarthoffEC,RehmanI,AzzouziAR, Cussenot O,MeuthM,etal.FGFR3mutationsindicatebettersurvival ininvasiveupperurinarytractandbladdertumours.EurUrol 2009;55(3):650—8.

[15]vanRhijnBW,vanderKwast TH,Vis AN,Kirkels WJ,Boevé ER,JöbsisAC,etal.FGFR3andP53characterizealternative geneticpathwaysinthepathogenesisofurothelialcellcarci- noma.CancerRes2004;64(6):1911—4.

[16]vanRhijnBW,vanderKwastTH,VisAN,KirkelsWJ,BoevéER, JöbsisAC,etal.Moleculargradingofurothelialcellcarcinoma withﬁbroblastgrowthfactorreceptor3andMIB-1issuperior topathologicgradeforthepredictionofclinicaloutcome.J ClinOncol2003;21(10):1912—21.

[17]KompierLC,Lurkin I,vanderAa MNM,vanRhijnBWG,van der Kwast TH, Zwarthoff EC, et al. FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations mutations in bladder cancer and theirpotentialas biomarkersfor surveillance andther- apy.PLoSOne2010;5(11):e13821,http://dx.doi.org/10.1371/

journal.pone.0013821.

[18]Burger M, van der Aa MNM, van Oers JMM, et al. Predic- tionofprogressionofnon-muscle-invasivebladdercancerby WHO1973and2004gradingandbyFGFR3mutationstatus:a prospectivestudy.EurUrol2008;54(4):835—44.

[19]Hernández S, López-Knowles E, Lloreta J, Kogevinas M, Amorós A, Tardón A, et al. Prospective study of FGFR3 mutations as a prognostic factor in nonmuscle invasive urothelial bladder carcinomas. J Clin Oncol 2006;24(22):

3664—71.

[20]vanRhijnBW,LiuL,VisAN,BostromPJ,ZuiverloonTC,Flesh- nerNE,etal.Prognosticvalueofmolecularmarkers,sub-stage and European Organisation for the Researchand Treatment ofCancer riskscores inprimary T1 bladdercancer.BJU Int 2012;110(8):1169—76.

[21]Lamont FR, Tomlinson DC, CooperPA, Shnyder SD, Chester JD,KnowlesMA.SmallmoleculeFGFreceptorinhibitorsblock FGFR-dependenturothelialcarcinoma growthinvitroandin vivo.BrJCancer2011;104(1):75—82.

[22]Slaton JW, Benedict WF, Dinney CP.P53 in bladder cancer:

mechanismofaction,prognosticvalue,andtargetfortherapy.

Urology2001;57(5):852—9.

[23]Knowles MA. The genetics of transitional cell carcinoma: progress and potential clinical application. BJU Int 1999;84(4):412—27.

[24]vanRhijnBW,CattoJW,GoebellPJ,KnüchelR,ShariatSF,van derPoelHG,etal.Molecularmarkersforurothelialbladder cancerprognosis:towardimplementationinclinicalpractice.

UrolOncol2014;32(7):1078—87.

[25]Malats N, Bustos A, Nascimento CM, FernandezF, Rivas M, Puente D, et al. P53 as a prognostic marker for bladder cancer:ameta-analysisandreview.LancetOncol2005;6(9):

678—86.

[26]Spruck3rdCH,OhneseitPF,Gonzalez-ZuluetaM,EsrigD,Miyao N,etal.Twomolecularpathwaystotransitionalcellcarcinoma ofthebladder.CancerRes1994;54(3):784—8.

[27]GoebellPJ,KnowlesMA.Bladdercancerorbladdercancers?

Geneticallydistinct malignantconditions oftheurothelium.

UrolOncol2010;28(4):409—28.

[28]RossRL,McPhersonHR,KettlewellL, ShnyderSD, HurstCD, AlderO,etal.PIK3CAdependenceandsensitivitytotherapeu- tictargetinginurothelialcarcinoma.BMCCancer2016;16:553, http://dx.doi.org/10.1186/s12885-016-02570.

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[29]Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF, KnowlesMA.FGFR3andRasgenemutationsaremutuallyexclu- sive genetic events in urothelial cell carcinoma. Oncogene 2005;24(33):5218—25.

[30]Pietzak EJ, Bagrodia A, Cha EK, Drill EN, Iyer G, Isharwal S, et al. Next-generation sequencing of nonmuscleinvasive bladdercancerrevealspotentialbiomarkersandrationalther- apeutictargets.EurUrol2017;72(6):952—9.