Abstract: Cisplatin-based chemotherapy is the standard of care for patients with muscle-invasive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant cisplatin-based chemotherapy is associated with improved survival, although molecular determinants of cisplatin response are incompletely understood. We performed whole-exome sequencing on pretreatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma who received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis “responders,” 25 pT2+ “nonresponders”) to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q & lt; 0.01). Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle-invasive urothelial carcinoma. Significance: Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemosensitivity in muscle-invasive urothelial carcinoma, and clinically identified mutations lead to cisplatin sensitivity in vitro. Nucleotide excision repair pathway defects may drive exceptional response to conventional chemotherapy. Cancer Discov; 4(10); 1140–53. ©2014 AACR. See related commentary by Turchi et al., p. 1118 This article is highlighted in the In This Issue feature, p. 1103

Abstract: Urothelial carcinoma (UC) is a major cause of morbidity and mortality for which there are no approved molecularly targeted agents and few good treatment options beyond cisplatin-based chemotherapy. As part of The Cancer Genome Atlas (TCGA) Project, we analyzed 131 chemotherapy-naive, muscle-invasive UC tumors for somatic 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 (TCGA Research Network, Nature, in press). Whole-exome sequencing showed 29 recurrently mutated genes. Potential therapeutic targets include 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 22 arm-level CNVs and 27 focally amplified or deleted regions. CDKN2A was deleted in 47%. Low-pass whole genome sequencing identified FGFR3-TACC3 fusions. Viral DNA was identified in 6% (CMV, HHV6B, HPV16, BK polyoma), and viral transcripts were identified in 4% (CMV, BK polyoma, HPV16). . mRNA-seq identified 4 tumor clusters. Cluster I shows papillary morphology and FGFR3 dysregulation. Clusters I and II express high HER2 (ERBB2) and estrogen receptor beta signaling signature, sharing features with Luminal A breast cancer. Cluster III shows similarities to Basal-like breast and squamous cell head and neck carcinomas. . Integrated analyses confirm alteration of multiple pathways, including cell cycle regulation (93%), kinase and PI3-K signaling (72%), and epigenetic regulation (histone-modifiers: 89%; SWI/SNF nucleosome remodeling complex: 64%). Recurrent alterations in the PI3-kinase/AKT/mTOR pathway (42%) and RTK/RAS pathway (44%) are potentially actionable. . Overall, this study and others have identified multiple druggable targets in UC. FGFR3 is activated by mutation, gene fusion, and overexpression, suggesting clinical trials of FGFR3 inhibitors. PI3-kinase/mTOR/AKT/TSC1 pathway alterations are frequent, and mutation in TSC1 has been associated with response to mTOR inhibitors. ERBB2 amplifications and activating mutations may be targetable with agents such as trastuzumab, trastuzumab-DM1, lapatinib, and neratinib. The frequent alterations in epigenetic regulatory pathways suggest trials of agents such as the bromodomain inhibitors. The project is now being updated on the basis of data on 117 additional tumors. Citation Format: John N. Weinstein, Jaegil Kim, Chad J. Creighton, Rehan Akbani, Katherine A. Hoadley, William Y. Kim, Margaret B. Morgan, Toshinori Hinoue, Andrew Cherniack, Xiaoping Su, Andrew J. Mungall, Michael C. Ryan, Jonathan E. Rosenberg, Dean F. Bajorin, Bogdan Czerniak, Donna Hansel, Victor E. Reuter, Brian D. Robinson, Hikmat A. Al-Ahmadie, Jeffrey S. Damrauer, Wei Zhang, Yuexin Liu, Dmitry Gordenin, Joshua M. Stuart, Nikolaus Schultz, Gordon Robertson, Raju Kucherlapati, Peter W. Laird, Gordon B. Mills, David J. Kwiatkowski, Seth P. Lerner, representing TCGA's Bladder Cancer Working Group. Comprehensive characterization of urothelial bladder cancer: a TCGA Project update. [abstract] . In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 987. doi:10.1158/1538-7445.AM2014-987

Abstract: In 2014, as part of The Cancer Genome Atlas (TCGA) Project, we reported molecular profiling of 131 chemotherapy-naive, muscle-invasive urothelial bladder cancers for DNA copy number variants (CNVs), somatic mutations by whole exome sequencing (WES), DNA methylation, mRNA expression, microRNA expression, protein expression and phosphorylation, transcript splicing, gene fusion, viral integration, pathway perturbation, clinical correlates, and histopathology (TCGA Research Network, Nature 507:315, 2014). Since that time, the number of tumors (from 19 tissue source sites) profiled comprehensively has doubled, and the final analyzed cohort will total 412 by February 2015. Unsupervised consensus clustering for the data from miRNAseq on the first 310 tumor samples shows five robust clusters, with miR-99a, miR-100, and miR-200 family members differentially abundant across the clusters. mRNA clusters I-IV remained stable as well. For the 266 tumour samples for which we have both miRNA and mRNA data, potential miRNA-mRNA targeting relationships supported by functional validation publications include miR-100-5p targeting FGFR3. The data also highlight the miR-200 family, miR-29abc, miR-17-92 and 106b-25 complexes, as well as miR-34a, miR-155, and miR-21. At this time, mutation analysis of WES data has been completed for 238 samples (including the first 131). Seven additional significantly mutated genes (using MutSig) have been identified: ASXL2 (11%), HSP90AA1 (7%), PSIP1 (5%), ZFP36L2 (5%), ZNF513 (5%), PTEN (4%), CEBPB (2%) (mutant frequency in parentheses). A sample with a POLE exonuclease domain mutation (P286R) exhibited an ultra-mutant phenotype (mutation rate ∼100 per MB). Many of the 38 (total) SMGs have not previously been described in bladder cancer. Combining copy number variation and somatic mutation data, 69% of tumors harbor one or more potentially actionable targets. Three mutation clusters were identified and characterized as: 1) “Focally amplified” - enriched in focal copy number alterations (e.g., 3p loss/PPARG) and MLL2 mutations; 2) Enriched for TP53 and RB1 mutations, E2F3 amplifications; and 3) Papillary histology, FGFR3 mutant CDKN2A-deficient. This work was supported by the following grants from the United States National Institutes of Health: U54HG003273, U54 HG003067, U54 HG003079, U24 CA143799, U24 CA143835, U24CA143840, U24 CA143843, U24 CA143845, U24 CA143848, U24 CA143858, U24CA143866, U24 CA143867, U24 CA143882, U24 CA143883, U24 CA144025 and P01 CA120964, as well as multiple other funding sources for the individual authors. Citation Format: John N. Weinstein, Jaegil Kim, Chad J. Creighton, Rehan Akbani, Katherine A. Hoadley, William Y. Kim, Margaret B. Morgan, Toshinori Hinoue, Andrew Cherniack, Xiaoping Su, Andrew J. Mungall, Michael C. Ryan, Dean F. Bajorin, Jonathan E. Rosenberg, Bogdan Czerniak, Donna Hansel, Victor E. Reuter, Brian D. Robinson, Hikmat A. Al-Ahmadie, Jeffrey S. Damrauer, Wei Zhang, Yuexin Liu, Dmitry R. Gordenin, Joshua M. Stuart, Nikolaus Schultz, Gordon Robertson, Steven JM Jones, Raju R. Kucherlapati, David J. McConkey, Peter W. Laird, Gordon B. Mills, David J. Kwiatkowski, Seth P. Lerner, TCGA Bladder Cancer Working Group, TCGA Research Network. Progress in The Cancer Genome Atlas bladder cancer project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2015-2969

Abstract: Introduction: In 2014, TCGA's Bladder Cancer Working Group presented a preliminary integrated molecular analysis of 131 muscle-invasive urothelial carcinomas (Nature 507:315, 2014). We now report on the entire cohort of 412 fresh-frozen, chemotherapy-naïve tumors. Included in the analysis were paired blood and/or tumor-adjacent tissue samples. This is the largest sequencing project on bladder cancer to date. After strict clinical and pathologic quality control, tumors were analyzed for DNA copy number variants, somatic mutations, DNA methylation, mRNA, microRNA and (phospho-) protein expression, transcript splicing, gene fusions, viral integration, APOBEC mutagenesis, pathway perturbation, clinical correlates, and histopathology. Results: There was a high overall somatic mutation rate (8.0/Mb), with a median of 245 and mean of 348 coding-region mutations per sample. That is the third highest mutation rate among the cancer types profiled by TCGA (after cutaneous melanoma and non-small cell lung cancers). We identified 54 genes as significantly mutated, compared with 32 in the original report on 131 tumors. TP53 mutations were the most common (49%), and also quite common were mutations in a number of chromatin-modifying genes, including MLL2 (29%), KDM6A (26%), ARID1A (25%), MLL3 (19%), EP300 (15%), CREBBP (12%), and MLL (11%). Other cancer-related genes showing frequent mutations included PIK3CA (22%), RB1 (17%), FGFR3 (14%), STAG2 (14%), ATM (14%), ELF3 (12%), FAT1 (12%), SPTAN1 (12%), ERBB2 (12%), ERBB3 (11%), ASXL2 (10%), ERCC2 (9%), CDKN1A (9%), TSC1 (8%), CDKN2A (7%), RHOB (6%), NFE2L2 (6%), PARD3 (6%), FAM47C (5%), RBM10 (5%),HRAS (5%), KRAS (4%), and PTEN (3%). High mutation burden was associated with improved outcome (p = 0.0004). APOBEC mutagenesis explained 70% of the mutation burden and was associated with survival. Gene silencing by promoter hypermethylation was identified in 167 genes with at least 5% frequency in the cohort. The previously identified four mRNA expression subtypes were again found in the complete set of 412 tumors, and the proportions of samples in each subtype were similar to the previous proportions. Reverse-phase proteomic array analysis of 344 of the samples revealed clusters associated with diagnostic subtype, pathological stage, and grade but not with smoking history or non-muscle invasive status. Conclusions: This integrated molecular analysis of 412 TCGA tumor samples largely validates and considerably extends observations from the initial cohort of 131 patients. The larger cohort significantly increased our power to detect lower-frequency aberrations that were not identified in the original cohort. The results provide a robust basis for further functional studies of bladder cancer biology and also provide additional incisive information for the identification of molecular targets for therapy. Citation Format: John N. Weinstein, Seth P. Lerner, David J. Kwiatkowski, Gad Getz, Jaegil Kim, Hikmat A. Al-ahmadie, Andrew D. Cherniack, Guangwu Guo, Rehan Akbani, Katherine A. Hoadley, William Y. Kim, Gordon Robertson, Andrew J. Mungall, Toshinori Hinoue, Peter W. Laird, Jonathan E. Rosenberg, Joaquim Bellmunt, Dean F. Bajorin, Margaret B. Morgan, Chad J. Creighton, Dmitry Gordenin, Joshua M. Stuart, Xiaoping Su, Michael C. Ryan, Jeffrey S. Damrauer, Wei Zhang, Yuexin Liu, Yiling Lu, Nikolaus Schultz, Raju Kucherlapati, Gordon B. Mills, Donna E. Hansel, Brian D. Robinson, Bodgen A. Czerniak, Victor E. Reuter. Comprehensive molecular characterization of 412 muscle-invasive urothelial bladder carcinomas: final analysis of The Cancer Genome Atlas (TCGA) project. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 128.

Abstract: Despite increasing knowledge of tumorigenesis, the identity of the cancer cell-of-origin, i.e. the normal cell type that acquired the cancer-initiating event, remains largely unknown. Our approach of identifying the cell-of-origin is based on two observations: (1) the chromatin structure is cell-specific; and (2) the density of somatic mutations along the genome is associated with the regional profile of chromatin modifications. We have previously developed a method that quantifies the ability to predict the mutational distribution along the cancer genome from the profile of epigenetic modifications in different normal cell types. Here we present the largest application of our method using 2,550 whole genomes representing 32 distinct cancer types. To identify the cell-of-origin, we determined the correlation between the observed density of mutations along the genome and the predicted values based on chromatin modifications from 104 different normal tissue types. The normal cell type that showed the strongest correlation with a specific cancer mutational landscape was the candidate cell-of-origin. We found that in almost all cancer types the cell-of-origin can be characterized solely from DNA sequences. Interestingly, we found that the fallopian tube was the best match for high-grade serous ovarian cancer, providing independent evidence that this is the cancer’s site of origin. For breast cancer we found that the four distinct subtypes best-matched cells from the luminal cell lineage: basal-like breast cancer likely originates from luminal progenitors, whereas all other subtypes from luminal mature cells. This association holds true even when accounting for different alterations in the homologous recombination repair pathway, suggesting that subtypes are more determined by the cell-of-origin than the specific DNA repair defect. In addition, we found that we could identify the cell-of-origin using metastatic samples – a finding that may help in difficult clinical diagnoses. Moreover, we demonstrate that cancer drivers, both germline risk alleles and somatically mutated drivers, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings indicate that many of the somatic mutations accumulated while the cells maintained a chromatin structure similar to the cell-of-origin (likely occurring prior to transformation). Therefore, this historical record, captured in the DNA, can be used to identify, the often elusive, cancer cell-of-origin. Our approach can ultimately help better understand the potential of particular normal cell types to transform and initiate cancer, as well as the association of the cell-of-origin with tumor subtypes and sensitivity to treatment. Citation Format: Kirsten Kubler, Rosa Karlic, Nicholas J. Haradhvala, Kyungsik Ha, Jaegil Kim, Maja Kuzman, Wei Jiao, Sitanshu Gakkhar, Kent W. Mouw, Lior Z. Braunstein, Olivier Elemento, Andrew V. Biankin, Ilse Rooman, Mendy Miller, Christopher D. Nogiec, Edward Curry, Mari Mino-Kenudson, Leif W. Ellisen, Robert Brown, Alexander Gusev, Cristian Tomasetti, Hong-Gee Kim, Hwajin Lee, Kristian Vlahovicek, Charles Sawyers, Katherine A. Hoadley, Edwin Cuppen, Amnon Koren, Peter F. Arndt, David N. Louis, Lincoln Stein, William D. Foulkes, Paz Polak, Gad Getz. The premalignant state captured in the landscape of somatic mutations can reveal the cancer cell-of-origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2727.

Abstract: Purpose Lung squamous cell carcinoma (SqCC) is the second most prevalent type of lung cancer. Currently, no targeted-therapeutics are approved for treatment of this cancer, largely due to a lack of systematic understanding of the molecular pathogenesis of the disease. To identify therapeutic targets and perform comparative analyses of lung SqCC, we probed somatic genome alterations of lung SqCC cases from Korean patients. Patients and Methods We performed whole-exome sequencing of DNA from 104 lung SqCC samples from Korean patients and matched normal DNA. In addition, copy number analysis and transcriptome analysis were conducted for a subset of these samples. Clinical association with cancer-specific somatic alterations was investigated. Results This cancer cohort is characterized by a very high mutational burden with an average of 261 somatic exonic mutations per tumor and a mutational spectrum showing a signature of cigarette-smoke exposure. Seven genes demonstrated statistical enrichment for mutation (TP53, RB1, PTEN, NFE2L2, KEAP1, MLL2 and PIK3CA). Comparative analysis between Korean and North American lung SqCC demonstrated similar spectrum of alterations in these two populations, in contrast to the differences seen in lung adenocarcinoma. We also uncovered recurrent occurrence of therapeutically actionable FGFR3-TACC3 fusion in lung SqCC. Conclusion These findings provide new steps towards the identification of genomic target candidates for precision medicine in lung SqCC, a disease with a significant unmet medical need. Note: This abstract was not presented at the meeting. Citation Format: Youngwook Kim, Peter S. Hammerman, Jaegil Kim, Gad Getz, Matthew Meyerson, Keunchil Park. Integrative and comparative genomic analysis of East-Asian lung squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2014-1526

Abstract: Osteosarcoma is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of osteosarcoma spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that osteosarcoma tumors show a high frequency of somatic copy-number alterations (SCNA), affecting key oncogenes and tumor-suppressor genes. The across-breed results are similar to what has been observed for human osteosarcoma, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported) and 11 significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human osteosarcoma. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in osteosarcoma. This study points to the likely importance of histone modifications in osteosarcoma and highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine osteosarcoma may serve as an excellent model for developing treatment strategies in both species. Significance: Canine osteosarcoma genomics identify SETD2 as a possible oncogenic driver of osteosarcoma, and findings establish the canine model as a useful comparative model for the corresponding human disease. Cancer Res; 78(13); 3421–31. ©2018 AACR.

Abstract: The advent of PD-1/PD-L1 agents has transformed the therapeutic landscape of many advanced cancers, including non-small cell lung cancer (NSCLC). However, our understanding of the genomic biomarkers underlying effective treatment response remain limited. Here we provide updated results from our ongoing effort, the Stand Up To Cancer Lung (SU2C-Lung)/Mark Foundation EXTOLConsortium, a multi-institution collaboration to expand our understanding of the molecular determinants of immunotherapy response in NSCLC. Comprising a set of nearly 400 patients, this cohort enables evaluation of both genomic and transcriptomic factors associated with checkpoint blockade response. In addition to validating previously known associations including TMB and neoantigen burden, we examined transcriptional predictors associated with response. We generated a list of differentially expressed genes with respect to best overall response (BOR), and performed dimensionality reduction using semi-supervised Bayesian Non-Negative Factorization (ssBNMF). We identified 3 distinct clusters with strong sample membership. Characterization of these subtypes revealed varying levels of immune infiltrate, histologic composition, and response rates to checkpoint blockade. Of these three subtypes, two were associated with low response rates to PD-1/PD-L1 blockade, suggesting the existence of distinct avenues toward resistance. To further characterize these transcriptional subtypes, we used ssBNMF marker genes to classify publicly available NSCLC samples from The Cancer Genome Atlas (TCGA), along with gene expression from a smaller cohort of large-cell neuroendocrine (LCNE) samples. Histologic composition showed good concordance with our SU2C samples, and redemonstrated observations within our smaller SU2C cohort of distinct immuno-suppressive and immuno-depleted milieus associated with resistance. Citation Format: Monica B. Arniella, Arvind Ravi, Justin Gainor, Chip Stewart, Sam Freeman, Mark Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan W. Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Nir Hacohen, Naiyer Rizvi, Roy Herbst, Victor E. Velculescu, Julie Brahmer, Kurt Schalper, Pasi Jänne, Jedd Wolchok, Alice Shaw, Gad Getz, Matthew D. Hellman. An SU2C-Mark Foundation Lung collaborative update: integrative genomics identifies distinct transcriptional states associated with checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for C ancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB197.

Abstract: Purpose: Squamous cell carcinoma of the anal canal (ASCC) accounts for 2% to 4% of gastrointestinal malignancies in the United States and is increasing in incidence; however, genomic features of ASCC are incompletely characterized. Primary treatment of ASCC involves concurrent chemotherapy and radiation (CRT), but the mutational landscape of resistance to CRT is unknown. Here, we aim to compare mutational features of ASCC in the pre- and post-CRT setting. Experimental Design: We perform whole-exome sequencing of primary (n = 31) and recurrent (n = 30) ASCCs and correlate findings with clinical data. We compare genomic features of matched pre- and post-CRT tumors to identify genomic features of CRT response. Finally, we investigate the mutational underpinnings of an extraordinary ASCC response to immunotherapy. Results: We find that both primary and recurrent ASCC tumors harbor mutations in genes, such as PIK3CA and FBXW7, that are also mutated in other HPV-associated cancers. Overall mutational burden was not significantly different in pre- versus post-CRT tumors, and several examples of shared clonal driver mutations were identified. In two cases, clonally related pre- and post-CRT tumors harbored distinct oncogenic driver mutations in the same cancer gene (KRAS or FBXW7). A patient with recurrent disease achieved an exceptional response to anti-programmed death (PD-1) therapy, and genomic dissection revealed high mutational burden and predicted neoantigen load. Conclusions: We perform comprehensive mutational analysis of ASCC and characterize mutational features associated with CRT. Although many primary and recurrent tumors share driver events, we identify several unique examples of clonal evolution in response to treatment. Clin Cancer Res; 23(12); 3214–22. ©2016 AACR.

Abstract: Approximately 20,000 new cases of muscle-invasive bladder cancer (MIBC), a localized but potentially lethal stage of disease, are diagnosed annually in the US alone. Standard-of-care therapy for MIBC includes neoadjuvant cisplatin-based chemotherapy followed by definitive bladder resection. In prior work, we identified and validated genomic alterations in DNA repair genes such as ERCC2, which predict response to cisplatin-based chemotherapy (Van Allen et al. Cancer Discovery 2014; Liu et al. JAMA Oncology 2016). However, the majority of patients have disease resistant to chemotherapy with a poor prognosis of & lt;40% survival at 5 years, and the genomic basis of chemotherapy resistance has not been well-characterized. In this study, our goal was to analyze matched pre-treatment and resistant post-chemotherapy cystectomy tumor samples to identify genomic correlates of cisplatin-based chemotherapy exposure and resistance. We identified 56 MIBC patients with matched pre-chemotherapy biopsy samples and resistant tumor samples from cystectomy. Along with matched normal samples (blood), we performed whole exome sequencing (WES) on these “trios” of pre, post, and normal tissue, and called somatic variants using standardized pipelines including single nucleotide variants (SNVs), short insertions and deletions, allelic copy number alterations (CNAs), tumor purity and ploidy, and purity- and ploidy-corrected copy number variants. After quality control, including contamination estimation & lt; 5%, mean target coverage & gt; 50x, and a tumor purity minimum threshold of 10%, we had data from 30 trios available for analysis. We hypothesized that DNA-damaging chemotherapy may lead to increased mutational load in the post-treatment tumor. However, we observed that while some tumors gained mutations, others lost mutations, with no overall change (mean change = -17.3 mutations, paired t-test p = 0.20) in total mutational load. We found that clonal mutations (found in all tumor cells) were virtually unchanged from matched pre- to post-treatment tumors. In contrast, subclonal mutations (found only in a subset of tumor cells) were private to pre- and post-treatment tumors. These pre- and post-treatment mutation differences may reflect tumor sampling heterogeneity (i.e. taking from different parts of the tumor), but may be also due to selection pressure from therapy (e.g. loss of subclones) and cisplatin-induced mutations.To investigate the latter possibility, we adapted a non-negative matrix factorization (NMF) approach (Lee and Seung Nature 1999) to discover mutational signatures (Alexandrov et al Nature 2013) in the mutations unique to post-treatment tumors. Along with signatures known to be operant in bladder cancers, we discovered a mutational signature dissimilar to any other previously described mutational signature which accounted for ~15% of post-treatment mutations. This signature exhibited a transcriptional strand bias consistent with known mechanisms of cisplatin-induced DNA damage and repair, and was enriched in subclonal mutations consistent with the relatively short time frame between cisplatin exposure and cystectomy. This signature also exhibited similar activity to a cisplatin-induced mutational signature derived in a preclinical model (DT40) exposed to cisplatin therapy (Pearson rho = 0.95, empiric p = 0.004). Finally, we were able to validate this signature in a separate cohort of pre- and post-cisplatin treated bladder cancers (Faltas et al Nature Genetics 2016). We further hypothesized that the degree of tumor heterogeneity itself may be a prognostic factor. We calculated two different measures of intratumor heterogeneity: (1) the proportion of mutations in each tumor that was subclonal; and (2) the number of unique subclones in each tumor, and examined the association of survival with these measures of intratumor heterogeneity using Cox survival analyses. We found that overall survival was associated with heterogeneity, with a 6.6% increase in mortality rate for each 10% increase in post-treatment proportion of subclonal mutations (p=0.013), and 64% increase in mortality rate for each additional inferred subclone (p=0.02). Tumor heterogeneity continued to be associated with survival after adjusting for clinical covariates (p=0.03, p=0.014, respectively).Finally, we analyzed our tumors for genomic alterations associated with resistance. While we did not discover highly recurrent post-treatment mutations in specific genes, we found drivers of cell cycle progression (E2F3 amplification, JUN amplification), biallelic loss of FBXW7 (regulator of protein degradation of multiple onco-proteins including c-MYC, Notch, Cyclin E, and c-JUN), and focal amplification of PD-L1/2 in individual post-treatment resistant tumors.In this study, we found that cisplatin-based chemotherapy did not induce a large increase in the number of mutations. Thus, while there is good empiric data for the efficacy of combination of chemotherapy and immune checkpoint inhibition in specific tumor type and clinical settings (e.g. platinum-doublet therapy + ICB in first-line therapy of non-small cell lung cancer (NSCLC)), our data suggests that alternative mechanisms other than increased neoantigen burden are responsible. We discovered a cisplatin-induced mutational signature in post-treatment tumors which has subsequently been found in other cisplatin-treated tumors (e.g. NSCLC and ovarian cancer). Interestingly, the proportion of mutations inferred to be cisplatin-induced was quite different between resistant tumors, and an area for further inquiry is whether these differences could be associated with different mechanisms of resistance (e.g. upregulation of efflux pumps vs. anti-apoptotic adaptations). Tumor heterogeneity, which has been associated with worse outcomes and resistance in multiple contexts, was prognostic for survival in our cohort, suggesting that this may be clinically useful as part of a prognostic biomarker. We discovered additional association of drivers of cell-cycle progression with resistance, and further identified acquisition of a focal amplification in a region containing PD-L1/PD-L2, suggesting a potential biomarker for a subset of bladder cancers for response to immune checkpoint blockade. Broadly, this study represents the development of algorithms to dissect genomic features associated with survival and resistance in a carefully curated cohort of matched patient tumors within a specific clinical context. These types of approaches can be applied across tumor types, therapies, and clinical contexts to shed light onto biological mechanisms underpinning response and resistance and inform the development of biomarkers to guide clinical management. Citation Format: David Liu, Philip Abbosh, Daniel Keliher, Brendan Reardon, Diana Miao, Kent Mouw, Amaro Weiner-Taylor, Stephanie Wankowicz, Garam Han, Min-Yuen Teo, Catharine Cipolla, Jaegil Kim, Gopa Iyer, Hikmat Al-Ahmadie, Essel Dulaimi, David Y.T. Chen, R. Katherine Alpaugh, Jean Hoffman-Censits, Levi A. Garraway, Gad Getz, Scott L. Carter, Joaquim Bellmunt, Elizabeth Plimack, Jonathan E. Rosenberg, Eliezer M. Van Allen. Dissecting genomic correlates of response and resistance to chemotherapy in bladder cancer through clinical computational oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY05-03.