4512 Background: Biomarkers of survival and resistance in chemotherapy-resistant muscle-invasive bladder cancer (MIBC) are not well-characterized, but may inform management in this setting. Methods: Matched pre- and post-neoadjuvant cisplatin-based chemotherapy (NAC) tumor samples were obtained from 30 MIBC patients with gross residual disease (≥ pT2) at cystectomy, followed by whole exome sequencing of these “trios” (pre- and post-NAC tumor with matched germline samples). Phylogenetic analysis of matched tumor samples was performed to identify subclones, their associated mutations, and the corresponding enrichment in post-treatment tumors. Intratumoral heterogeneity was assessed by the proportion of mutations that were subclonal; the number of inferred subclones; and associated with overall survival using a Cox Proportional Hazards model. Results: Increased proportion of subclonal mutations in post-treatment tumors was associated with worse overall survival (HRR 1.86 [95% CI 1.12-3.06], p = 0.02), whereas pre-treatment proportion of subclonal mutations was only borderline statistically significant (HRR 1.48 [95% CI 0.99-2.20], p = 0.052). The total number of inferred tumor subclones in pre- or post-treatment tumor (or both) was associated with overall survival (HRR 1.60 [95% CI 1.05-2.43], p = 0.03), interpreted as a 60% increase in death rate per additional inferred subclone. While no single gene was statistically significantly enriched for new alterations in the post-chemotherapy resistant samples, we observed new post-treatment amplifications in cell-cycle genes ( E2F3, c-JUN), biallelic events in cell-cycle regulators ( FBXW7), and amplification of immune checkpoint genes ( PDL1/2). Conclusions: These results suggest that intratumoral heterogeneity (particularly post-therapy) predicts survival in a chemotherapy-resistant cohort. Further, alterations in cell cycle regulation may contribute to the mechanism of chemotherapy resistance. Finally, we observe evidence of immune checkpoint gene amplification post-treatment, suggesting that testing immune checkpoint blockade during NAC or, in high risk patients, following NAC may be warranted.
