Comprehensive molecular characterization of urothelial bladder carcinoma

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive landscape of molecular alterations. There were statistically significant recurrent mutations in 32 genes, including multiple genes involved in cell-cycle regulation, chromatin regulation, and kinase signalling pathways, as well as 9 genes not previously reported as significantly mutated in any cancer. RNA sequencing revealed four expression subtypes, two of which (papillary-like and basal/squamous-like) were also evident in microRNA sequencing and protein data. Whole-genome and RNA sequencing identified recurrent in-frame activating FGFR3–TACC3 fusions and expression or integration of several viruses (including HPV16) that are associated with gene inactivation. Our analyses identified potential therapeutic targets in 69% of the tumours, including 42% with targets in the phosphatidylinositol-3-OH kinase/AKT/mTOR pathway and 45% with targets (including ERBB2) in the RTK/MAPK pathway. Chromatin regulatory genes were more frequently mutated in urothelial carcinoma than in any other common cancer studied so far, indicating the future possibility of targeted therapy for chromatin abnormalities.


Introduction
Urothelial carcinoma of the bladder is a major cause of morbidity and mortality worldwide, causing an estimated 150,000 deaths per year 1 . Previous studies have identified multiple regions of somatic copy number alteration, including amplification of PPARG, E2F3, EGFR, CCND1 and MDM2, as well as loss of CDKN2A and RB1 2,3 . Sequencing of candidate pathways has identified recurrent mutations in TP53, FGFR3, PIK3CA, TSC1, RB1 and HRAS 2,3 . More recently, a candidate gene study identified mutations at >10% frequency in several chromatin remodeling genes: KDM6A, CREBBP, EP300, and ARID1A 4 . Focused molecular analyses 5,6 have delineated tumour subtypes and identified kinase-activating FGFR3 gene fusions 7,8 .
We report here a comprehensive, integrated study of 131 high-grade muscle-invasive urothelial bladder carcinomas as part of The Cancer Genome Atlas (TCGA) project. Included are data on DNA copy number, somatic mutation, mRNA and microRNA expression, protein and phosphorylated protein expression, DNA methylation, transcript splice variation, gene fusion, viral integration, pathway perturbation, clinical correlates and histopathology to characterize the molecular landscape of urothelial carcinoma. This study identifies a number of mutations and regions of copy number variation that involve genes not previously reported as altered in a significant fraction of bladder cancers. It also identifies potential therapeutic targets in most of the samples analyzed.

Demographic, clinical and pathological data
Samples, (from 19 tissue source sites), consisted of 131 chemotherapy-naïve, muscleinvasive, high-grade urothelial tumours (T2-T4a, Nx, Mx), as well as peripheral blood (n=118) and/or tumour-adjacent, histologically normal-appearing bladder tissue (n=23). Cases were retained only if they met the following criteria: tumour nuclei constituted ≥ 60% of all nuclei, tumour necrosis was ≤ 20% of the specimen, and variant histologies (squamous or small cell) were ≤50% (Supplementary Text S1). Clinical and demographic characteristics are described in Supplementary Data File S1.1. Five expert genitourinary pathologists re-reviewed all of the cases for multiple parameters, including the extent of variant histology (Supplementary Text S1 and Fig. S1.1a).
Fifty-one per cent of mutations overall were Tp*C->(T/G) (Supplementary Table S2.1.1), a class of mutation recently reported to be mediated by one of the DNA cytosine deaminases, APOBEC 19,20 . APOBEC3B was expressed at high levels in all of the tumours, suggesting a major role for APOBEC-mediated mutagenesis in bladder carcinogenesis (Supplementary Figs. S12.1 and S12.2).  Table S2.8.1), suggesting that mutations in the two genes have redundant downstream effects on carcinogenesis or that the combined loss is synthetically lethal.

Chromosomal rearrangements and viral integration
To identify structural variations and pathogen sequences, we used low-pass, paired-end, whole-genome sequencing (WGS; 6-8× coverage) of 114 tumours and RNA sequencing of all tumours. We detected 2,529 structural aberrations, including 1,153 that involve genegene fusions. Among the translocations, 379 were inter-chromosomal, 237 were intrachromosomal, 274 were the result of inversions and 263 resulted from deletions (Supplementary Table S3.1). We found several recurrent translocations of likely pathogenic significance, including an intra-chromosomal translocation on chromosome 4 involving FGFR3 and TACC3 (n=3). The breakpoints were in intron 16 (2 cases) or exon 17 (1 case) of FGFR3 and intron 10 of TACC3 (confirmed by DNA sequencing and RNA-seq). All three lead to fusion mRNA products whose predicted proteins include the N-terminal 758 amino acids of FGFR3 fused with the C-terminal 191 amino acids of TACC3 (Fig. 2a). Based on the structure of the FGFR3-TACC3 fusion protein, we predict that it can autodimerize, leading to constitutive activation of the kinase domain of FGFR3. FGFR3-TACC3 fusion, which was recently described in both glioblastoma 21  represents a promising therapeutic target. The ERBB2 gene was also involved in translocations in four tumours, all with different fusion partners and all confirmed by DNA sequencing, RNA-Seq or both. In one case, exons 4 to 29 of ERBB2 were fused to the promoter plus exon 1 of DIP2B, and the fusion product was amplified (Fig. 2b). Two other fusion products resulted in novel mRNA products whose biological significance is not known.
We identified viral DNAs in 7 of 122 tumours (6%), and viral transcripts in 5 of 122 (4%). Three tumours expressed cytomegalovirus (CMV) transcripts (encoding RL5A, RNA2.7, RL9A, RNA1.2, UL5 and UL22A), one expressed BK polyoma virus and one expressed human papilloma virus 16 (HPV16). HPV16 and human herpes virus 6B DNA were each identified in one other sample but without expression. None of the tumours expressing CMV showed evidence of CMV integration into the host genome, suggesting the presence of a stable episome. In the BK-positive tumour, two BK genes were integrated into GRB14, a signaling adapter protein for receptor tyrosine kinases. In the HPV 16-expressing case, the virus integrated into BCL2L1, an apoptosis-regulating gene (Fig. 2c). In that tumour, BCL2L1 was amplified (∼6×) and overexpressed (∼10× median; > 2× any of the other samples). Overall, those findings suggest that viral infection may play a role in the development of a small percentage of urothelial carcinomas.
Reverse-phase protein array (RPPA) data indicate that clusters I and II express high HER2 (ERBB2) levels and an elevated estrogen receptor beta (ESR1) signaling signature, indicating potential targets for hormone therapies such as tamoxifen or raloxifene (Fig. 3d). In fact, the HER2 protein levels in a subset of the tumours are comparable to those found in TCGA HER2-positive breast cancers 23 .
For comparison, we asked whether any of the four clusters show gene signatures similar to those identified in any other tumour type(s) among the first 11 analyzed by TCGA. We found that the signature of bladder cancer cluster III ('basal/squamous-like') is similar to that of basal-like breast cancers, as well as squamous cell cancers of the head and neck and lung ( Supplementary Fig. S4.2) 24,25 . All four of those cancer types express characteristic epithelial lineage genes, including KRT14, KRT5, KRT6 and EGFR. Basal-like subtype 26 and squamous cell subtype 27  were recently reported to mark stem/progenitor cells 26 . Some of those samples also show a level of variant squamous histology (Fig. 3b). Bladder clusters I and II show features similar to those of Luminal A breast cancer, with high mRNA and protein expression of luminal breast differentiation markers, including GATA3 and FOXA1 (Fig. 3c). Markers of urothelial differentiation such as the uroplakins are also highly expressed in clusters I and II, as are the epithelial marker E-cadherin and members of the miR-200 family of microRNAs (which target multiple regulators of epithelial-mesenchymal transition) 28 (Fig. 3c). Taken together, those observations suggest that, despite their diverse tissue origins, some bladder, breast, head and neck and lung cancers share common pathways of tumour development.
To determine if the expression-based clusters could be seen in other datasets, we used the muscle-invasive bladder cancer samples from Sjodahl, et al. 27 , hierarchically clustering them with the genes used in our analysis. From the sample dendrogram, we identified four groups (Supplemental Figure S4.3a). The four groups identified in the Sjodahl data set correlated well with the four clusters identified in our TCGA data. (Supplemental Figure  S4.3b).
When we analyzed the RNA-seq data for transcript splice variation using SpliceSeq 29 (Supplemental Material S11), one finding of interest was an average of 3% PKM1 and 97% PKM2 transcripts in the tumour samples. The PKM2 isoform of pyruvate kinase is the principal driver of a shift to aerobic glycolysis in tumours (the Warburg effect) 30 . Therefore, urothelial bladder cancers (and other cancer types) may prove sensitive to inhibition of glycolysis or related metabolic pathways.

Pathway analysis and therapeutic targeting
Integrated analysis of the mutation and copy-number data revealed three main pathways as frequently dysregulated in bladder cancer: cell cycle regulation (altered in 93% of cases); kinase and PI3K signaling (72%); chromatin remodeling, including mutations/SCNAs in histone modifying genes (89%); and components of the SWI/SNF nucleosome remodeling complex (64%) (Fig. 4a). To complement those results for well-defined pathways, we applied network analysis methods to examine other possible interactions between genes and pathways (Fig. 4b). In particular, we used the TieDIE algorithm to search for causal regulatory interactions within the PARADIGM network, which connects mutated genes to active transcriptional hubs 31,32 . The analysis identified a sub-network linking mutated histone-modifying genes to a large array of activated transcription factors, suggesting potential far-reaching effects of histone modification on other pathways (Supplementary Fig.  S8.2.1), converging on MYC/MAX regulation. Both MYC and MAX showed similar levels of pathway activity, independent of mutations in chromatin genes, suggesting that mutations in histone-modifying genes provide just one mechanism for disruption of the MYC/MAX hub. In contrast, tumours with chromatin-related mutations showed differential activity of transcription factors FOXA2 and SP1, implicating de-differentiation processes as a result of the mutations. Our network analysis also identified HSP90AA1 as a critical signaling hub, suggesting that inhibitors of HSP90 may have therapeutic value in urothelial carcinoma. Although the linkages between mutations and transcriptional changes were statistically significant in terms of their proximity in the network (as determined by permutation tests; Page 7 Nature. Author manuscript; available in PMC 2014 September 20. see Supplementary Fig. S8.2), further studies will be needed to assess the biological relevance of the findings.
Integrated analysis also identified mutations, copy number alterations or RNA expression changes affecting the PI3-kinase/AKT/mTOR pathway in 42% of the tumours (Fig.5a). Included were activating point mutations in PIK3CA (17%; potentially responsive to PI3K inhibitors), mutation or deletion of TSC1 or TSC2 (9%; potentially responsive to mTOR inhibitors) and overexpression of AKT3 (10%; potentially responsive to AKT inhibitors). We also observed mutations, genomic amplifications or gene fusions that affect the RTK/RAS pathway in 44% of the tumours (Fig. 5b). Included were events that can activate FGFR3 (17%; potentially responsive to FGFR inhibitors or antibodies), amplification of EGFR (9%; potentially responsive to EGFR antibodies or inhibitors), mutations of ERBB3 (6%; potentially sensitive to ERBB kinase inhibitors) and mutation or amplification of ERBB2 (9%; potentially sensitive to ERBB2 kinase inhibitors or antibodies). ERBB3 mutations in bladder cancer have been noted previously 4 , but statistically significant mutation of ERBB2 in bladder cancer has not been reported. Both genes are potential therapeutic targets in other diseases [33][34][35] (Fig. 5c). Interestingly, ERBB2 alterations were approximately as frequent in this study as in TCGA breast cancers, but with fewer amplifications and more mutations (Fig. 5d) 24 .

Discussion
This integrated study of 131 invasive urothelial bladder carcinomas provides numerous novel insights into disease biology and delineates multiple potential opportunities for therapeutic intervention. Treatment for muscle-invasive bladder cancer has not advanced beyond cisplatin-based combination chemotherapy and surgery in the past 30 years 36 , and no new drugs for the disease have been approved in that time. Median survival for patients with recurrent or metastatic bladder cancer remains 14-15 months with cisplatin-based chemotherapy, and there is no widely recognized second-line therapy 37 . With the exception of a single case report, there is also no known benefit from treatment with newer, targeted agents 38 . Several of the genomic alterations identified in this study, particularly those involving the PI3-kinase/AKT/mTOR, CDKN2A/CDK4/CCND1 and RTK/RAS pathways, as well as ERBB2 (Her-2), ERBB3 and FGFR3, are amenable in principle to therapeutic targeting. Clinical trials based on patients with relevant druggable genomic alterations are warranted.
FGFR3 mutation is a common feature of low-grade non-invasive papillary urothelial bladder cancer, but it occurs at a much lower frequency in high-grade invasive bladder cancer. The cluster analysis in Figure 3 highlights multiple mechanisms of FGFR3 activation, and its strong association with papillary morphology. The data presented here suggest a subset of muscle-invasive cancers that can potentially be targeted through FGFR3. Similarly, ERBB2 amplification may be targetable by strategies used in breast cancer, by small-molecule tyrosine kinase inhibitors or by novel immunotherapeutic approaches (NCT01353222) 34 . The data here provide further support for several ongoing ERBB2-targeted trials in bladder cancer and further define the subpopulation of cancers suited to that approach. Finally, cluster III of the integrated expression profiling analysis reveals the existence of a urothelial carcinoma subtype with high cancer stem cell content (including KRT14/5), perhaps providing another avenue for therapeutic targeting.
The alterations identified in epigenetic pathways also suggest new possibilities for bladder cancer treatment. Ninety-nine (76%) of the tumours analyzed here had an inactivating mutation in one or more of the chromatin regulatory genes, and 53 (41%) had at least two such mutations. Overall, the bladder cancers showed a mutational spectrum highly enriched with mutations in chromatin regulatory genes (Supplementary Table S2.10). Further, integrated network analyses revealed a profound impact of those mutations on the activity levels of various transcription factors and pathways implicated in cancer. Recent development of drugs that bind competitively to acetyl-lysine recognition motifs (i.e., bromodomains) might prove useful for treatment of the subset of bladder tumours that have abnormalities in chromatin-modifyingenzymes 39 . Our findings suggest that bladder cancer is a prime candidate for further exploration of that approach to therapy.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Red, activating genetic alterations; blue, inactivating genetic alterations. Percentages shown denote activation or inactivation of at least one allele. b, The network connecting mutated histone-modifying genes to transcription factors with differential activity (methodology and larger implicated network in Supplementary Fig. 8.2.1). Each gene is depicted as a multiring circle with various levels of data, plotted such that each 'spoke' in the ring represents a single patient sample (same sample ordering for all genes). 'PARADIGM' ring: bioinformatically inferred levels of gene activity (red, higher activity); 'Transcriptional Activity': mean mRNA levels of all of the targets of each transcription factor; 'Expression': mRNA levels relative to normal (red, high); 'Mutation in gene': somatic mutation; 'Mutation in histone modifier genes': somatic mutation in at least one such gene; 'IPL anticorrelation': genes with PARADIGM Integrated Pathway Levels (IPLs) inversely correlated with histone-gene mutation status. Gene-gene relationships inferred using public resources.