Low T-cell Receptor Diversity, High Somatic Mutation Burden, and High Neoantigen Load as Predictors of Clinical Outcome in Muscle-invasive Bladder Cancer

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Bladder

Cancer

Low

T-cell

Receptor

Diversity,

High

Somatic

Mutation

Burden,

and

High

Neoantigen

Load

as

Predictors

of

Clinical

Outcome

in

Muscle-invasive

Bladder

Cancer

Noura

J. Choudhury,

Kazuma

Kiyotani,

Kai

Lee

Yap,

Alexa

Campanile,

Tatjana

Antic,

Poh

Yin

Yew,

Gary

Steinberg,

Jae

Hyun

Park,

Yusuke

Nakamura

*

,

Peter

H. O’Donnell

*

UniversityofChicago,Chicago,IL,USA

a v ai l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n al h o m e p a g e : w w w . e u r o p e an u r o l o g y . c o m / e u f o c u s Articleinfo Articlehistory: AcceptedSeptember19,2015 AssociateEditor: JamesCatto Keywords: T-cellreceptor Neoantigens Bladdercancer

Immuneresponsestocancer Molecularprognosis

Abstract

Background: Thesuccessofcancerimmunotherapieshashighlightedthepotentability oflocaladaptiveimmuneresponsestoeradicatecancercellsbytargetingneoantigens generated by somatic alterations. However, how thesefactors interacttodrive the naturalhistoryofmuscle-invasivebladdercancer(MIBC)isnotwellunderstood.

Objective: ToinvestigatetheroleofimmuneregulationinMIBCdiseaseprogression,we performedmassivelyparallelT-cellreceptor(TCR)sequencingoftumor-inﬁltratingT cells (TILs), in silico neoantigen prediction from exome sequences, and expression analysisofimmune-relatedgenes.

Design, setting,and participants: We analyzed 38 MIBC tissues from patients who underwentdeﬁnitivesurgerywithaminimumclinicalfollow-upof2yr.

Outcomemeasurements andstatisticalanalysis: Recurrence-free survival(RFS)was determined.TCRdiversitywasquantiﬁedusingSimpson’sdiversityindex.Themain analysesinvolvedtheMann-WhitneyUtest,Kaplan-Meiersurvivalanalysis,andCox proportionalhazardsmodels.

Results and limitations: Low TCRb chain diversity, correlating with oligoclonal TIL expansion, was signiﬁcantly correlated withlonger RFS, even after adjustment for pathologic tumorstage,nodestatus, and receiptofadjuvant chemotherapy(hazard ratio 2.67, 95% conﬁdence interval 1.08–6.60; p=0.03). Patients with both a high numberofneoantigensandlowTCRbdiversityhadlongerRFScomparedtothosewith fewerneoantigensandhighTCRdiversity(medianRFS275vs30wk;p=0.03).Higher expression of immune cytolytic genes was associated with nonrecurrence among patientswithlowTCRdiversityorfewerneoantigens.Limitationsincludethesample sizeandtheinabilitytodistinguishCD8+_and_CD4+_T_cells_using_TCR_sequencing.

Conclusions: Thesefindingsarethefirsttoshowthatdetailedtumorimmune-genome analysis atdefinitive surgery canidentifymolecular patternsof antitumor immune responsecontributingtobetterclinicaloutcomesinMIBC.

Patientsummary: WediscoveredthatclonalexpansionofcertainTcellsintumortissue, possiblytargetingcancer-speciﬁcantigens,contributestopreventionofbladdercancer recurrence.

#2015EuropeanAssociationofUrology.PublishedbyElsevierB.V.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

* Correspondingauthors.UniversityofChicago,KCBD6130,900East57thStreet,

Chicago,IL60637,USA.

E-mail addresses:[email protected](Y. Nakamura),

[email protected](P.H. O’Donnell).

http://dx.doi.org/10.1016/j.euf.2015.09.007

2405-4569/# 2015 European Association of Urology. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://

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1. Introduction

Muscle-invasive bladder cancer (MIBC), unlike the more favorablenon–muscle-invasiveform(NMIBC), hasdismal prognosis anda highrecurrence rate. Extensive genomic profilingofurothelialcancersuggeststhatMIBCisdrivenby distinctgeneticdifferencesfromNMIBC[1–5].Inaddition, urothelialcarcinomahasahighsomaticmutationburden, third in frequency onlyafter lung cancer and melanoma

[2].Recentevidenceindicatesthatsomaticmutationsarethe basisforthegenerationofpotentialneoantigensrecognized byantitumorTlymphocytes[6,7],whichcanbeeffectively rescued from an exhausted state by immune checkpoint blockadetherapyincertainpatients[8].Tumor-infiltrating lymphocytes(TILs)carryprognosticsignificanceinurothelial cancer,withhighCD8expressionintumorsassociatedwith prolongeddisease-freeandoverallsurvival[9].Giventhese results,ourgoalistoimproveunderstandingoftheroleof immune regulation in MIBC disease progression. More specifically, we investigated how potential neoantigens derivedfromsomaticnonsynonymousmutationsinfluence thediversityofTILsinMIBCtoimpactrecurrencestatusand thelengthofrecurrence-freesurvival(RFS).

Next-generationsequencingofexpressedT-cellreceptor (TCR)transcriptsallowsforquantificationofT-celldiversity

[10,11] via calculation of Simpson’s diversity index (DI)

fromsequencingreads,withlowDIindicatingoligoclonal T-cellexpansion[12].Tocomprehensivelyaddress correla-tionsbetweenTCRdiversityandthemutationalneoantigen landscape of a tumor, we performed TCR sequencing, whole-exomesequencingwithneoepitopeprediction,and immune-related gene expression analysis for 38 chemo-therapy-naı¨ve MIBC tumors from patients with clinical follow-upofatleast2years.Tothebestofourknowledge, thisin-depthlevelofintegrationoftheimmuneandgenetic landscapewithclinicaloutcomeshasnotbeeninvestigated inMIBC.

2. Materialsandmethods

2.1. Studydesign

UnderaprotocolapprovedbytheUniversityofChicagoinstitutional

review board (#15550B), tumor tissue and available adjacent normal

frozen tissue were collected from chemotherapy-naı¨ve MIBC patients at

the time of deﬁnitive surgical resection of the primary tumor. Since the

great majority of MIBC patients who experience recurrence (denoted

Rec)willdosowithin2yr,wedeﬁnednonrecurrent(NR)patientsas

thosewhowereknowntoberecurrence-freeatleast2yrafterdeﬁnitive

surgery (median length of follow-up for NR patients 262 59.3 wk;

Supplementary Table 1). We collected all samples with at least 2 yr of

clinical follow-up from the tissue bank organized by one urologist (G.S.). Of

these, we included only tissues that yielded high-quality and sufﬁcient DNA

andRNAafterextraction,resultinginatotalof38cases.

2.2. TCRsequencing

cDNA libraries were prepared using previously described methods

(Supplementarymaterial)[11].TheBowtie2alignerwasusedtomap

sequencing reads to the human TCR loci and a previously described

algorithm was applied to decompose the V-(D)-J regions of the CDR3s

[11]. Simpson’s DI was calculated to quantify the clonality of the TCR a

and b repertoires[12]according to DI ¼ Pi¼1K

niði1Þ NðN1Þ

h i1

, where N is the

total number of sequences, niis the number of sequences belonging to

the ith clonotype, and K is the total number of clonotypes.

2.3. AssessmentofT-cellinfiltration

Tumor sections were stained with hematoxylin and eosin, and T-cell

inﬁltration into tumor and stroma was qualitatively assessed by an

attendinggenitourinarypathologist(T.A.)whowasblindedtotheTCRDI

scores.Alltumorsweregivenaholisticnumericscoreincomparisonto

other samples. After TCR sequencing, a subset of samples with high and

low TCR diversity were also subjected to immunohistochemistry (IHC) for

CD3, CD4, and CD8 to investigate the correlation between T cell number

and DI score. The IHC slides were scored (T.A.) in the same holistic manner.

2.4. Geneexpression

Gene expression analysis was performed on tumor cDNAs using TaqMan

gene expressionassays (ThermoFisherScientiﬁc,Carlsbad, CA, USA)

according to the manufacturer’s instructions. GAPDH (assay

Hs02758991_g1) was used as a housekeeping gene. Ampliﬁcation of

FOXP3 and CD4 within 40 cycles failed for two samples (A18 and A30), so

these were excluded from the analysis.

2.5. Whole-exomesequencing

Libraries were prepared and analyzed as previously described

(Supple-mentary material)[13], with peripheral blood serving as a normal control.

2.6. Bioinformaticsanalysis

2.6.1. Exomesequencing

Results have already been reported for 28 of the 36 whole-exome

sequencing samples[13]. Eight additional samples were sequenced and

analyzedinthesamemanner(Supplementarymaterial).

2.6.2. Neoantigenprediction

Neoantigenswerepredictedforeachnonsynonymousvariantbydeﬁning

all novel 8- to 11-mers resulting from the mutation and determining

whether the predicted binding afﬁnity to human leukocyte antigen (HLA)

class I alleles was <500 nM[14,15] using NetMHCpanv2.8 software

[16,17]. A total of 32 samples underwent neoantigen prediction. Four

samples wereexcludedowingtounavailabilityoftheHLAtype.Two

additionalsampleslackednormalcontrolsforwhole-exomesequencing.

2.7. Statisticalanalysis

ContinuousvariableswerecomparedusingtheMann-WhitneyUtest.

Survival analysis was conducted using both Kaplan-Meier log-rank

analysis and Cox proportional hazards models. Analysis was carried out

using GraphPad Prism 6 software (GraphPad, San Diego, CA, USA), and R

version 3.2.0 (R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. TCRsequencingconclusivelydistinguishesTILsfrom normal-tissueT-cellpopulations

For20patients,weperformedTCRsequencingonbothtumor and adjacent normal tissues. An attending genitourinary oncology pathologist (T.A.) verified that tumor tissue

EUROPEAN UROLOGYFOCUS 2(2016)445–452

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containedahighpercentageoftumornucleiandthatnormal tissueexcludedtumorcells.AnalysisofV-(D)-Jcombinations with complementarity-determining region 3 (CDR3) sequences demonstrated that unique TCR clonotypes in tumor tissues were absent or hardly detectable in the adjacent normal tissues (frequency <0.01; Fig. 1A and SupplementaryFig.1). WeconcludedthatthetumorTCR repertoires were significantly different from those of adjacentnormaltissues,andthenfocusedourTCRanalysis onlyontumortissuefortheremaining18patientsbecauseof ourinterestinTILdiversity.

TCRsequencingwasperformedonatotalof38tumors

(Table1, SupplementaryTable 1).To assess whether the

number of T cells influences DI quantification, we per-formedIHCforCD3,CD4,andCD8onasubsetofsamples (Fig.1BandSupplementaryFig.2).Apathologistblindedto thesampleDIsindependentlyandqualitativelyscoredthe degree of T-cell infiltration into tumor centers and the surroundingstroma.Weobservedthatevenpatientswith verylowDIvalues(DI<10)hadvaryingdegreesofT-cell infiltration into tumor centers and surrounding stroma, ensuringthattheselowDIscoreswerenotsimplydueto lowTILnumbers.Sinceitwasnotedthathigh-DIsamples (DI>50)also hadsimilar variationsinTILnumbers,DIis trulyarepresentationoftheclonalexpansionofTcellsand notsimplyareflectionofthetotalnumberofTcells.

3.2. TCRdiversityinprimarytumorsforeshadowsrecurrence risk

Notably,weobservedthattumorsfromNRpatientshada lower TCRb DI than tumors from Rec patients (mean standarddeviation[SD]36.131.8vs129.8159;p=0.08,

Fig.2A).Wepursuedthisfindingbyclassifyingtumorsinto binaryDIgroupsusingthemedianDIasacut-point. Kaplan-MeiersurvivalanalysisrevealedthatpatientswithlowTCRb DIhadsignificantlylongerRFSthanpatientswithhighTCRb DI(mediannotreached vs34.7wk;log-rank testp=0.018,

Fig.2BandSupplementaryFig.3).Therelationshipbetween RFS and TCRb DI remained significant after adjusting for pathologictumorstage,nodestatus,andreceiptofadjuvant chemotherapyinaCoxproportionalhazardsmodel(p=0.033, hazard ratio [HR] 2.67, 95% confidence interval [CI] 1.08– 6.60).Wealso stratifiedpatients accordingtotumorstage/ nodestatusandobservedapositive correlationbetweenDI and tumorstage/node status whenassessed without other covariates (analysis of variance p=0.028; Fig. 2C). These results indicate that primary tumors at a more advanced pathologic stage at the time of surgery already have less oligoclonalTcellexpansion.

WehypothesizedthatinRecpatientswithlowTCRbDI, immunosuppressive regulatory T (Treg) cells rather than cytolytic CD8+ _T _cells _may _be _expanding. _We _therefore

Fig.1–T-cellreceptor(TCR)sequencingprovidesgranulardataonTCRrepertoiresbeyondimmunohistochemistry(IHC).(A)Representativeheatmaps ofthetop15clonotypesidentifiedforaandbchainsfortwosamples,comparingtumor(T)andnormal(N)distributions.Whitespacesindicate particularclonotypesnotfoundinthecorrespondingtissue.TheSupplementarymaterialprovidesheatmapsfor18additionalsamples.Sampleswere sortedaccordingtothemostabundantclonotypesineachchainandcomparedtocorrespondingtumorornormaltissue.Uniqueclonotypesfoundin tumortissuewereabsentorhardlydetectable(frequency<0.1%)incorrespondingnormaltissueformostsamples.(B)RepresentativeIHCexamples forCD8inmuscle-invasivebladdercancertumorswithhighTCRdiversity.TumorA4(DI95.3)hasminimalCD8stainingintumorandstroma,while tumorA6(DI120.6)hasmoderatestainingintumorandminimalstaininginstroma.(C)CD8stainingoftwotumorswithlowTCRdiversity.Tumor A24(DI11.6)hasminimalstainingintumorandstroma,whiletumorA9(DI10.5)hasminimaltumorstainingandmoderatestromalstaining.TCRDI scoresdidnotcorrelatewithqualitativescoringofT-cellinfiltrationaccordingtoIHC(performedbyapathologistblindedtoDIscores).DI=Simpson’s diversityindex.

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classifiedourcasesintogroupswithhighorlowCD8/FOXP3 expressionusingmedianexpressioninalltumors(equalto 3.9)asthecut-point.AmongRecpatients,thosewithlower TCRDIscorestypicallyalsohadlowCD8/FOXP3expression, although the difference was not statistically significant (SupplementaryFig.4A).Moreinterestingly,weobserveda significantly positive correlation between CD8/FOXP3 ex-pressionandDIamongRecpatientsthatwasnotseeninNR patients (R2₌_0.57; _p_<_0.001, _{Supplementary} _Fig. _4B).

These two observations indicate that Rec patients with

low T-cell diversity may have dominant expansion of certainTregcellsand,similarly,RecpatientswithhighCD8/ FOXP3ratiosmaylackclonalexpansionofantitumorCD8+_T

cells,asevidencedbytheircomparativelyhigherTCRbDI values.

3.3. Predictedneoantigensimpactonclinicaloutcome

Whole-exome sequencing of 36 of the 38 MIBC tumors revealed a higher somatic non-synonymous mutation

Table 1 – Patient demographic data: detailed clinical variables associated with prognosis in the 38 patients with muscle-invasive bladder

cancerincludedinthestudy

Patient Rec Time to Gender Age at pT pN Histology Site of recurrence Adjuvant

ID FU or surgery therapy

Rec (wk) (yr)

A3 Y 6.1 F 72 pT4a pN0 Adenocarcinoma Pulmonarynodules,

gastricmasses

N

A4 Y 23.1 M 70 pT3b pN0 UC Iliacmasses N

A10 Y 30.0 M 65 pT3b pN1 UC Bone Y

A11 Y 36.9 F 74 pT3a pN0 UC Pulmonarynodules N

A14 Y 5.4 M 59 pT4a pN2 UC Liver,lungs,LN,bone Y

A15 Y 29.4 M 57 pT3a pN1 UCwithsarcomatoid

features

PelvicmassandLN Y

A18 Y 188.1 M 77 pT2a pN0 UC Pelvicmass,liver N

A20 Y 27.0 M 50 pT3b pN0 UC Pelvicmass,LN Y

A24 Y 48.7 M 45 pT4a pN1 UC RPLN Y

A25 Y 86.6 M 70 pT4a pN2 UCwithsmall-cellor

NEfeatures

PelvicLN,brain Y

A26 Y 68.4 F 78 pT3a pN1 UC Bone N

A27 Y 62.3 M 70 pT4a pN2 UC Bone,pelvicLN Y

A28 Y 39.3 M 68 pT4a pN2 UC PelvicmassandLN Y

A29 Y 9.7 F 80 pT4a pN2 UC Lungs,mediastinum,

hila,liver,RPLN

N

A30 Y 12.3 M 49 pT4a pN2 Adenocarcinoma Bowel N

A32 Y 22.4 M 76 pT3b pN2 SCC Pelvicmass,

periportalLN

N

A34 Y 138.1 M 62 pT3a pN2 UC RPLN Y

A36 Y 16.9 M 68 pT3a pN1 UC Unspeciﬁedsites Y

A37 Y 25.1 M 70 pT3b pN0 UC Pulmonarynodules N

A38 Y 3.6 M 59 pT4 pN0 SCC Pelvic,lower

abdominalLN N A1 N 360.6 M 78 pT3b pN0 SCC NA N A2 N 287.1 M 57 pT2b pN0 UCwithsquamous features NA N A5 N 261.7 M 67 pT2b pN0 UC NA N A6 N 262.6 F 68 pT3a pN0 UC NA N A7 N 347.7 M 74 pT4a pN0 UC NA N

A8 N 239.0 F 60 pT3a pN0 UCwithsquamous

features NA N A9 N 166.6 F 43 pT2b pN0 UCwithsquamous features NA N A12 N 265.4 M 59 pT2b pN0 UC NA N A13 N 234.1 M 55 pT3a pN0 UC NA N A16 N 220.4 M 84 pT3a pN0 UC NA N A17 N 71.9 M 68 pT2a pN0 UC NA N A19 N 289.7 F 74 pT2b pN2 UC NA Y A21 N 249.1 M 39 pT3a pN0 UC NA Y A22 N 330.9 M 57 pT2a pN0 UC NA N A23 N 209.6 M 56 pT3a pN0 UC NA N

A31 N 212.6 M 78 pT3a pN1 UCwithsquamous

features

NA N

A33 N 378.3 M 74 pT3a pN1 UC NA Y

A35 N 323.7 M 52 pT3b pN1 UC NA Y

REC=recurrence;FU=follow-up;M=male;F=female;pT=pathologicprimarytumorstage;pN=pathologicnodestatusasdeﬁnedbytheAmericanJoint CommitteeonCancer;UC=urothelialcarcinoma; NE=neuroendocrine;SCC=squamouscellcarcinoma; RP=retroperitoneal;LN=lymphnode;Y=yes; N=no;NA=notapplicable.

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burdeninNRcomparedto Rectumors,afindingthathas been validated in other tumor types (mean SD 217152 vs103.494; p=0.007, Supplementary Fig. 5). Weperformed insilico potentialneoantigen or neoepitope prediction from these nonsynonymous mutations [8,18,19]

for32ofour38MIBCtumors(SupplementaryTables2and3). The number of somatic nonsynonymous mutations had a tightly linear positive correlation with the number of predictedneoantigens(R2₌_0.89;_p_<_0.0001,_{Supplementary}

Fig.6).TumorsfromNRpatientshadahigheraveragenumber ofpredictedneoantigens(177.8vs103.9;p=0.032,Fig.3A). Toensureweselectedonlythoseneoantigensmostlikelytobe presented to T-cells, we applied two additional filtering criteria: (1) HLA-binding affinity of >500nM for the corresponding wild-type peptide; and (2) affinity at least three times greaterfor the mutant thanfor the wild-type peptide (larger numbers indicate weaker affinity). The relationship betweenfilteredneoantigen load andRFSwas not statistically significant (median RFS not reached vs 52.6 wk for high vs low neoantigen group; log-rank p=0.11,Fig.3B).Wenextcomparedtherelationshipbetween filteredneoantigenloadandDI,andobservedthattherewas norelationshipbetweenneoantigen loadandTCRdiversity (p=0.9)intheNRgroup,butapositivecorrelationintheRec group(p=0.07),withastatisticallysignificantdifferencein slope between the two cohorts (p=0.038; Fig. 3C). This indicates that NR tumors maintain low TCR diversity independentofthenumberofpredictedneoantigens,while inRectumorsTCRdiversitysimplyincreasesinthepresence ofmoreneoantigens.

Finally,wecomparedRFSusingstratificationaccording tobothneoantigenloadandTCRbdiversity.Patientswitha highneoantigenloadandlowTCRbdiversityhadlongerRFS comparedtothosewithlowneoantigenloadandhighTCR diversity(medianRFS275vs30wk;p=0.03, Supplemen-taryFig.7).

3.4. Immune-relatedgeneexpressionsignaturedifferentiates clinicaloutcome

WeperformedgeneexpressionassaysforCD4,CD8,FOXP3, indoleamine2,3-dioxygenase1(IDO1),granzymeA(GZMA), andperforin-1(PRF1).HighexpressionofCD8,GZMA,and PRF1 is associated with an immunostimulatory profile, whilehighexpressionofCD4,FOXP3,andIDO1isassociated with immunoregulation. For each of these factors, we calculatedtheNR/Recexpressionratio.WefoundthatNR tumorshadhigherlevelsofCD8/CD4expression(p<0.01,

Table 2). Among tumors with either low DI or low

neoantigenload,NRpatientshadsignificantlyhigherlevels ofCD8,CD8/CD4,GZMA,andIDO1comparedtoRecpatients (Fig.4A).Forvisualrepresentation,wegeneratedaheatmap of immunostimulatory gene expression according to recurrence, diversity, and neoantigen classification (Fig. 4B).Ofnote,therewasahighlylinearpositivecorrelation between CD8 and IDO1 expression (R2₌_0.82, _p_<_0.001);

IDO1expressionisprobablyupregulatedinresponsetoCD8 upregulation in a negative feedback mechanism [20,21]. These data suggest that a degree of both intratumoral oligoclonal T-cell expansion and high expression of

Fig.2–LowT-cellreceptor(TCR)diversityindex(DI)correlateswithclinicaloutcome.(A)Patientswithoutdiseaserecurrence(NR;n=17)hadlower averageTCRbDIcomparedtopatientswhoexperiencedrecurrence(Rec;n=21)(36.1W31.8vs129.8W159,p=0.08).(B)Kaplan-MeiercurveofRFS, withpatientsdividedintogroupswithhighandlowTCRDIgroups(medianDI40.4;pvaluebylog-ranktest).(C)Stratificationaccordingtotumor sizeandnodalstatus(pvaluebyanalysisofvariance).

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immunostimulatory genes may be necessary to protect againstrecurrence.

4. Discussion

Ourstudyisthefirsttoexaminetherelationshipbetween predictedneoantigenloadandTCRdiversitywithclinical outcomes in chemotherapy-naı¨ve MIBC. No study has quantitativelycharacterizedtheTILreceptorrepertoirein depth via TCR sequencing in MIBC. Our results demon-strate that DI is a measure of the expansion of T cells that captures the functionalityof TILs in a mannerthat cannot be achieved with IHC quantification alone. TCR

sequencing may therefore more objectively capture the immune environment within the entire tumor sample and be a novel method for predictingdisease coursein MIBC.

The finding that TCRDI remains associated withRFS even after correcting for prognostic clinical variables implies that it is the immune response to genetic (and epigenetic) alterations, rather than simply the genetic alterationsthemselves,thatinfluencesclinicaloutcome.As evidence,TCRDIwasassociatedwithRFS,butneoantigen load alonewas not. Suchahypothesis isvalidatedby a recent trial demonstrating a strongclinical response to anti-PDL1 therapy in MIBC [22]. Just as boosting the

Fig.3–AcombinationofahighnumberofneoantigensandlowT-cellreceptor(TCR)diversityindex(DI)mediatessurvivalandnonrecurrence. (A)Tumorsfrompatientswithoutrecurrence(NR)hadahighernumberofpredictedneoantigensthantumorsfrompatientswhoexperience recurrence(Rec;177.8vs103.9;p=0.032).(B)Kaplan-Meiersurvivalanalysisrevealsanonsignificantrecurrence-freesurvival(RFS)benefitfor patientswithtumorswithahighnumberofpredictedneoantigens(RFSmediannotreachedvs52.6wkforhighvslowneoantigens;p=0.11).(C) TumorsfromNRpatientsexhibitnoassociationbetweenneoantigennumberandTCRDI(p=0.9),whilethereisapositivecorrelationforRecpatients (p=0.07).Thedifferenceinslopeissignificant(p=0.038).

Table 2 – Expression ratios for immunocytolytic genes according to clinical outcomea

Allpatients Lowdiversity LowerNNA Highdiversity HigherNNA

(n = 36) (n = 17) (n = 13) (n = 19) (n = 16)

NR/Rec pvalue NR/Rec pvalue NR/Rec pvalue NR/Rec pvalue NR/Rec pvalue

CD8 2.97 0.342 11.85 0.06 10.55 0.029 1.90 0.898 0.75 1

CD8/CD4 2.17 0.003 4.52 0.027 6.96 0.007 1.60 0.179 0.70 0.562

GZMA 10.22 0.071 26.12 0.027 41.99 0.019 4.90 0.416 2.10 0.635

IDO1 27.62 0.077 57.17 0.048 149.86 0.019 2.00 0.373 2.10 0.679

CD8/FOXP3 1.43 0.232 2.42 0.45 3.35 0.029 1.50 0.21 0.70 0.635

NNA=numberofneoantigens;NR/Rec=expressionratiobetweenpatientswithoutrecurrence(NR)andpatientswithrecurrence(Rec). a

Expressionlevelsofthegeneslistedinthetabledifferedsigniﬁcantlybyclinicaloutcomeaccordingtogeneticanddiversityvariables.Insampleswithlow diversity,relativeincreasesinCD8,GZMA,andIDO1expressionwereobservedintumorsfromRecpatients.Similartrendsareseenfortumorswithfewerthan themediannumberofneoantigens(lowneoantigenload).Thesetrendswerenotseenfortumorswithhighdiversityorthosewithhighnumbersofneoantigens. TheMann-WhitneyUtestwasusedtodeterminepvalues.

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immuneresponseviaanti-PDL1therapiesresultsinclinical improvement in MIBC, our study shows that patients whointrinsicallyhavemorerobustintratumoralimmune responses independently have improved long-term clinical outcomes. Patients withlow TCRDI atthe time of cystectomy may therefore be optimal candidates for immunotherapy.

ThelackofcorrelationbetweenRFSandneoantigenload alsosuggeststhatwhileahighermutationburdenincreases thelikelihoodof generating relevantneoantigens [8], NR tumorsmay simplyhave‘‘wontheneoepitopelottery’’to generate those few true neoantigens that elicit strong oligoclonal T-cell expansion, probably via high-affinity binding to both TCR and HLA class I molecules [23]. In other words, having a greater number of neoantigens increases the probability, but without guarantee, of capturingthosestronglyimmunogenicneoantigenscapable ofstimulatingaveryeffectiveantitumorimmuneresponse. T-cell anergymay also contribute to the inabilityof Rec tumors to mount an effective immune response to presented neoantigens. Assessment of PD1 or PDL1 may therefore clarify the relationship between TCR DI and neoantigenload.Intheinterim,identificationofpotential criticalneoantigens in MIBC, or those alreadycapable of

inducing strongT cell–mediated responses, could poten-tiallyserveasabasisforpeptidevaccines.

Thelimitationsofourstudyincludeitssmallsamplesize, makingitcriticaltovalidatetheresultsbyanalyzingfuture MIBCpatientcohortswithappropriateclinicalfollow-upto improveourunderstandingoftheserelationships.Second, variant tumor histology is a potential confounderin our analysis that could not be corrected. Third, our TCR sequencingapproachcannotdistinguishsubsetsofTcells; however,flowcytometrycannotbeusedtoperformthese analysesowingtothelimitedavailabilityofcancertissues andtechnicaldifficultyinseparatingsubsetsofcellsinsolid tumors.Finally,next-generationsequencingdoesnotallow definition of the TCR a and b pair that recognizes neoantigens. Single-cell sequencing, by contrast, could potentially help to identify the pair of TCR chains that respondtoneoantigens.

5. Conclusions

Takentogether,ourresultshaveimportantimplicationsfor prognosticpredictioninMIBC,aswellasforthefutureuse of immunotherapies. In particular, identification of the neoepitopes capable of stimulating oligoclonal T-cell

Fig.4–Patientswithoutrecurrence(NR)exhibithigherlevelsofimmunocytolyticgeneexpressionthanpatientswhoexperiencedrecurrence(Rec) whenstratifiedbydiversityandneoantigenload.(A)Foreachgroupindicated,wecalculatedtherelativeexpressionofCD8,CD8/CD4,GZMA,andCD8/ FOXP3fortumorsfromNRpatientsoverRecpatients.T-cellpopulationswithhighCD8/FOXP3expressionhavehighratiosofcytolyticCD8+_T_cells_to Tregcells,whileT-cellpopulationswithalowCD8/FOXP3expressionratiocorrespondtoalowCD8+_T_cell_to_Treg_ratio._Bars_marked_with_asterisks reachedstatisticalsignificance(p<0.05).Thedifferenceinimmunocytolyticgeneexpressionisgreatestfortumorswithalownumberofpredicted neoantigens(n=5NR,n=9Rec).IncalculatingtheNR/Recgeneexpressionratio,weusedthestandarderrorofthemeantorepresenterrorbars. HD=highdiversity;LD=lowdiversity;HNa=highneoantigenload;LNa=lowneoantigenload.(B)Heatmapofgeneexpressionofimmune-related factorsaccordingtorecurrencestatus,TCRdiversity,andneoantigenclassification.Redindicatesthehighestexpressionandgreenindicatesthe lowest.Thescaleisnormalizedforeachgene.NRtumorswithlowdiversitytendedtohavehigherexpressionofimmunostimulatoryfactorsand IDO1.H=high;L=low.

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expansion could offer new avenues for adaptive T-cell therapyandpeptide vaccinesfor varioustypesof human tumorbyeitherboostingthenaturalantitumorsystemor bycompensatingfordeficienciesingeneticalterations.Our results demonstrate that integration of the genetic and immune landscapes of bladder tumors provides valuable prognosticinformationontheclinicalcourseofMIBCthat canbeharnessedfortherapeuticmeasures.

Author contributions: Yusuke Nakamura had full access to all the data in

the study and takes responsibility for the integrity of the data and the

accuracy of the data analysis.

Study concept and design: Park, O’Donnell, Nakamura.

Acquisition of data: Choudhury, Kiyotani, Yew, Antic, Steinberg, KL Yap.

Analysis and interpretation of data: Choudhury, Park, Kiyotani, Nakamura,

O’Donnell.

Drafting of the manuscript: Choudhury.

Critical revision of the manuscript for important intellectual content:

Nakamura, O’Donnell, Choudhury, KL Yap, JH Park.

Statistical analysis: Choudhury, Kiyotani, KL Yap.

Obtaining funding: Choudhury, O’Donnell, Nakamura, Park, Steinberg.

Administrative,technical,ormaterialsupport:Campanile.

Supervision:Nakamura,O’Donnell.

Other: None.

Financialdisclosures:Yusuke Nakamuracertiﬁesthat all conﬂictsof

interest, including speciﬁc ﬁnancial interests and relationships and

afﬁliations relevant to the subject matter or materials discussed in the

manuscript (eg, employment/afﬁliation, grants or funding,

consultan-cies, honoraria, stock ownership or options, expert testimony, royalties,

or patents ﬁled, received, or pending), are the following: None.

Funding/Support and role of the sponsor: Funding for this project was

provided by the following sources: Pritzker School of Medicine Pritzker

Fellowship and Alpha Omega Alpha Carolyn L. Kuckein Research

Fellowship (N.C.),JohnsHopkins Greenberg BladderCancer Institute

(P.H.O’D.), Cancer Research Foundation Young Investigator Award

(P.H.O’D.), a subaward from NIH N02C007009-65 (G.S.), and a University

of Chicago Comprehensive Cancer Center Support Grant (CCSG P30

CA014599). The sponsors played no direct role in the study.

Acknowledgments: The authors would like to thank Drs. Rui Yamaguchi,

Seiya Imoto, and Satoru Miyano at the Human Genome Center, Institute

of Medical Science, The University of Tokyo, for developing the algorithm

for decomposing the TCR repertoire; Magdeline Montoya for library

preparation;andtheHumanGenomeCenter,theInstituteofMedical

Science, The University of Tokyo for providing the supercomputer

resource used for sequencing analysis.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbe found,intheonlineversion,atdoi:10.1016/j.euf.2015.09.

007.

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