Clonal Relatedness and Mutational Differences between ... · Precision Medicine and Imaging Clonal Relatedness and Mutational Differences between Upper Tract and Bladder Urothelial

Precision Medicine and Imaging

Clonal Relatedness and Mutational Differencesbetween Upper Tract and Bladder UrothelialCarcinomaFrancois Audenet1, Sumit Isharwal1, Eugene K. Cha1, Mark T.A. Donoghue2,3,Esther N. Drill2, Irina Ostrovnaya2, Eugene J. Pietzak1, John P. Sfakianos4,Aditya Bagrodia5, Paari Murugan6, Guido Dalbagni1, Timothy F. Donahue1,Jonathan E. Rosenberg7, Dean F. Bajorin7, Maria E. Arcila3,8, Jaclyn F. Hechtman8,Michael F. Berger3,8, Barry S. Taylor2,3, Hikmat Al-Ahmadie3,8, Gopa Iyer3,7,9,Bernard H. Bochner1,9, Jonathan A. Coleman1, and David B. Solit3,7,9

Abstract

Purpose: To investigate genomic differences betweenurothelial carcinomas of the upper tract (UTUC) and bladder(UCB), with a focus on defining the clonal relatedness oftemporally distinct tumors.

Experimental Design:We prospectively sequenced tumorsand matched germline DNA using targeted next-generationsequencingmethods. The cohort included 195 UTUC patientsand 454 UCB patients. For a subgroup of 29 patients withUTUC and a history of a subsequent UCB, both tumors wereanalyzed to assess their clonal relatedness.

Results:With the progression to higher UTUC clinical state,there were fewer alterations in the RTK/RAS pathway butmorealterations in TP53/MDM2. Compared with UCB, TP53, RB1,and ERBB2 were less frequently altered in UTUC (26% vs.46%, 3% vs. 20%, 8% vs. 19%, respectively; Q < 0.001),whereas FGFR3 and HRAS were more frequently altered

(40% vs. 26%, 12% vs. 4%, respectively; Q < 0.001). On thebasis of an integrated analysis of tumor mutational burden,MSIsensor score and mutational signature, 7.2% of UTUCtumors were classified as MSI-high/MMR-deficient(MSI-H/dMMR). The risk of bladder recurrence after UTUCwas significantly associated with mutations in FGFR3,KDM6A, CCND1, and TP53. Comparison of UCB withcorresponding UTUC tumors from the same patient supportstheir clonal relatedness.

Conclusions:UTUCandUCBexhibit significant differencesin the prevalence of common genomic alterations. In individ-ual patients with a history of both tumors, UCB and UTUCwere always clonally related. Genomic characterization ofUTUC provides information regarding the risk of bladderrecurrence and can identify tumors associated with Lynchsyndrome.

IntroductionUrothelial carcinoma (UC) is the sixth most common cancer

type in the United States (1). UCs of the bladder (UCB) accountfor the majority of UCs (90%–95%; ref. 2), whereas UCs of theupper tract (UTUC) are significantly less common, accounting for5% to 10% (1, 3). Although both UCB andUTUC arise within theurothelium and have a similar histologic appearance, they havedistinct clinical characteristics (4). UTUC is uniquely associatedwith several environmental risk factors (5–7) and is more com-mon than UCB in patients with Lynch syndrome (8).

Due to the relative rarity of UTUC compared with UCB, mostclinical and biological studies have focused predominantly orexclusively on bladder cancers, with the results and clinicalimplications extrapolated to UTUC. To date, relatively few UTUChave been examined using newer next-generation sequencing(NGS) methods and The Cancer Genome Atlas (TCGA) study ofUCs excluded UTUC patients (9, 10). Small retrospective caseseries have, however, suggested differences in the prevalence ofoncogenic driver mutations between UCB and UTUC (11, 12).

Here, we leveraged a prospective molecular characterizationinitiative to explore the potential clinical utility of tumor

1Urology Service, Department of Surgery, Memorial Sloan Kettering CancerCenter, New York, New York. 2Department of Epidemiology and Biostatistics,Memorial Sloan Kettering Cancer Center, New York, New York. 3Human Oncol-ogy and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, NewYork, New York. 4Department of Urology, Mount Sinai Hospital, New York, NewYork. 5Department of Urology, UT Southwestern Medical Center, Dallas, Texas.6Department of Laboratory Medicine and Pathology, University of Minnesota,Minneapolis, Minnesota. 7Department of Medicine, Memorial Sloan KetteringCancer Center, New York, NewYork. 8Department of Pathology, Memorial SloanKettering Cancer Center, New York, New York. 9Weil Cornell Medical College,New York, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

F. Audenet and S. Isharwal contributed equally to this article.

Corresponding Authors: Francois Audenet, Memorial Sloan Kettering CancerCenter, NewYork, NY 10065. Phone: 646-888-2646; Fax: 646-888-3266; E-mail:[email protected]; Jonathan A. Coleman, Urology Service,Department of Surgery, Kimmel Center for Prostate and Urologic Cancers,Memorial Sloan Kettering Cancer Center, 353 East 68th Street, New York, NY10065. Phone: 646-422-4432; Fax: 212-452-3323; E-mail: [email protected];andDavid B. Solit, Memorial Sloan Kettering Cancer Center, 417 East 68th Street,New York, NY 10065. Phone: 646-888-2646; Fax: 646-888-3266; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-18-2039

�2018 American Association for Cancer Research.

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genomic characterization in patients with UTUC, in particularits ability to guide clinical management and to identify patientswhose tumors could be attributed to a diagnosis of Lynchsyndrome. We were particularly interested in determiningwhether differences in clinical characteristics reflect differencesin the spectrum of genetic drivers between UTUC and UCB.Furthermore, as UTUC and UCB often co-occur in individualpatients, we sought to address an ongoing controversy in thefield as to whether temporally distinct tumors arising in indi-vidual patients are clonally related recurrences or representdistinct primary tumors.

Materials and MethodsStudy samples

Written consent was obtained from all participating patients.The study was conducted in accordance with International EthicalGuidelines for Biomedical Research Involving Human Subjects,Good Clinical Practice guidelines, the Declaration of Helsinki,and local laws, and approved by an institutional review board. Intotal, 195UTUCandmatched germlineDNA samples fromblood[including 84 UTUC samples from a prior retrospective study(ref. 11) and 111 samples profiled prospectively] were analyzedusing a targeted NGS platform (MSK-IMPACT; SupplementaryMethods; refs. 13, 14). Representative hematoxylin and eosinslides for each tumor were reviewed by a board-certified genito-urinary pathologist (H. Al-Ahmadie) to confirm grade, stage, andurothelial histology. Patients with a predominant variant histol-ogy in the reviewed specimen were excluded. For comparativeanalyses, we used a cohort of 454 patients with UCB (including102 UCB samples from the prior study, ref. 11) and no priorhistory of UTUCwhowere similarly analyzed byMSK-IMPACT atour center. For a subgroup of 29 patients withUTUC and a historyof a subsequent UCB, we analyzed both tumors to define theirclonal relatedness.

Statistical analysisWeusedc2 and exact Fisher tests todefine statistical significance

of differences between clinical and demographic categorical

variables, and Wilcoxon and Kruskal–Wallis tests for continuousvariables comparisons. Tumormutational burdenwas adjusted toaccount for the number of genes included in the version ofMSK-IMPACT used for individual patients (from 230 to 468;Supplementary Table S1). To determine allelic configurations,total and allele-specific copy-number states were inferred usingFACETS (version 0.5.6; ref. 15). Microsatellite instability wasassessed using the MSIsensor algorithm (version 0.2; ref. 16). Totest tumors for clonal relatedness, we used somaticmutation data,including silent mutations (17). Q-values were used to indicatethe P values adjusted for multiple comparisons using theBonferroni method to control the false discovery rate. A P valueof <0.05 was considered statistically significant. Analyseswere conducted using R v.3.4.3 (https://cran.r-project.org).

ResultsGenomic landscape of UTUC

To explore the genomic landscape of UTUC across thedisease spectrum, we analyzed 195 UTUC tumors, including84 samples analyzed retrospectively and reported previously(11) and 111 samples collected and sequenced within thecontext of a prospective molecular characterization initiative.Overall, the UTUC samples included 84 tumors from patientswith no muscle invasion (<pT2, 43%), 76 from patients withmuscle invasion (�pT2, 39%) and 35 from patients withmetastatic disease (18%) at the time of tumor collection(Table 1). Eighty-five percent of tumors were high-grade. Weperformed targeted NGS analysis of matched tumor andgermline DNA of at least 275 and up to 468 cancer-associatedgenes. The most frequently mutated genes included FGFR3(40%), KMT2D (37%), KDM6A (32%), TP53 (26%), andARID1A (23%; Fig. 1). We further grouped mutations bycanonical pathway or functional significance (18).

Translational Relevance

To date, relatively few urothelial carcinomas of the uppertract (UTUC) have been examined using next-generationsequencing (NGS) methods. In this study, we sequenced195 UTUC using a targeted NGS platform and compared theresults to 454 urothelial carcinomas of the bladder (UCB). Weidentified significant differences in the prevalence of commongenomic alterations between UTUC and UCB. Targeted NGSwas a robust methodology for identifying UTUC patientswhose tumors were microsatellite instability-high/mismatch-repair-deficient, the vast majority of which arose in patientswith a pathogenic germline mutation in a Lynch syndrome–associated gene. In individual patients with a history of both,we found that the UCB and UTUC were always clonallyrelated. The latter results justify the development of methodsto prevent lower tract seeding during surgery and the adoptionof risk-adapted surveillance for bladder recurrence in patientswith UTUC.

Table 1. Patient characteristics

VariablesUTUC(N ¼ 195)

Bladder(N ¼ 454) P

Median age (IQR), years 67.1 (58.1–74.5) 67.5 (60.1–74.4) 0.692SexMale 121 (62%) 367 (81%) <0.001Female 74 (38%) 87 (19%)

Smoking statusActive 22 (11%) 63 (14%) 0.619Former 112 (58%) 246 (54%)Never 61 (31%) 143 (32%)

LocationRenal pelvis 154 (79%) NAUreter 41 (21%) NA

Clinical state<pT2 84 (43%) 151 (33%) 0.055�pT2 76 (39%) 213 (47%)Metastatic 35 (18%) 90 (20%)

Tumor gradeLow grade 30 (15%) 26 (6%) <0.001High grade 165 (85%) 428 (94%)

Tumor stage<pT2 86 (44%) 170 (37%) 0.186�pT2 82 (42%) 226 (50%)Metastasis 27 (14%) 58 (13%)

Presence of pathologic variantYes 23 (12%) 126 (28%) <0.001No 172 (88%) 328 (72%)

NOTE: P values in bold are statistically significant.Abbreviation: IQR, interquartile range.

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In patients with higher stage disease, there were fewer altera-tions in the RTK/RAS pathway (P < 0.001) but more alterations inTP53/MDM2 (P < 0.001). The frequency of activating alterationsin FGFR3 and HRAS was lower in patients with higher stagedisease (63% vs. 26% and 17% vs. 3%, respectively; P <0.001). Conversely, the frequency of alterations in TP53 andMDM2 was significantly increased in patients with higher stagedisease (11% vs. 31% and 4% vs. 17%, respectively; P < 0.001).The frequency of alterations in only two other genes was signif-icantly greater in patients with lower stage UTUC tumors: STAG2,a component of the cohesin complex that plays a role in meiosisandmitosis (24%vs. 6%,P<0.001), and FBXW7, a component ofthe SCF complex involved in regulating protein degradation ofseveral oncogenes including cyclin E and c-Myc (12% vs. 0%, P <0.001). When comparing the genomic profiles of UTUC based onthe location of the primary tumor (renal pelvis vs. ureter), onlyKMT2C and KMT2D, two genes with a purported role in chro-matin regulation, were significantly more frequently altered inureteral tumors as compared with renal pelvic tumors afteradjusting for multiple comparison (41% vs. 18%,Q¼ 0.023 and59% vs. 32%, Q ¼ 0.034, respectively).

Genomic comparison between UTUC and bladder tumorsTo identify differences in the mutational landscape between

upper and lower tract urothelial tumors, we compared the 195UTUC tumors with 454 UCB tumors from patients who had noprior history of UTUC (Table 1). The spectrum of mutationsidentified by MSK-IMPACT in these bladder tumors was compa-rable to those reported by the TCGA (9, 10). Overall, TP53, RB1,and ERBB2 were significantly more frequently altered in UCB ascompared with UTUC (46% vs. 26%, 20% vs. 3%, 19% vs. 8%,respectively; Q < 0.001), whereas FGFR3 and HRAS were signif-icantly more frequently altered in UTUC (40% vs. 26%, 12% vs.4%, respectively; Q < 0.001; Fig. 2). When grouped by pathway/function, genes in the cell-cycle and TP53/MDM2 pathways weresignificantly more frequently altered in UCB compared withUTUC (56% vs. 43%, Q ¼ 0.01 and 55% vs. 35%, Q < 0.001,respectively).

We next sought to determine if the prevalence of alterations inindividual genes varied as a function of clinical disease state inUTUCandUCB (Fig. 3). For clinical state<pT2,HRAS andKMT2Cwere significantly more frequently altered in UTUC (17% vs. 3%,Q ¼ 0.003 and 30% vs. 12%, Q ¼ 0.008, respectively). When

Figure 1.

Overview of the genomic landscape of UTUC, stratified by clinical state and molecular pathways. The genes with significant differences in genomic alterationfrequencies across clinical states are highlighted by red boxes.

Clonality and Differences between UTUC and UCB

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limiting the analysis to only high-grade samples <pT2,HRASwasstill significantly more frequently altered in UTUC as comparedwith UCB (22% vs. 4%,Q¼ 0.001). For clinical state�pT2, TP53and RB1were more frequently altered in UCB (62% vs. 39%,Q¼0.008 and 26% vs. 3%, Q < 0.001), whereas HRAS was morefrequently altered in UTUC (12% vs. 2%, Q ¼ 0.003). The TP53/MDM2 pathway was significantly more frequently altered in UCB(69% vs. 51%,Q¼ 0.022). For patients withmetastatic disease atthe timeof tumor collection,RB1, ERBB2, and cell-cycle pathway–associated genes as a group were all more frequently altered inUCB (27% vs. 3%, Q ¼ 0.031; 27% vs. 0%, Q ¼ 0.007; 71% vs.43%, Q ¼ 0.015, respectively).

Patients with Lynch syndrome have germline mutations inmismatch-repair (MMR)–associated genes, which results in anincreased risk for the development of tumors with microsatelliteinstability (MSI) and hypermutation. UCbelongs to the spectrumof Lynch syndrome, but UTUC is much more frequent than UCBin patients with Lynch syndrome (8). To assess for evidence ofMMR deficiency in the two groups, we calculated tumor muta-tional burden and MSIsensor scores, a measure of somatic alter-ation of microsatellite regions (Supplementary Methods; refs. 16,19). The median number of somatic mutations/Mb was signifi-cantly higher in UTUC compared with UCB [13.2 (7.4–19.1) vs.8.8 (5.9–15.4); P < 0.001]. Overall, 6.2% (12/194) of the UTUC

Figure 2.

Genomic differences between UTUC and UCB. A, Comparison of the differences in the prevalence of genomic alterations between UTUC and UCB, for thegenes present at a frequency of �10% in any clinical state. The genes with significant differences after multiple comparison testing using the Bonferronimethod are highlighted by red boxes.B,Volcanoplot of the frequencyof gene alterations [log2(OR)] by significance [�log10(P)] in urothelial carcinomasof the uppertract (UTUC) or bladder. The horizontal dotted line indicates an adjusted P < 0.001. C, Comparison of genomic alteration frequencies for molecular pathwayscommonly mutated in UCs.

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sampleswereMSI-high (defined as anMSIsensor score�10),with3.1% (6/194) having an indeterminate MSIsensor score (3–10).As expected, the number of mutations/Mb was significantlyhigher in tumors with MSIsensor scores �10 (median: 49.3) ascompared with tumors with MSIsensor scores between 3 and 10(median: 13.0; P¼ 0.002) or MSIsensor scores <3 (median: 6; P <0.001). Four patients with UCB (1.1%) had an MSIsensor score�10, including one patient known to have Lynch syndrome andno prior history of UTUC.

UTUC is the third most prevalent cancer in Lynch syndrome(20), and current European guidelines recommend that germ-line DNA sequencing be considered for all UTUC patientsyounger than age 60 and those with a personal or familialhistory of Lynch-related cancers (21). However, no optimalscreening strategy has been validated. In our cohort of 195UTUC, consent to assess for germline mutations was obtainedin 47 patients, typically those in whom there was clinicalsuspicion of a hereditary syndrome or a high MSIsensor score.Analysis of the germline sequencing data revealed that 12of these 47 patients had a pathogenic or likely pathogenicgermline mutation in a Lynch syndrome–associated gene

(Supplementary Table S2). Notably, all tumors from thesepatients had a high tumor mutational burden (range, 22–472,median: 54.9, IQR: 32.4–67.0). Only 10 of 12 Lynch patients,however, had an MSIsensor score of �10 (Fig. 4; SupplementaryTable S2). Furthermore, of the 12 tumorswithMSIsensor scores of�10, 10 had a Lynch syndrome–associated germline mutation,one a personal history strongly suggestive of Lynch syndrome butno consent for germline analysis (patient 2; Fig. 4), and one asomatic Lynch-like MSI-high tumor (patient 1; Fig. 4).

To further characterize those tumors with evidence of hyper-mutation but indeterminate or low MSIsensor scores, we per-formed mutational signature decomposition analysis (22, 23)on all tumor samples with 10 or more single-nucleotide somat-ic mutations. Notably, only 6 of 12 tumors from patients withLynch syndrome had a predominant MMR/MSI signature bymutational signature decomposition analysis, whereas theothers exhibited a predominant mitotic clock (aging) signature.In the broader cohort of UTUC samples with 10 or moremutations without an MMR germline mutation, we identifiedevidence of a predominant AID/APOBEC signature in 20 of 30,a smoking signature in 2 of 30 and a POLE signature in 1 of 30.

Figure 3.

Stage-by-stage comparison of the differences in the prevalence of genomic alterations between UTUC and UCB, for the genes identified at a frequencyof �10% at any clinical state. The genes with significant differences after multiple comparisons using the Bonferroni method are highlighted by red boxes.






As above, two patients with MSIsensor scores <10 had a Lynchsyndrome–associated UTUC (patients 3 and 4; Fig. 4).

In sum, the data indicate that no singlemetric could identify allMMR-deficient UTUC tumors and that the positive and negativepredictive value of current clinical germline testing guidelines islow (Supplementary Table S3). The data thus suggest that anintegrated approach that incorporates tumor mutational burden,MSIsensor score, mutational signature analysis, and somatic andgermline mutation of Lynch syndrome–associated genes is need-ed to accurately classify the MMR deficiency status of UTUCtumors. Given the limitations of current clinical germline testing

guides, our results also support universal tumor/normal geneticprofiling in all patients with UTUC to identify those with a Lynchsyndrome–associated tumor.

Clonal relatedness of lower tract recurrences inUTUC patients

Of the 195 patients with UTUC, 137 underwent radicalnephroureterectomy, with 57 (42%) later developing a UCB aftera median time of 7.3 months (IQR: 4.1–13.7; SupplementaryTable S4). After adjusting for clinical factors associated withbladder recurrence, including sex, history of prior bladder cancer,

Figure 4.

Use of NGS to identify microsatellite instability-high (MSI-H)/mismatch-repair–deficient (dMMR) UTUC tumors. A, MSIsensor scores of UTUC samples werecalculated using MSIsensor (version 0.2) and are displayed as a function of the number of somatic mutations/Mb, and its association with Lynch syndrome asdetermined by analysis of germline DNA. B, FACETS analysis of case 1 showing MSH2 loss of heterozygosity. This patient had 29 somatic mutations/Mb,an MSIsensor score of 23.7, a predominant MMR/MSI signature by mutational decomposition analysis, and loss of MSH2 and MSH6 expression by IHC in theabsence of a germline mutation in these two genes, all consistent with a somatic Lynch-like MMR-deficient tumor. C, Mutational decomposition analysis of theUTUC samples with more than 10 somatic mutations. Case 2 had no consent for germline analysis. However, the patient's personal history of colon cancer, thetumor mutational burden (TMB) of 21 somatic mutations/Mb, the elevated MSIsensor score of 17.4, and the predominant MMR/MSI signature by mutationaldecomposition indicated that this patient had anMMR-deficient tumor, and thus germline testingwould havebeen indicated. Notably, one patientwith no clinical riskfactors for hereditary UTUC but with an MSI-high tumor was found to have Lynch syndrome. Two of the 12 patients with pathogenic/likely pathogenicgermline mutations in Lynch syndrome–associated genes had low or intermediate MSIsensor scores but elevated tumor mutational burden in the setting of apredominant MMR/MSI signature by mutational decomposition. Case 3 was 45 years old at diagnosis and had a TMB of 22 somatic mutations/Mb, an MSIsensorscore of 3.8, a germline mutation in MSH6, absence of both MSH6 and MSH2 expression by IHC, and a predominant MMR/MSI signature. Case 4 had a familialhistory of Lynch-related cancers, a UTUC with 343 somatic mutations/Mb, an MSIsensor score of 1.1, a germline mutation in MSH2, absence of MSH2and MSH6 expression by IHC, and a predominant MMR/MSI signature.

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location, grade, and tumor stage (24), alterations in FGFR3 (HR¼3.00; 95% CI, 1.58–5.68; P ¼ 0.001), KDM6A (HR ¼ 2.27; 95%CI, 1.29–4.02; P ¼ 0.005), and CCND1 (HR ¼ 3.10; 95% CI,1.17–8.21; P ¼ 0.023) were significantly associated with a higherrisk of developing a subsequent bladder tumor, whereas TP53alterations were associated with a lower risk (HR¼ 0.32; 95% CI,0.13–0.80; P ¼ 0.014; Supplementary Table S5).

To determinewhether the bladder tumors that arose in patientswith a prior history of UTUC represented clonal recurrences orsecond primary tumors, we analyzed temporally distinct pairs ofUTUC and UCB from 29 patients (Supplementary Table S6).Clonal relatedness between the tumors was investigated using allsomatic mutation data, including synonymous mutations, aspreviously described (17). Sixteen patients (55%) had no priorhistory ofUCB. All pairs ofUTUCandUCBwere deemed tohave ashared clonal origin (P < 0.005). We did, however, observe somelesion-to-lesion heterogeneity with only 86% of somatic muta-tions being present in both the initial UTUC and the subsequentUCB recurrence (Fig. 5). In the 13 patients with a prior history ofUCB, 75% of the mutations were found to be similar in both theUTUC and subsequent bladder tumors (Supplementary Fig. S1).Again, all pairs of UTUC and recurrent UCB were clonal (P <

0.005). For the 7 bladder tumors collected before radicalnephroureterectomy with tissue available for sequencing, 69%ofmutationswere shared between the initialUCBand subsequentUTUC,with all the tumor pairs again being of shared clonal origin(P < 0.005).

DiscussionAlthough they share a similar urothelial histologic appearance,

UTUC and UCB have several epidemiologic and clinical differ-ences (4). By comparing the mutational profiles of UTUC andUCB in 649 urothelial cancer samples (195UTUC and 454UCB),we sought to determine whether differences in somatic muta-tional patterns underlie the clinical differences between the twodiseases. Although the spectrum of genomic alterations wassimilar between UTUC and UCB, significant differences in theprevalence of mutations in individual genes were observed,including a higher frequency of FGFR3 andHRAS alterations anda lower frequency ofTP53, RB1, andERBB2 alterations inUTUCascompared with UCB. Furthermore, we found that significantdifferences in themutational frequency of several genes remainedafter adjusting for clinical disease state.

Figure 5.

Oncoprints and Venn diagrams for the matched-pair comparison between UTUC and UCB tumors in patients with no prior history of a bladder tumor beforeresection of the UTUC. The arrows show the time between sample collection in months. Patient � underwent radical nephroureterectomy for pT2N0 UTUC andhad a bladder recurrence 4 months later (pTa high grade). He was followed regularly for 65 months after the initial RNU and ultimately progressed tometastatic disease. The molecular profile of the metastasis was closer to the bladder tumor than to the radical nephroureterectomy specimen. However, allthree tumor samples shared a clonal origin.






Consistent with prior studies (25), FGFR3 alterations weremore common in low-grade tumors. FGFR3 mutations inurothelial cancer typically arise in one of several hotspot loca-tions and have been shown to be oncogenic and thus representpotential therapeutic targets. Notably, 31% of high-gradeUTUC had FGFR3 alterations, a frequency significantly higherthan that observed in high-grade UCB (21%). As FGFR3 andTP53 tend to be mutually exclusive, our results lend furthersupport to the model that high-grade tumors often arise de novo,in the absence of a prior low-grade lesion, whereas some low-grade tumors with FGFR3 alterations, more frequently in theupper tract than in the bladder, progress over time to high-grade disease (26).

RB1 alterations were rarely observed in our high-grade UTUCcohort (2%), whereas RB1 inactivation is common in MIBC,where it has been associated with genomic instability (27). It isnoteworthy that no single pathognomonic molecular event hasbeen identified in UC. Overall, we found that the RTK/RAS, PIK3/AKT, cell-cycle, and TP53/MDM2 pathways were all altered inaround half the tumors, with RTK/RAS and TP53/MDM2 altera-tions being largely mutually exclusive. Recently, a whole-exomesequencing analysis of 27 UTUC samples confirmed that therewas a significantly higher frequency of mutations in the p53 andrelated pathways in high-grade tumors, leading to genomic insta-bility, copy-number alterations, and disruption of the cell-cycleand apoptotic pathways, which was corroborated by proteinanalysis (12). However, given the complex co-mutation patternof UC and the lack of in-depth studies on the biological role ofmost of the oncogenes and tumor suppressors identified in thisdisease, a full understanding of the key co-mutational eventsrequired for progression to a high-grade, lethal phenotype willrequire further study.

Tumor genomic analysis can provide information that could beused by physicians to guide the clinical management of UTUCpatients. For example, clinical tumor genomic profiling can pro-spectively identify potential therapeutic targets and thus guidetreatment selection (28). Overall, over half of the urothelialtumors in the current study as well as the TCGA had potentiallyactionable genomic alterations, including ERBB2 amplificationsandmutations and FGFR3 hotspot mutations (29). Furthermore,patients with Lynch syndrome are at increased risk for the devel-opment of UTUC, and the identification of patients with Lynchsyndrome–associated tumors has treatment and screening impli-cations for both affected patients and unaffected family members(30). Our data suggest that screening for hereditary UTUCbased on clinical risk assessment or MSI status alone can misssome cases of Lynch syndrome (Supplementary Table S3). Withthe implementation of NGS technology in daily practice, inte-gration of tumor mutational burden, analysis of microsatelliteregions (MSIsensor score) and mutational signature couldmore robustly identify MMR-deficient tumors. Recently,upfront tumor sequencing in colorectal cancer has been shownto have greater sensitivity than the prior multitest approachesfor Lynch syndrome screening (31). Our results support thisstrategy in UTUC as well. Furthermore, given the strongassociation between mutational burden and immunotherapyresponse (32), UTUC patients with microsatellite instability inthe setting of MMR deficiency may represent a subpopulationof patients with a greater likelihood of benefiting from bothlocal and systemic immunotherapies (33).

Intravesical recurrence after radical nephroureterectomy iscommon, with a median rate of recurrence of 22% to 47%(24), and several patient-specific and tumor-specific clinicopath-ologic predictors of recurrence have been identified (24).Here, weidentified a strong association in multivariable analysis betweenthe risk of recurrence and alterations in FGFR3, KDM6A, CCND1,and TP53. These findings could help identify those patients ingreatest need for close monitoring or early instillation of mito-mycin C (34). The pathogenesis of bladder tumors that arise aftercurative intent treatment of UTUC is an ongoing controversy inthe field. Two different theories have been proposed: UCB couldresult from intraluminal seeding from the prior UTUC (35) orrepresent a second primary tumor in the setting of a toxin-induced, field-cancerization effect (36). Initial studies usingloss-of-heterozygosity analysis reported conflicting results(37, 38). To our knowledge, this is the first study to use contem-porary NGS methods to assess the clonality or multifocality ofbladder tumors in patients with a history of UTUC. Our resultsclearly demonstrate the clonal origin of UTUC and UCB tumorsthat arise sequentially in individual patients with a history of bothtumors. The results justify the exploration of methods to preventlower tract seeding during radical nephroureterectomy or uretero-scopy andmore rigorous surveillance for bladder recurrences aftersurgery, particularly in those patients with genetic features asso-ciated with lower tract recurrence (TP53-wild-type, FGFR3/KDM6A-mutant). Due to the limited efficacy of a single postop-erative intravesical dose of mitomycin C (34), studies of adjuvanttreatments designed to prevent bladder recurrences are alsowarranted in patients at highest risk for subsequent lower tractrecurrence.

Although this study is the largest cohort of UTUC tumorsgenomically characterized to date, it has several limitations. Lessfrequent mutational events and structural alterations not coveredin the MSK-IMPACT assay could not be detected and may haveimportant prognostic implications, at least for individualpatients. However, the majority of the genomic alterations iden-tified by whole-exome sequencing from 27 patients with UTUC(12) could be identified using our gene panel. Furthermore,differences in gene expression due to changes in the epigeneticregulation of genes may provide added prognostic information, apossibility not addressed by our targeted DNA-sequencingapproach. Finally, individual mutations in specific genes mayhave different biological consequences. Integration of genome,transcriptome, and proteomic analyses may thus provide fur-ther insights into the factors that drive UTUC initiation andprogression.

In conclusion, a genomic comparison of UTUC and UCB in acohort of 649 patients revealed significant differences in theprevalence of common genomic alterations. However, in indi-vidual patients with a history of both tumors, we found that theUCB and UTUC were always clonally related. Genomic charac-terization of UTUC provides clinically relevant information,including identification of patients who could be candidates formolecularly driven clinical trials or for treatment off-label withagents approved for use in other cancer types; evaluation of therisk of bladder recurrence, which may help guide the selection ofadjuvant therapies; and identification of those patients in need ofgermline analysis for Lynch syndrome. Routine implementationof tumor genomic profiling in patients withUTUC should thus beconsidered and prospectively evaluated.

Audenet et al.





Disclosure of Potential Conflicts of InterestE.J. Pietzak is a consultant/advisory boardmember for Merck. J.E. Rosenberg

has ownership interests (including patents) at Illumina; reports receivingspeakers bureau honoraria fromChugai Pharma; is a consultant/advisory boardmember for Adicet Bio, Agensys/Astellas, AstraZeneca, Bayer, Fortress Biotech,Lilly, Merck, BioClin Therapeutics, and Sensei Biotherapeutics; reports receivingcommercial research grants from Novartis and Roche/Genentech; and reportsreceiving commercial research support from Astellas, Bayer, Mirati, Roche/Genentech, and Seattle Genetics. D.F. Bajorin reports receiving speakers bureauhonoraria fromMerck; is a consultant/advisory boardmember for AstraZeneca,Eli Lilly, Fidia Farmici, Genentech, Merck, Pfizer, and Urogen; and reportsreceiving commercial research grants from Merck and Novartis. J.F. Hechtmanreports receiving speakers bureau honoraria from Medscape. M.F. Berger is aconsultant/advisory board member for Roche. H. Al-Ahmadie is a consultant/advisory board member for Bristol-Myers Squibb and EMD Serono. G. Iyer is aconsultant/advisory board member for Bayer. B.H. Bochner is a consultant/advisory boardmember forGenentech.D.B. Solit is a consultant/advisory boardmember for Illumina, Loxo Oncology and Pfizer. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: F. Audenet, S. Isharwal, E.K. Cha, E.J. Pietzak, J.P.Sfakianos, A. Bagrodia, H. Al-Ahmadie, B.H. Bochner, J.A. Coleman, D.B. SolitDevelopment of methodology: F. Audenet, E.K. Cha, E.J. Pietzak, M.E. Arcila,J.F. Hechtman, B.S. Taylor, H. Al-Ahmadie, J.A. Coleman, D.B. SolitAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Audenet, S. Isharwal, E.K. Cha, E.J. Pietzak, J.P.

Sfakianos, A. Bagrodia, P. Murugan, T.F. Donahue, J.E. Rosenberg, D.F. Bajorin,M.E. Arcila, J.F. Hechtman, H. Al-Ahmadie, J.A. Coleman, D.B. SolitAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Audenet, S. Isharwal, E.K. Cha, M.T.A. Donoghue,E.N. Drill, I. Ostrovnaya, E.J. Pietzak, J.P. Sfakianos, A. Bagrodia, T.F. Donahue,J.E. Rosenberg, M.E. Arcila, M.F. Berger, B.S. Taylor, H. Al-Ahmadie, G. Iyer,J.A. Coleman, D.B. SolitWriting, review, and/or revision of the manuscript: F. Audenet, S. Isharwal,E.K. Cha, M.T.A. Donoghue, E.N. Drill, E.J. Pietzak, J.P. Sfakianos, A. Bagrodia,P. Murugan, J.E. Rosenberg, D.F. Bajorin, M.E. Arcila, J.F. Hechtman,H. Al-Ahmadie, G. Iyer, B.H. Bochner, J.A. Coleman, D.B. SolitAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.J. Pietzak, G. Dalbagni, J.E. Rosenberg,B.S. Taylor, J.A. Coleman, D.B. SolitStudy supervision: E.K. Cha, J.A. Coleman, D.B. Solit

AcknowledgmentsThis work was supported by NIH R01-CA182587-05, the Marie-Jos�ee and

Henry R. Kravis Center for Molecular Oncology, Cycle for Survival, and theThompson Family Foundation.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 27, 2018; revised August 20, 2018; accepted October 19, 2018;published first October 23, 2018.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin

2018;68:7–30.2. Babjuk M, B€ohle A, Burger M, Capoun O, Cohen D, Comp�erat EM, et al.

EAU guidelines on non-muscle-invasive urothelial carcinoma of the blad-der: update 2016. Eur Urol 2017;71:447–61.

3. Munoz JJ, Ellison LM. Upper tract urothelial neoplasms: incidence andsurvival during the last 2 decades. J Urol 2000;164:1523–5.

4. Green DA, Rink M, Xylinas E, Matin SF, Stenzl A, Roupret M, et al.Urothelial carcinoma of the bladder and the upper tract: disparate twins.J Urol 2013;189:1214–21.

5. ChiouHY, Chiou ST, Hsu YH, Chou YL, Tseng CH,WeiML, et al. Incidenceof transitional cell carcinoma and arsenic in drinking water: a follow-upstudy of 8,102 residents in an arseniasis-endemic area in northeasternTaiwan. Am J Epidemiol 2001;153:411–8.

6. Grollman AP, Shibutani S, Moriya M, Miller F, Wu L, Moll U, et al.Aristolochic acid and the etiology of endemic (Balkan) nephropathy.Proc Natl Acad Sci USA 2007;104:12129–34.

7. Colin P, Koenig P, Ouzzane A, Berthon N, Villers A, Biserte J, et al.Environmental factors involved in carcinogenesis of urothelial cell carci-nomas of the upper urinary tract. BJU Int 2009;104:1436–40.

8. Win AK, Lindor NM, Young JP, Macrae FA, Young GP, Williamson E, et al.Risks of primary extracolonic cancers following colorectal cancer in lynchsyndrome. J Natl Cancer Inst 2012;104:1363–72.

9. The Cancer Genome Atlas Research Network. Comprehensive molecularcharacterization of urothelial bladder carcinoma. Nature 2014;507:315–22.

10. Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD,et al. Comprehensive molecular characterization of muscle-invasive blad-der cancer. Cell 2017;171:540–556.e25.

11. Sfakianos JP, Cha EK, Iyer G, Scott SN, Zabor EC, Shah RH, et al. Genomiccharacterization of upper tract urothelial carcinoma. Eur Urol 2015;68:970–7.

12. Moss TJ, Qi Y, Xi L, Peng B, Kim T-B, Ezzedine NE, et al. Comprehensivegenomic characterization of upper tract urothelial carcinoma. Eur Urol2017;72:641–9.

13. Cheng DT, Mitchell TN, Zehir A, Shah RH, Benayed R, Syed A, et al.Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable

Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn 2015;17:251–64.

14. Zehir A, Benayed R, Shah RH, Syed A, Middha S, KimHR, et al. Mutationallandscape of metastatic cancer revealed from prospective clinical sequenc-ing of 10,000 patients. Nat Med 2017;23:703–13.

15. Shen R, Seshan VE. FACETS: allele-specific copy number and clonalheterogeneity analysis tool for high-throughput DNA sequencing. NucleicAcids Res 2016;44:e131.

16. Niu B, Ye K, Zhang Q, Lu C, Xie M, McLellan MD, et al. MSIsensor:microsatellite instability detection using paired tumor–normal sequencedata. Bioinformatics 2014;30:1015–6.

17. Ostrovnaya I, Seshan VE, Begg CB. Using somatic mutation data to testtumors for clonal relatedness. Ann Appl Stat 2015;9:1533–48.

18. Iyer G, Al-Ahmadie H, Schultz N, Hanrahan AJ, Ostrovnaya I, Balar AV,et al. Prevalence and co-occurrence of actionable genomic alterations inhigh-grade bladder cancer. J Clin Oncol 2013;31:3133–40.

19. Middha S, Zhang L, Nafa K, Jayakumaran G, Wong D, Kim HR, et al.Reliable pan-cancer microsatellite instability assessment by using targetednext-generation sequencing data. JCO Precis Oncol 2017;1–17.

20. Roupret M, Yates DR, Comperat E, Cussenot O. Upper urinary tracturothelial cell carcinomas and other urological malignancies involved inthe hereditary nonpolyposis colorectal cancer (lynch syndrome) tumorspectrum. Eur Urol 2008;54:1226–36.

21. RoupretM, BabjukM, Comp�erat E, Zigeuner R, Sylvester RJ, BurgerM, et al.European Association of Urology guidelines on upper urinary tract urothe-lial carcinoma: 2017 update. Eur Urol 2018;73:111–22.

22. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, BiankinAV, et al. Signatures of mutational processes in human cancer. Nature2013;500:415–21.

23. Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR.Deciphering signatures ofmutational processes operative in human cancer.Cell Rep 2013;3:246–59.

24. Seisen T, Granger B, Colin P, L�eon P, Utard G, Renard-Penna R, et al. Asystematic review and meta-analysis of clinicopathologic factors linked tointravesical recurrence after radical nephroureterectomy to treat upper tracturothelial carcinoma. Eur Urol 2015;67:1122–33.






25. Liu X, Zhang W, Geng D, He J, Zhao Y, Yu L. Clinical significance offibroblast growth factor receptor-3 mutations in bladder cancer:a systematic review and meta-analysis. Genet Mol Res 2014;13:1109–20.

26. Lindgren D, Sj€odahl G, Lauss M, Staaf J, Chebil G, L€ovgren K, et al.Integrated genomic and gene expression profiling identifies two majorgenomic circuits in urothelial carcinoma. PLoS One 2012;7:e38863.

27. Hurst CD, Tomlinson DC, Williams SV, Platt FM, Knowles MA. Inactiva-tion of the Rb pathway and overexpression of both isoforms of E2F3 areobligate events in bladder tumours with 6p22 amplification. Oncogene2008;27:2716–27.

28. Cheng ML, Berger MF, Hyman DM, Solit DB. Clinical tumour sequencingfor precision oncology: time for a universal strategy. Nat Rev Cancer2018;18:527–8.

29. Loriot Y, Necchi A, Park SH, García-Donas J, Huddart RA, Burgess EF, et al.Erdafitinib (ERDA; JNJ-42756493), a pan-fibroblast growth factor receptor(FGFR) inhibitor, in patients (pts) with metastatic or unresectable urothe-lial carcinoma (mUC) and FGFR alterations (FGFRa): Phase 2 continuousversus intermittent dosing. J Clin Oncol 2018;36:411–411.

30. Audenet F, Colin P, Yates DR, Ouzzane A, Pignot G, Long J-A, et al. Aproportion of hereditary upper urinary tract urothelial carcinomas aremisclassified as sporadic according to a multi-institutional databaseanalysis: proposal of patient-specific risk identification tool. BJU Int2012;110:E583–9.

31. Hampel H, Pearlman R, Beightol M, Zhao W, Jones D, Frankel WL, et al.Assessment of tumor sequencing as a replacement for lynch syndromescreening and current molecular tests for patients with colorectal cancer.JAMA Oncol 2018;4:806–13.

32. Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, Balar AV,Necchi A, et al. Atezolizumab in patients with locally advanced andmetastatic urothelial carcinoma who have progressed following treatmentwith platinum-based chemotherapy: a single-arm, multicentre, phase 2trial. Lancet 2016;387:1909–20.

33. Iyer G, Audenet F, Middha S, CarloMI, Regazzi AM, Funt S, et al. Mismatchrepair (MMR) detection in urothelial carcinoma (UC) and correlationwithimmune checkpoint blockade (ICB) response. J Clin Oncol 2017;35:15_suppl:4511–4511.

34. O'Brien T, Ray E, Singh R, Coker B, Beard R, British Association ofUrological Surgeons Section of Oncology. Prevention of bladder tumoursafter nephroureterectomy for primary upper urinary tract urothelial carci-noma: a prospective, multicentre, randomised clinical trial of a singlepostoperative intravesical dose of mitomycin C (the ODMIT-C Trial). EurUrol 2011;60:703–10.

35. Habuchi T, Takahashi R, Yamada H, Kakehi Y, Sugiyama T, Yoshida O.Metachronous multifocal development of urothelial cancers by intralum-inal seeding. Lancet 1993;342:1087–8.

36. Jones TD,WangM, Eble JN,MacLennanGT, L�opez-Beltr�anA, Zhang S, et al.Molecular evidence supporting field effect in urothelial carcinogenesis.Clin Cancer Res 2005;11:6512–9.

37. Hafner C, Knuechel R, Zanardo L, Dietmaier W, Blaszyk H, Cheville J, et al.Evidence for oligoclonality and tumor spread by intraluminal seeding inmultifocal urothelial carcinomas of the upper and lower urinary tract.Oncogene 2001;20:4910–5.

38. Wang Y, Lang MR, Pin CL, Izawa JI. Comparison of the clonality ofurothelial carcinoma developing in the upper urinary tract and thosedeveloping in the bladder. Springerplus 2013;2:412.


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2019;25:967-976. Published OnlineFirst October 23, 2018.Clin Cancer Res François Audenet, Sumit Isharwal, Eugene K. Cha, et al. Tract and Bladder Urothelial CarcinomaClonal Relatedness and Mutational Differences between Upper

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