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4500 Background: We reported the integrated molecular analysis of 131 tumors in 2014 (Nature 507:315, 2014) and now report on the entire cohort of 412 tumors from the TCGA project in chemotherapy-naïve, muscle-invasive urothelial bladder cancer. Methods: Following strict clinical and pathologic quality control, tumors were analyzed for DNA copy number variants, somatic mutations (WES), DNA methylation, mRNA, non-coding RNA (lncRNA and miRNA) and (phospho-) protein expression, gene fusions, viral integration, pathway perturbation, clinical correlates, outcomes, and histopathology. Results: There was a high overall somatic mutation rate (8.2/Mb), as previously reported. There were 58 significantly mutated genes (SMGs) (MutSig_2CV), increased from 32 in the original report. We identified 5 mutation signatures including APOBEC-a and b, ERCC2, C > T_CpG, and a single ultra-mutated sample with a functional POLE mutation. APOBEC mutagenesis explained 70% of the mutation burden and was associated with survival (p = 0.0013). High mutation burden and neoantigen load were also associated with improved outcome (p = 0.00014 and 0.00078). The previously identified four mRNA subtypes were predicted on the larger set and also identified a novel poor-survival ‘neuronal’ subtype that nevertheless lacked small cell or neuroendocrine histology. Clustering converged for mRNA, lncRNA and miRNA expression, and for inferred activity of gene sets associated with regulator expression. We identified subsets with differential epithelial-mesenchymal transition scores, carcinoma-in-situ scores, and survival, with implications for distinct therapeutic potential. Conclusions: This integrated analysis of 412 TCGA patient samples validates and extends observations from the first 131 patients and significantly increases our power to detect additional low-frequency aberrations. The results provide unique insights into mechanisms of bladder cancer development, and identify novel subsets of MIBC that may benefit from differential treatment approaches.

405 Background: We reported the integrated molecular analysis of 131 tumors in 2014 (Nature 507:315, 2014) and now report on the entire cohort of 412 tumors from the TCGA project in chemotherapy-naïve muscle invasive urothelial bladder cancer. Methods: Following strict clinical and pathologic quality control, tumors were analyzed for DNA copy number variants, somatic mutations (WES), DNA methylation, mRNA, microRNA and (phospho-) protein expression, transcript splicing, gene fusions, viral integration, pathway perturbation, clinical correlates, and histopathology Results: There was a high overall somatic mutation rate (8.0/Mb), as previously reported, with a median of 245 and mean of 348 coding region mutations per sample. There were 54 significantly mutated genes (SMGs) (MutSig_2CV), increased from 32 in the original report. TP53 remained the most commonly mutated gene, and chromatin-modifying genes (MLL2, ARID1A, KDM6A) were also frequently mutated. KRAS, ERBB2, RB1, and ELF3 showed significant increased frequencies of mutation. High mutation burden was associated with improved outcome (p = 0.0004). APOBEC mutagenesis explained 70% of the mutation burden and also was associated with survival. 167 genes were silenced by promoter DNA hypermethylation in at least 5% of tumors. Several SMGs were also epigenetically silenced at various frequencies (ZNF773, CDKN2A, FAT1, CASP8). The previously identified four mRNA subtypes were predicted on the larger set identifying a similar proportion of samples in each subtype. We identified 5 expression subtypes based on reverse-phase protein arrays(344 tumors). Five miR expression subtypes were strongly associated with 4 mRNA-based subtypes (P = 3.8E-37) and associated with overall survival (P = 0.05). Conclusions: This integrated analysis of 412 TCGA patient samples validates and extends observations from the first 131 patients and significantly increases our power to detect additional low-frequency aberrations. The results provide a robust basis for functional studies to further our understanding of the biology of bladder cancer, and aid in the development of more precisely targeted therapies.

4509 Background: Urothelial carcinoma (UC) is a major cause of mortality with no approved molecularly targeted agents or good treatment options beyond cisplatin-based chemotherapy. Methods: For The Cancer Genome Atlas (TCGA), we profiled muscle-invasive UCs for mutations, DNA copy number variants (CNVs), mRNA and microRNA expression, protein expression and phosphorylation, DNA methylation, transcript splicing, gene fusion, viral integration, pathway perturbation, clinical correlates, and histopathology. The finding here are based on the first 131 tumors, but >250 more are being processed as of 1/2014. Results: Whole-exome sequencing revealed 29 recurrently mutated genes. Potential therapeutic targets included altered PIK3CA, ERBB2, FGFR3, TSC1, and ERBB3, plus mutated chromatin-regulating genes MLL, MLL2, MLL3, CREBBP, CHD7, SRCAP, ARID1A, KDM6A (UTX), and EP300. There were 27 focal CNVs, including CDKN2A deletion in 47%. Low-pass whole-genome sequencing identified 3 tumors with FGFR3-TACC3 fusions. Viral...