Urothelial carcinoma of the bladder (UCB) is genomically heterogeneous, with frequent alterations in genes regulating chromatin state, cell cycle control, and receptor kinase signaling. To identify prognostic genomic markers in high-grade UCB, we used capture-based massively parallel sequencing to analyze 109 tumors. Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases. The presence of a recurrent phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation was associated with improved recurrence-free survival (RFS) (hazard ratio [HR]: 0.35; p = 0.014) and improved cancer-specific survival (CSS) (HR: 0.35; p = 0.040) in patients treated with radical cystectomy (RC). In multivariable analyses controlling for pT and pN stages, PIK3CA mutation remained associated with RFS (HR: 0.39; p = 0.032). The most frequent alteration, TP53 mutation (57%), was more common in extravesical disease (69% vs 32%, p = 0.005) and lymph node–positive disease (77% vs 56%, p = 0.025). Patients with cyclin-dependent kinase inhibitor 2A (CDKN2A)–altered tumors experienced worse RFS (HR: 5.76; p < 0.001) and worse CSS (HR: 2.94; p = 0.029) in multivariable analyses. Mutations in chromatin-modifying genes were highly prevalent but not associated with outcomes. In UCB patients treated with RC, PIK3CA mutations are associated with favorable outcomes, whereas TP53 and CDKN2A alterations are associated with poor outcomes. Genomic profiling may aid in the identification of UCB patients at highest risk following RC.
Using next-generation sequencing, we identified genomic subsets of high-grade urothelial bladder cancer associated with favorable and unfavorable outcomes. These findings may aid in the selection of patients most likely to benefit from novel combined modality approaches.
Keywords: Bladder cancer, Genomics, Clinical outcomes, PIK3CA, Mutation.
Despite multimodality treatment, patients with bladder cancer who undergo radical cystectomy (RC) remain at high risk for recurrence. We hypothesized that the significant variability in outcomes of patients with urothelial carcinoma of the bladder (UCB) partly results from differences in the genetic changes mediating UCB development and progression , , , and . With the goal of identifying prognostic genomic alterations, we used capture-based next-generation sequencing to analyze high-grade UCB tumors for somatic mutations in cancer-associated genes.
Tissue samples and matched germline blood from 109 patients with high-grade UCB were sequenced using MSK-IMPACT, as previously described (details in Supplemental Materials and Methods) . Cohort characteristics are listed in Supplemental Table 1. Average sequence coverage was 579× across all targeted exons. An average of 10 mutations was detected per tumor (range: 0–46). Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases (Fig. 1). As validation, we reanalyzed all samples using an orthogonal sequencing platform including all coding exons of STAG2, KDM6A, ARID1A, and KMT2D (also known as MLL2). Using this method, the vast majority of mutations in these genes identified by MSK-IMPACT (99.3%) were confirmed.
Most commonly altered genes in 109 high-grade urothelial carcinoma of the bladder tumors analyzed by MSK-IMPACT. The number of patients with alterations is depicted on the right. Alterations were categorized by type, as recurrent missense mutations, novel missense mutations, truncating mutations (frameshift, nonsense, and splice site), and copy number amplifications or deletions. The oncoprint shows the distribution of alterations across the sequenced samples. The top bar graph illustrates the number of MSK-IMPACT gene alterations per sample.
We analyzed mutations and copy number alterations independently and in the context of cancer-related pathways. The most common alteration was TP53 mutation, identified in 62 patients (57%) (Supplemental Fig. 1a). Alterations in genes that regulate entry into the S phase of the cell cycle were also prevalent (46%) (Supplemental Fig. 1b). Consistent with previous reports, mutations in chromatin-modifying genes (CMGs) were highly prevalent, occurring in 83% of patients (Supplemental Fig. 1c) , , and . KDM6A was mutated in 45 patients (41%), with 41 of 45 truncating mutations. ARID1A alterations (28%), similarly enriched for truncating mutations (30 of 31), were mutually exclusive with SMARCA4 alterations, suggesting potential overlapping functionality. The histone acetyltransferase genes CREBBP and EP300 also exhibited a pattern of mutually exclusive truncating mutations (13% and 15%, respectively).
Alterations in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway, mutationally activated in many cancers, are potential therapeutic targets in UCB . We identified PI3K/Akt pathway alterations in 38 patients (35%) (Supplemental Fig. 2a) Recurrent missense mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), which encodes the α subunit of PI3-kinase, were detected in 23 patients (21%); the vast majority of the mutations were functionally validated activating helical domain mutations (Supplemental Fig. 2b). Additional recurrently mutated PI3K/Akt pathway genes included PTEN (6%), AKT1 (2%), and TSC1 (6%).
To identify prognostic genomic markers, we analyzed associations between alteration status, clinicopathologic characteristics, and outcomes in 89 patients whose tumors were obtained at RC. For these analyses, we included 22 alterations (Supplemental Table 2) with putative functional significance (oncogenes: recurrent missense mutations and amplifications; tumor suppressors: nonsense mutations, frameshift indels, and deletions). TP53 mutations were more common in extravesical than organ-confined disease (69% vs 32%; p = 0.005), and p53 pathway alterations were more common in node-positive disease (77% vs 56%; p = 0.025) (Supplemental Table 2).
The presence of a recurrent missense mutation in PIK3CA was associated with significantly improved recurrence-free survival (RFS) and cancer-specific survival (CSS) (p = 0.014 and 0.040, respectively) (Fig. 2a). A similar improvement was observed on analyzing PI3K/Akt pathway genes as a functionally related group (hazard ratio [HR]: 0.34, p = 0.006 and HR: 0.29, p = 0.01) (Fig. 2b). After controlling for pT stage and nodal involvement, PIK3CA mutation and PI3K/Akt pathway alteration remained significant predictors of improved RFS (HR: 0.39, p = 0.032 and HR: 0.37, p = 0.017, respectively) (Supplemental Table 3). It is notable that PIK3CA alterations have been associated with favorable disease-specific outcomes in other malignancies  and . Although their disease-specific outcomes were relatively favorable, UCB patients with PIK3CA-mutant tumors still experienced a significant rate of disease recurrence (44% at 5 yr). Adjuvant trials of selective PI3-kinase inhibitors in this genetically defined subset of patients would thus be rational.
Associations with PI3K/Akt pathway alterations and clinical outcomes in 89 patients with urothelial carcinoma of the bladder treated with radical cystectomy. Recurrence-free survival and cancer-specific survival stratified by (A) PIK3CA mutation status and (B) PI3K/Akt pathway alteration status. The p values reflect the log-rank test.
Patients with p53 pathway alterations had worse RFS (HR: 1.92; p = 0.048) and CSS (HR: 2.94; p = 0.013), but these numbers did not remain statistically significant after adjustment for pT and pN stages (Supplemental Table 3). Patients with cyclin-dependent kinase inhibitor 2A (CDKN2A) alterations experienced worse RFS and CSS in multivariable analyses (HR: 5.76, p < 0.001 and HR: 2.94, p = 0.029, respectively). The significantly poor outcome of patients with CDKN2A-altered tumors provides rationale for the study of selective CDK4 inhibitors in this genomic subgroup.
We identified higher CMG mutation rates than previously reported . Methodological differences may account for this outcome, as our deep sequencing approach (mean coverage: 579×) likely provided greater sensitivity. However, we did not find an association between CMG mutations and outcomes. Given the presence of these mutations in nearly all UCB tumors (83% in this series), we speculate that epigenetic alterations may be necessary events in UCB pathogenesis, and thus CMG mutations are not associated with outcomes.
Studies investigating the prognostic significance of alterations in the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in UCB have reported contradictory findings. In one study, STAG2 alterations were associated with worse survival ; another study found that loss of STAG2 expression was associated with worse disease characteristics and outcomes following RC . In contrast, a third study reported that loss of STAG2 expression was associated with improved outcomes . In our study, we observed no association between STAG2 mutations and outcomes.
Given the genomic heterogeneity of bladder cancer, one limitation of the current study is its sample size. We may have also underestimated the genetic variation present within individual tumors by sampling a single site. Although our assay included all highly mutated genes identified by recent whole exome studies, less frequently mutated genes and structural alterations were not detectable with our capture-based approach. Finally, our targeted DNA sequencing approach does not address epigenetic differences and/or differences in gene expression . Given these limitations of the current study, a larger and more comprehensive analysis may identify co-mutation patterns that are more predictive of disease outcome.
In summary, we performed capture-based next-generation sequencing in a large cohort of high-grade UCB patients with the goal of identifying prognostic genomic alterations. Patients with TP53-mutant tumors experienced worse outcomes, but TP53 mutations were not significantly associated with outcome in multivariable analyses, given their association with locally advanced and/or node-positive disease. In contrast, PIK3CA and/or PI3K/Akt pathway alterations were associated with improved disease-specific outcomes independent of tumor and lymph node stage, whereas CDKN2A alterations were independently associated with worse outcomes. These findings, pending validation, may inform patient selection for novel combined modality approaches.
Author contributions: Bernard H. Bochner 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: Kim, Cha, Iyer, Al-Ahmadie, Solit, Bochner.
Acquisition of data: Kim, Cha, Sfakianos, Sun, Yee, Al-Ahmadie.
Analysis and interpretation of data: Kim, Cha, Sfakianos, Iyer, Zabor, Scott, Ostrovnaya, Ramirez, Shah, Schultz, Berger, Solit, Bochner.
Drafting of the manuscript: Kim, Cha, Solit, Bochner.
Critical revision of the manuscript for important intellectual content: Kim, Cha, Sfakianos, Iyer, Zabor, Berger, Al-Ahmadie, Solit, Bochner.
Statistical analysis: Zabor, Ostrovnaya.
Obtaining funding: Solit, Bochner.
Administrative, technical, or material support: None.
Supervision: Ostrovnaya, Reuter, Bajorin, Rosenberg, Berger, Solit, Bochner.
Other (specify): None.
Financial disclosures: Bernard H. Bochner certifies that all conflicts of 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, consultancies, 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: The design and conduct of this study and the collection, management, analysis, and interpretation of the data were funded by the National Institutes of Health, the Michael and Zena Wiener for Therapeutics Program in Bladder Cancer, Cycle for Survival, and Pin Down Bladder Cancer.
-  Y. Gui, G. Guo, Y. Huang, et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat Genet. 2011;43:875-878 Crossref
-  G. Guo, X. Sun, C. Chen, et al. Whole-genome and whole-exome sequencing of bladder cancer identifies frequent alterations in genes involved in sister chromatid cohesion and segregation. Nat Genet. 2013;45:1459-1463
-  Cancer Genome Atlas Research, Network. Comprehensive molecular characterization of urothelial bladder, carcinoma. Nature. 2014;507:315-322
-  G. Iyer, H. Al-Ahmadie, N. Schultz, et al. Prevalence and co-occurrence of actionable genomic alterations in high-grade bladder cancer. J Clin Oncol. 2013;31:3133-3140 Crossref
-  N. Wagle, M.F. Berger, M.J. Davis, et al. High-throughput detection of actionable genomic alterations in clinical tumor samples by targeted, massively parallel sequencing. Cancer Discov. 2012;2:82-93 Crossref
-  K. Kalinsky, L.M. Jacks, A. Heguy, et al. PIK3CA mutation associates with improved outcome in breast cancer. Clin Cancer Res. 2009;15:5049-5059 Crossref
-  H. Shigaki, Y. Baba, M. Watanabe, et al. PIK3CA mutation is associated with a favorable prognosis among patients with curatively resected esophageal squamous cell carcinoma. Clin Cancer Res. 2013;19:2451-2459 Crossref
-  D.A. Solomon, J.S. Kim, J. Bondaruk, et al. Frequent truncating mutations of STAG2 in bladder cancer. Nat Genet. 2013;45:1428-1430
-  C. Balbas-Martinez, A. Sagrera, E. Carrillo-de-Santa-Pau, et al. Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy. Nat Genet. 2013;45:1464-1469
-  G. Sjodahl, M. Lauss, K. Lovgren, et al. A molecular taxonomy for urothelial carcinoma. Clin Cancer Res. 2012;18:3377-3386 Crossref
a Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
b Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
c Weill Medical College of Cornell University, New York, NY, USA
d Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA
e Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
f Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
g Department of Computational Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Corresponding author. Department of Surgery, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Tel. +1 646 422 4387; Fax: +1 212 988 0759.
These authors contributed equally.
© 2014 European Association of Urology, Published by Elsevier B.V.