Purpose:

To investigate genomic differences between urothelial carcinomas of the upper tract (UTUC) and bladder (UCB), with a focus on defining the clonal relatedness of temporally distinct tumors.

Experimental Design:

We prospectively sequenced tumors and matched germline DNA using targeted next-generation sequencing methods. The cohort included 195 UTUC patients and 454 UCB patients. For a subgroup of 29 patients with UTUC and a history of a subsequent UCB, both tumors were analyzed to assess their clonal relatedness.

Results:

With the progression to higher UTUC clinical state, there were fewer alterations in the RTK/RAS pathway but more alterations 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 the basis of an integrated analysis of tumor mutational burden, MSIsensor score and mutational signature, 7.2% of UTUC tumors were classified as MSI-high/MMR-deficient (MSI-H/dMMR). The risk of bladder recurrence after UTUC was significantly associated with mutations in FGFR3, KDM6A, CCND1, and TP53. Comparison of UCB with corresponding UTUC tumors from the same patient supports their clonal relatedness.

Conclusions:

UTUC and UCB exhibit significant differences in the prevalence of common genomic alterations. In individual patients with a history of both tumors, UCB and UTUC were always clonally related. Genomic characterization of UTUC provides information regarding the risk of bladder recurrence and can identify tumors associated with Lynch syndrome.

This article is featured in Highlights of This Issue, p. 897

Translational Relevance

To date, relatively few urothelial carcinomas of the upper tract (UTUC) have been examined using next-generation sequencing (NGS) methods. In this study, we sequenced 195 UTUC using a targeted NGS platform and compared the results to 454 urothelial carcinomas of the bladder (UCB). We identified significant differences in the prevalence of common genomic alterations between UTUC and UCB. Targeted NGS was a robust methodology for identifying UTUC patients whose tumors were microsatellite instability-high/mismatch-repair-deficient, the vast majority of which arose in patients with 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 clonally related. The latter results justify the development of methods to prevent lower tract seeding during surgery and the adoption of risk-adapted surveillance for bladder recurrence in patients with UTUC.

Urothelial carcinoma (UC) is the sixth most common cancer type in the United States (1). UCs of the bladder (UCB) account for the majority of UCs (90%–95%; ref. 2), whereas UCs of the upper tract (UTUC) are significantly less common, accounting for 5% to 10% (1, 3). Although both UCB and UTUC arise within the urothelium and have a similar histologic appearance, they have distinct clinical characteristics (4). UTUC is uniquely associated with several environmental risk factors (5–7) and is more common than UCB in patients with Lynch syndrome (8).

Due to the relative rarity of UTUC compared with UCB, most clinical and biological studies have focused predominantly or exclusively on bladder cancers, with the results and clinical implications extrapolated to UTUC. To date, relatively few UTUC have been examined using newer next-generation sequencing (NGS) methods and The Cancer Genome Atlas (TCGA) study of UCs excluded UTUC patients (9, 10). Small retrospective case series have, however, suggested differences in the prevalence of oncogenic driver mutations between UCB and UTUC (11, 12).

Here, we leveraged a prospective molecular characterization initiative to explore the potential clinical utility of tumor genomic characterization in patients with UTUC, in particular its ability to guide clinical management and to identify patients whose tumors could be attributed to a diagnosis of Lynch syndrome. We were particularly interested in determining whether differences in clinical characteristics reflect differences in the spectrum of genetic drivers between UTUC and UCB. Furthermore, as UTUC and UCB often co-occur in individual patients, we sought to address an ongoing controversy in the field as to whether temporally distinct tumors arising in individual patients are clonally related recurrences or represent distinct primary tumors.

Study samples

Written consent was obtained from all participating patients. The study was conducted in accordance with International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, the Declaration of Helsinki, and local laws, and approved by an institutional review board. In total, 195 UTUC and matched germline DNA samples from blood [including 84 UTUC samples from a prior retrospective study (ref. 11) and 111 samples profiled prospectively] were analyzed using a targeted NGS platform (MSK-IMPACT; Supplementary Methods; refs. 13, 14). Representative hematoxylin and eosin slides for each tumor were reviewed by a board-certified genitourinary pathologist (H. Al-Ahmadie) to confirm grade, stage, and urothelial histology. Patients with a predominant variant histology in the reviewed specimen were excluded. For comparative analyses, we used a cohort of 454 patients with UCB (including 102 UCB samples from the prior study, ref. 11) and no prior history of UTUC who were similarly analyzed by MSK-IMPACT at our center. For a subgroup of 29 patients with UTUC and a history of a subsequent UCB, we analyzed both tumors to define their clonal relatedness.

Statistical analysis

We used χ2 and exact Fisher tests to define statistical significance of differences between clinical and demographic categorical variables, and Wilcoxon and Kruskal–Wallis tests for continuous variables comparisons. Tumor mutational burden was adjusted to account for the number of genes included in the version of MSK-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 using FACETS (version 0.5.6; ref. 15). Microsatellite instability was assessed using the MSIsensor algorithm (version 0.2; ref. 16). To test tumors for clonal relatedness, we used somatic mutation data, including silent mutations (17). Q-values were used to indicate the P values adjusted for multiple comparisons using the Bonferroni method to control the false discovery rate. A P value of <0.05 was considered statistically significant. Analyses were conducted using R v.3.4.3 (https://cran.r-project.org).

Genomic landscape of UTUC

To explore the genomic landscape of UTUC across the disease spectrum, we analyzed 195 UTUC tumors, including 84 samples analyzed retrospectively and reported previously (11) and 111 samples collected and sequenced within the context of a prospective molecular characterization initiative. Overall, the UTUC samples included 84 tumors from patients with no muscle invasion (<pT2, 43%), 76 from patients with muscle invasion (≥pT2, 39%) and 35 from patients with metastatic disease (18%) at the time of tumor collection (Table 1). Eighty-five percent of tumors were high-grade. We performed targeted NGS analysis of matched tumor and germline DNA of at least 275 and up to 468 cancer-associated genes. The most frequently mutated genes included FGFR3 (40%), KMT2D (37%), KDM6A (32%), TP53 (26%), and ARID1A (23%; Fig. 1). We further grouped mutations by canonical pathway or functional significance (18).

Table 1.

Patient characteristics

VariablesUTUC (N = 195)Bladder (N = 454)P
Median age (IQR), years 67.1 (58.1–74.5) 67.5 (60.1–74.4) 0.692 
Sex 
 Male 121 (62%) 367 (81%) <0.001 
 Female 74 (38%) 87 (19%)  
Smoking status 
 Active 22 (11%) 63 (14%) 0.619 
 Former 112 (58%) 246 (54%)  
 Never 61 (31%) 143 (32%)  
Location 
 Renal pelvis 154 (79%) NA  
 Ureter 41 (21%) NA  
Clinical state 
 <pT2 84 (43%) 151 (33%) 0.055 
 ≥pT2 76 (39%) 213 (47%)  
 Metastatic 35 (18%) 90 (20%)  
Tumor grade 
 Low grade 30 (15%) 26 (6%) <0.001 
 High 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 variant 
 Yes 23 (12%) 126 (28%) <0.001 
 No 172 (88%) 328 (72%)  
VariablesUTUC (N = 195)Bladder (N = 454)P
Median age (IQR), years 67.1 (58.1–74.5) 67.5 (60.1–74.4) 0.692 
Sex 
 Male 121 (62%) 367 (81%) <0.001 
 Female 74 (38%) 87 (19%)  
Smoking status 
 Active 22 (11%) 63 (14%) 0.619 
 Former 112 (58%) 246 (54%)  
 Never 61 (31%) 143 (32%)  
Location 
 Renal pelvis 154 (79%) NA  
 Ureter 41 (21%) NA  
Clinical state 
 <pT2 84 (43%) 151 (33%) 0.055 
 ≥pT2 76 (39%) 213 (47%)  
 Metastatic 35 (18%) 90 (20%)  
Tumor grade 
 Low grade 30 (15%) 26 (6%) <0.001 
 High 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 variant 
 Yes 23 (12%) 126 (28%) <0.001 
 No 172 (88%) 328 (72%)  

NOTE: P values in bold are statistically significant.

Abbreviation: IQR, interquartile range.

Figure 1.

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

Figure 1.

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

Close modal

In patients with higher stage disease, there were fewer alterations in the RTK/RAS pathway (P < 0.001) but more alterations in TP53/MDM2 (P < 0.001). The frequency of activating alterations in FGFR3 and HRAS was lower in patients with higher stage disease (63% vs. 26% and 17% vs. 3%, respectively; P < 0.001). Conversely, the frequency of alterations in TP53 and MDM2 was significantly increased in patients with higher stage disease (11% vs. 31% and 4% vs. 17%, respectively; P < 0.001). The frequency of alterations in only two other genes was significantly greater in patients with lower stage UTUC tumors: STAG2, a component of the cohesin complex that plays a role in meiosis and mitosis (24% vs. 6%, P < 0.001), and FBXW7, a component of the SCF complex involved in regulating protein degradation of several oncogenes including cyclin E and c-Myc (12% vs. 0%, P < 0.001). When comparing the genomic profiles of UTUC based on the location of the primary tumor (renal pelvis vs. ureter), only KMT2C and KMT2D, two genes with a purported role in chromatin regulation, were significantly more frequently altered in ureteral tumors as compared with renal pelvic tumors after adjusting for multiple comparison (41% vs. 18%, Q = 0.023 and 59% vs. 32%, Q = 0.034, respectively).

Genomic comparison between UTUC and bladder tumors

To identify differences in the mutational landscape between upper and lower tract urothelial tumors, we compared the 195 UTUC tumors with 454 UCB tumors from patients who had no prior history of UTUC (Table 1). The spectrum of mutations identified by MSK-IMPACT in these bladder tumors was comparable to those reported by the TCGA (9, 10). Overall, TP53, RB1, and ERBB2 were significantly more frequently altered in UCB as compared with UTUC (46% vs. 26%, 20% vs. 3%, 19% vs. 8%, respectively; Q < 0.001), whereas FGFR3 and HRAS were significantly 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 were significantly more frequently altered in UCB compared with UTUC (56% vs. 43%, Q = 0.01 and 55% vs. 35%, Q < 0.001, respectively).

Figure 2.

Genomic differences between UTUC and UCB. A, Comparison of the differences in the prevalence of genomic alterations between UTUC and UCB, for the genes present at a frequency of ≥10% in any clinical state. The genes with significant differences after multiple comparison testing using the Bonferroni method are highlighted by red boxes. B, Volcano plot of the frequency of gene alterations [log2(OR)] by significance [−log10(P)] in urothelial carcinomas of the upper tract (UTUC) or bladder. The horizontal dotted line indicates an adjusted P < 0.001. C, Comparison of genomic alteration frequencies for molecular pathways commonly mutated in UCs.

Figure 2.

Genomic differences between UTUC and UCB. A, Comparison of the differences in the prevalence of genomic alterations between UTUC and UCB, for the genes present at a frequency of ≥10% in any clinical state. The genes with significant differences after multiple comparison testing using the Bonferroni method are highlighted by red boxes. B, Volcano plot of the frequency of gene alterations [log2(OR)] by significance [−log10(P)] in urothelial carcinomas of the upper tract (UTUC) or bladder. The horizontal dotted line indicates an adjusted P < 0.001. C, Comparison of genomic alteration frequencies for molecular pathways commonly mutated in UCs.

Close modal

We next sought to determine if the prevalence of alterations in individual genes varied as a function of clinical disease state in UTUC and UCB (Fig. 3). For clinical state <pT2, HRAS and KMT2C were significantly more frequently altered in UTUC (17% vs. 3%, Q = 0.003 and 30% vs. 12%, Q = 0.008, respectively). When limiting the analysis to only high-grade samples <pT2, HRAS was still significantly more frequently altered in UTUC as compared with UCB (22% vs. 4%, Q = 0.001). For clinical state ≥pT2, TP53 and RB1 were more frequently altered in UCB (62% vs. 39%, Q = 0.008 and 26% vs. 3%, Q < 0.001), whereas HRAS was more frequently altered in UTUC (12% vs. 2%, Q = 0.003). The TP53/MDM2 pathway was significantly more frequently altered in UCB (69% vs. 51%, Q = 0.022). For patients with metastatic disease at the time of tumor collection, RB1, ERBB2, and cell-cycle pathway–associated genes as a group were all more frequently altered in UCB (27% vs. 3%, Q = 0.031; 27% vs. 0%, Q = 0.007; 71% vs. 43%, Q = 0.015, respectively).

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 frequency of ≥10% at any clinical state. The genes with significant differences after multiple comparisons using the Bonferroni method are highlighted by red boxes.

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 frequency of ≥10% at any clinical state. The genes with significant differences after multiple comparisons using the Bonferroni method are highlighted by red boxes.

Close modal

Patients with Lynch syndrome have germline mutations in mismatch-repair (MMR)–associated genes, which results in an increased risk for the development of tumors with microsatellite instability (MSI) and hypermutation. UC belongs to the spectrum of Lynch syndrome, but UTUC is much more frequent than UCB in patients with Lynch syndrome (8). To assess for evidence of MMR deficiency in the two groups, we calculated tumor mutational burden and MSIsensor scores, a measure of somatic alteration of microsatellite regions (Supplementary Methods; refs. 16, 19). The median number of somatic mutations/Mb was significantly 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 samples were MSI-high (defined as an MSIsensor score ≥10), with 3.1% (6/194) having an indeterminate MSIsensor score (3–10). As expected, the number of mutations/Mb was significantly higher in tumors with MSIsensor scores ≥10 (median: 49.3) as compared with tumors with MSIsensor scores between 3 and 10 (median: 13.0; P = 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 and no prior history of UTUC.

UTUC is the third most prevalent cancer in Lynch syndrome (20), and current European guidelines recommend that germline DNA sequencing be considered for all UTUC patients younger than age 60 and those with a personal or familial history of Lynch-related cancers (21). However, no optimal screening strategy has been validated. In our cohort of 195 UTUC, consent to assess for germline mutations was obtained in 47 patients, typically those in whom there was clinical suspicion of a hereditary syndrome or a high MSIsensor score. Analysis of the germline sequencing data revealed that 12 of these 47 patients had a pathogenic or likely pathogenic germline mutation in a Lynch syndrome–associated gene (Supplementary Table S2). Notably, all tumors from these patients 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; Supplementary Table S2). Furthermore, of the 12 tumors with MSIsensor scores of ≥10, 10 had a Lynch syndrome–associated germline mutation, one a personal history strongly suggestive of Lynch syndrome but no consent for germline analysis (patient 2; Fig. 4), and one a somatic Lynch-like MSI-high tumor (patient 1; Fig. 4).

Figure 4.

Use of NGS to identify microsatellite instability-high (MSI-H)/mismatch-repair–deficient (dMMR) UTUC tumors. A, MSIsensor scores of UTUC samples were calculated using MSIsensor (version 0.2) and are displayed as a function of the number of somatic mutations/Mb, and its association with Lynch syndrome as determined 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 the absence of a germline mutation in these two genes, all consistent with a somatic Lynch-like MMR-deficient tumor. C, Mutational decomposition analysis of the UTUC samples with more than 10 somatic mutations. Case 2 had no consent for germline analysis. However, the patient's personal history of colon cancer, the tumor mutational burden (TMB) of 21 somatic mutations/Mb, the elevated MSIsensor score of 17.4, and the predominant MMR/MSI signature by mutational decomposition indicated that this patient had an MMR-deficient tumor, and thus germline testing would have been indicated. Notably, one patient with no clinical risk factors for hereditary UTUC but with an MSI-high tumor was found to have Lynch syndrome. Two of the 12 patients with pathogenic/likely pathogenic germline mutations in Lynch syndrome–associated genes had low or intermediate MSIsensor scores but elevated tumor mutational burden in the setting of a predominant MMR/MSI signature by mutational decomposition. Case 3 was 45 years old at diagnosis and had a TMB of 22 somatic mutations/Mb, an MSIsensor score 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 familial history of Lynch-related cancers, a UTUC with 343 somatic mutations/Mb, an MSIsensor score of 1.1, a germline mutation in MSH2, absence of MSH2 and MSH6 expression by IHC, and a predominant MMR/MSI signature.

Figure 4.

Use of NGS to identify microsatellite instability-high (MSI-H)/mismatch-repair–deficient (dMMR) UTUC tumors. A, MSIsensor scores of UTUC samples were calculated using MSIsensor (version 0.2) and are displayed as a function of the number of somatic mutations/Mb, and its association with Lynch syndrome as determined 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 the absence of a germline mutation in these two genes, all consistent with a somatic Lynch-like MMR-deficient tumor. C, Mutational decomposition analysis of the UTUC samples with more than 10 somatic mutations. Case 2 had no consent for germline analysis. However, the patient's personal history of colon cancer, the tumor mutational burden (TMB) of 21 somatic mutations/Mb, the elevated MSIsensor score of 17.4, and the predominant MMR/MSI signature by mutational decomposition indicated that this patient had an MMR-deficient tumor, and thus germline testing would have been indicated. Notably, one patient with no clinical risk factors for hereditary UTUC but with an MSI-high tumor was found to have Lynch syndrome. Two of the 12 patients with pathogenic/likely pathogenic germline mutations in Lynch syndrome–associated genes had low or intermediate MSIsensor scores but elevated tumor mutational burden in the setting of a predominant MMR/MSI signature by mutational decomposition. Case 3 was 45 years old at diagnosis and had a TMB of 22 somatic mutations/Mb, an MSIsensor score 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 familial history of Lynch-related cancers, a UTUC with 343 somatic mutations/Mb, an MSIsensor score of 1.1, a germline mutation in MSH2, absence of MSH2 and MSH6 expression by IHC, and a predominant MMR/MSI signature.

Close modal

To further characterize those tumors with evidence of hypermutation but indeterminate or low MSIsensor scores, we performed mutational signature decomposition analysis (22, 23) on all tumor samples with 10 or more single-nucleotide somatic mutations. Notably, only 6 of 12 tumors from patients with Lynch syndrome had a predominant MMR/MSI signature by mutational signature decomposition analysis, whereas the others exhibited a predominant mitotic clock (aging) signature. In the broader cohort of UTUC samples with 10 or more mutations without an MMR germline mutation, we identified evidence 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. As above, two patients with MSIsensor scores <10 had a Lynch syndrome–associated UTUC (patients 3 and 4; Fig. 4).

In sum, the data indicate that no single metric could identify all MMR-deficient UTUC tumors and that the positive and negative predictive value of current clinical germline testing guidelines is low (Supplementary Table S3). The data thus suggest that an integrated approach that incorporates tumor mutational burden, MSIsensor score, mutational signature analysis, and somatic and germline mutation of Lynch syndrome–associated genes is needed to accurately classify the MMR deficiency status of UTUC tumors. Given the limitations of current clinical germline testing guides, our results also support universal tumor/normal genetic profiling in all patients with UTUC to identify those with a Lynch syndrome–associated tumor.

Clonal relatedness of lower tract recurrences in UTUC patients

Of the 195 patients with UTUC, 137 underwent radical nephroureterectomy, with 57 (42%) later developing a UCB after a median time of 7.3 months (IQR: 4.1–13.7; Supplementary Table S4). After adjusting for clinical factors associated with bladder recurrence, including sex, history of prior bladder cancer, location, grade, and tumor stage (24), alterations in FGFR3 (HR = 3.00; 95% CI, 1.58–5.68; P = 0.001), KDM6A (HR = 2.27; 95% CI, 1.29–4.02; P = 0.005), and CCND1 (HR = 3.10; 95% CI, 1.17–8.21; P = 0.023) were significantly associated with a higher risk of developing a subsequent bladder tumor, whereas TP53 alterations were associated with a lower risk (HR = 0.32; 95% CI, 0.13–0.80; P = 0.014; Supplementary Table S5).

To determine whether the bladder tumors that arose in patients with a prior history of UTUC represented clonal recurrences or second primary tumors, we analyzed temporally distinct pairs of UTUC and UCB from 29 patients (Supplementary Table S6). Clonal relatedness between the tumors was investigated using all somatic mutation data, including synonymous mutations, as previously described (17). Sixteen patients (55%) had no prior history of UCB. All pairs of UTUC and UCB were deemed to have a shared clonal origin (P < 0.005). We did, however, observe some lesion-to-lesion heterogeneity with only 86% of somatic mutations being present in both the initial UTUC and the subsequent UCB recurrence (Fig. 5). In the 13 patients with a prior history of UCB, 75% of the mutations were found to be similar in both the UTUC 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 radical nephroureterectomy with tissue available for sequencing, 69% of mutations were shared between the initial UCB and subsequent UTUC, with all the tumor pairs again being of shared clonal origin (P < 0.005).

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 before resection of the UTUC. The arrows show the time between sample collection in months. Patient * underwent radical nephroureterectomy for pT2N0 UTUC and had a bladder recurrence 4 months later (pTa high grade). He was followed regularly for 65 months after the initial RNU and ultimately progressed to metastatic disease. The molecular profile of the metastasis was closer to the bladder tumor than to the radical nephroureterectomy specimen. However, all three tumor samples shared a clonal origin.

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 before resection of the UTUC. The arrows show the time between sample collection in months. Patient * underwent radical nephroureterectomy for pT2N0 UTUC and had a bladder recurrence 4 months later (pTa high grade). He was followed regularly for 65 months after the initial RNU and ultimately progressed to metastatic disease. The molecular profile of the metastasis was closer to the bladder tumor than to the radical nephroureterectomy specimen. However, all three tumor samples shared a clonal origin.

Close modal

Although they share a similar urothelial histologic appearance, UTUC and UCB have several epidemiologic and clinical differences (4). By comparing the mutational profiles of UTUC and UCB in 649 urothelial cancer samples (195 UTUC and 454 UCB), we sought to determine whether differences in somatic mutational patterns underlie the clinical differences between the two diseases. Although the spectrum of genomic alterations was similar between UTUC and UCB, significant differences in the prevalence of mutations in individual genes were observed, including a higher frequency of FGFR3 and HRAS alterations and a lower frequency of TP53, RB1, and ERBB2 alterations in UTUC as compared with UCB. Furthermore, we found that significant differences in the mutational frequency of several genes remained after adjusting for clinical disease state.

Consistent with prior studies (25), FGFR3 alterations were more common in low-grade tumors. FGFR3 mutations in urothelial cancer typically arise in one of several hotspot locations and have been shown to be oncogenic and thus represent potential therapeutic targets. Notably, 31% of high-grade UTUC had FGFR3 alterations, a frequency significantly higher than that observed in high-grade UCB (21%). As FGFR3 and TP53 tend to be mutually exclusive, our results lend further support 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 the upper tract than in the bladder, progress over time to high-grade disease (26).

RB1 alterations were rarely observed in our high-grade UTUC cohort (2%), whereas RB1 inactivation is common in MIBC, where it has been associated with genomic instability (27). It is noteworthy that no single pathognomonic molecular event has been identified in UC. Overall, we found that the RTK/RAS, PIK3/AKT, cell-cycle, and TP53/MDM2 pathways were all altered in around half the tumors, with RTK/RAS and TP53/MDM2 alterations being largely mutually exclusive. Recently, a whole-exome sequencing analysis of 27 UTUC samples confirmed that there was a significantly higher frequency of mutations in the p53 and related pathways in high-grade tumors, leading to genomic instability, copy-number alterations, and disruption of the cell-cycle and apoptotic pathways, which was corroborated by protein analysis (12). However, given the complex co-mutation pattern of UC and the lack of in-depth studies on the biological role of most of the oncogenes and tumor suppressors identified in this disease, a full understanding of the key co-mutational events required for progression to a high-grade, lethal phenotype will require further study.

Tumor genomic analysis can provide information that could be used by physicians to guide the clinical management of UTUC patients. For example, clinical tumor genomic profiling can prospectively identify potential therapeutic targets and thus guide treatment selection (28). Overall, over half of the urothelial tumors in the current study as well as the TCGA had potentially actionable genomic alterations, including ERBB2 amplifications and mutations and FGFR3 hotspot mutations (29). Furthermore, patients with Lynch syndrome are at increased risk for the development of UTUC, and the identification of patients with Lynch syndrome–associated tumors has treatment and screening implications for both affected patients and unaffected family members (30). Our data suggest that screening for hereditary UTUC based on clinical risk assessment or MSI status alone can miss some cases of Lynch syndrome (Supplementary Table S3). With the implementation of NGS technology in daily practice, integration of tumor mutational burden, analysis of microsatellite regions (MSIsensor score) and mutational signature could more robustly identify MMR-deficient tumors. Recently, upfront tumor sequencing in colorectal cancer has been shown to have greater sensitivity than the prior multitest approaches for Lynch syndrome screening (31). Our results support this strategy in UTUC as well. Furthermore, given the strong association between mutational burden and immunotherapy response (32), UTUC patients with microsatellite instability in the setting of MMR deficiency may represent a subpopulation of patients with a greater likelihood of benefiting from both local and systemic immunotherapies (33).

Intravesical recurrence after radical nephroureterectomy is common, with a median rate of recurrence of 22% to 47% (24), and several patient-specific and tumor-specific clinicopathologic predictors of recurrence have been identified (24). Here, we identified a strong association in multivariable analysis between the risk of recurrence and alterations in FGFR3, KDM6A, CCND1, and TP53. These findings could help identify those patients in greatest need for close monitoring or early instillation of mitomycin C (34). The pathogenesis of bladder tumors that arise after curative intent treatment of UTUC is an ongoing controversy in the field. Two different theories have been proposed: UCB could result from intraluminal seeding from the prior UTUC (35) or represent a second primary tumor in the setting of a toxin-induced, field-cancerization effect (36). Initial studies using loss-of-heterozygosity analysis reported conflicting results (37, 38). To our knowledge, this is the first study to use contemporary NGS methods to assess the clonality or multifocality of bladder tumors in patients with a history of UTUC. Our results clearly demonstrate the clonal origin of UTUC and UCB tumors that arise sequentially in individual patients with a history of both tumors. The results justify the exploration of methods to prevent lower tract seeding during radical nephroureterectomy or ureteroscopy and more rigorous surveillance for bladder recurrences after surgery, particularly in those patients with genetic features associated with lower tract recurrence (TP53-wild-type, FGFR3/KDM6A-mutant). Due to the limited efficacy of a single postoperative intravesical dose of mitomycin C (34), studies of adjuvant treatments designed to prevent bladder recurrences are also warranted in patients at highest risk for subsequent lower tract recurrence.

Although this study is the largest cohort of UTUC tumors genomically characterized to date, it has several limitations. Less frequent mutational events and structural alterations not covered in the MSK-IMPACT assay could not be detected and may have important prognostic implications, at least for individual patients. However, the majority of the genomic alterations identified 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 epigenetic regulation of genes may provide added prognostic information, a possibility not addressed by our targeted DNA-sequencing approach. Finally, individual mutations in specific genes may have different biological consequences. Integration of genome, transcriptome, and proteomic analyses may thus provide further insights into the factors that drive UTUC initiation and progression.

In conclusion, a genomic comparison of UTUC and UCB in a cohort of 649 patients revealed significant differences in the prevalence of common genomic alterations. However, in individual patients with a history of both tumors, we found that the UCB and UTUC were always clonally related. Genomic characterization of UTUC provides clinically relevant information, including identification of patients who could be candidates for molecularly driven clinical trials or for treatment off-label with agents approved for use in other cancer types; evaluation of the risk of bladder recurrence, which may help guide the selection of adjuvant therapies; and identification of those patients in need of germline analysis for Lynch syndrome. Routine implementation of tumor genomic profiling in patients with UTUC should thus be considered and prospectively evaluated.

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

Conception 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. Solit

Development 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. Solit

Acquisition 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. Solit

Analysis 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. Solit

Writing, 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. Solit

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.J. Pietzak, G. Dalbagni, J.E. Rosenberg, B.S. Taylor, J.A. Coleman, D.B. Solit

Study supervision: E.K. Cha, J.A. Coleman, D.B. Solit

This work was supported by NIH R01-CA182587-05, the Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Cycle for Survival, and the Thompson Family Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2018
.
CA Cancer J Clin
2018
;
68
:
7
30
.
2.
Babjuk
M
,
Böhle
A
,
Burger
M
,
Capoun
O
,
Cohen
D
,
Compérat
EM
, et al
EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2016
.
Eur Urol
2017
;
71
:
447
61
.
3.
Munoz
JJ
,
Ellison
LM
. 
Upper tract urothelial neoplasms: incidence and survival during the last 2 decades
.
J Urol
2000
;
164
:
1523
5
.
4.
Green
DA
,
Rink
M
,
Xylinas
E
,
Matin
SF
,
Stenzl
A
,
Rouprêt
M
, et al
Urothelial carcinoma of the bladder and the upper tract: disparate twins
.
J Urol
2013
;
189
:
1214
21
.
5.
Chiou
HY
,
Chiou
ST
,
Hsu
YH
,
Chou
YL
,
Tseng
CH
,
Wei
ML
, et al
Incidence of transitional cell carcinoma and arsenic in drinking water: a follow-up study of 8,102 residents in an arseniasis-endemic area in northeastern Taiwan
.
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 carcinomas 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 lynch syndrome
.
J Natl Cancer Inst
2012
;
104
:
1363
72
.
9.
The Cancer Genome Atlas Research Network
. 
Comprehensive molecular characterization 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 bladder cancer
.
Cell
2017
;
171
:
540
556
.
e25
.
11.
Sfakianos
JP
,
Cha
EK
,
Iyer
G
,
Scott
SN
,
Zabor
EC
,
Shah
RH
, et al
Genomic characterization 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
Comprehensive genomic characterization of upper tract urothelial carcinoma
.
Eur Urol
2017
;
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
,
Kim
HR
, et al
Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients
.
Nat Med
2017
;
23
:
703
13
.
15.
Shen
R
,
Seshan
VE
. 
FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing
.
Nucleic Acids 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 sequence data
.
Bioinformatics
2014
;
30
:
1015
6
.
17.
Ostrovnaya
I
,
Seshan
VE
,
Begg
CB
. 
Using somatic mutation data to test tumors 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 in high-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 targeted next-generation sequencing data
.
JCO Precis Oncol
2017
;
1
17
.
20.
Rouprêt
M
,
Yates
DR
,
Comperat
E
,
Cussenot
O
. 
Upper urinary tract urothelial cell carcinomas and other urological malignancies involved in the hereditary nonpolyposis colorectal cancer (lynch syndrome) tumor spectrum
.
Eur Urol
2008
;
54
:
1226
36
.
21.
Rouprêt
M
,
Babjuk
M
,
Compérat
E
,
Zigeuner
R
,
Sylvester
RJ
,
Burger
M
, et al
European Association of Urology guidelines on upper urinary tract urothelial carcinoma: 2017 update
.
Eur Urol
2018
;
73
:
111
22
.
22.
Alexandrov
LB
,
Nik-Zainal
S
,
Wedge
DC
,
Aparicio
SAJR
,
Behjati
S
,
Biankin
AV
, et al
Signatures of mutational processes in human cancer
.
Nature
2013
;
500
:
415
21
.
23.
Alexandrov
LB
,
Nik-Zainal
S
,
Wedge
DC
,
Campbell
PJ
,
Stratton
MR
. 
Deciphering signatures of mutational processes operative in human cancer
.
Cell Rep
2013
;
3
:
246
59
.
24.
Seisen
T
,
Granger
B
,
Colin
P
,
Léon
P
,
Utard
G
,
Renard-Penna
R
, et al
A systematic review and meta-analysis of clinicopathologic factors linked to intravesical recurrence after radical nephroureterectomy to treat upper tract urothelial carcinoma
.
Eur Urol
2015
;
67
:
1122
33
.
25.
Liu
X
,
Zhang
W
,
Geng
D
,
He
J
,
Zhao
Y
,
Yu
L
. 
Clinical significance of fibroblast growth factor receptor-3 mutations in bladder cancer: a systematic review and meta-analysis
.
Genet Mol Res
2014
;
13
:
1109
20
.
26.
Lindgren
D
,
Sjödahl
G
,
Lauss
M
,
Staaf
J
,
Chebil
G
,
Lövgren
K
, et al
Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma
.
PLoS One
2012
;
7
:
e38863
.
27.
Hurst
CD
,
Tomlinson
DC
,
Williams
SV
,
Platt
FM
,
Knowles
MA
. 
Inactivation of the Rb pathway and overexpression of both isoforms of E2F3 are obligate events in bladder tumours with 6p22 amplification
.
Oncogene
2008
;
27
:
2716
27
.
28.
Cheng
ML
,
Berger
MF
,
Hyman
DM
,
Solit
DB
. 
Clinical tumour sequencing for precision oncology: time for a universal strategy
.
Nat Rev Cancer
2018
;
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 urothelial carcinoma (mUC) and FGFR alterations (FGFRa): Phase 2 continuous versus 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
A proportion of hereditary upper urinary tract urothelial carcinomas are misclassified as sporadic according to a multi-institutional database analysis: proposal of patient-specific risk identification tool
.
BJU Int
2012
;
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 syndrome screening 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 and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial
.
Lancet
2016
;
387
:
1909
20
.
33.
Iyer
G
,
Audenet
F
,
Middha
S
,
Carlo
MI
,
Regazzi
AM
,
Funt
S
, et al
Mismatch repair (MMR) detection in urothelial carcinoma (UC) and correlation with immune 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 of Urological Surgeons Section of Oncology
. 
Prevention of bladder tumours after nephroureterectomy for primary upper urinary tract urothelial carcinoma: a prospective, multicentre, randomised clinical trial of a single postoperative intravesical dose of mitomycin C (the ODMIT-C Trial)
.
Eur Urol
2011
;
60
:
703
10
.
35.
Habuchi
T
,
Takahashi
R
,
Yamada
H
,
Kakehi
Y
,
Sugiyama
T
,
Yoshida
O
. 
Metachronous multifocal development of urothelial cancers by intraluminal seeding
.
Lancet
1993
;
342
:
1087
8
.
36.
Jones
TD
,
Wang
M
,
Eble
JN
,
MacLennan
GT
,
López-Beltrán
A
,
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 in multifocal 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 of urothelial carcinoma developing in the upper urinary tract and those developing in the bladder
.
Springerplus
2013
;
2
:
412
.