Abstract 3880: Next generation sequencing for discovery of somatic mutations and clonal relationships in platinum-resistant metastatic urothelial cancer
Authors: Faltas Bishoy M. , Beltran Himisha , Eng Kenneth , Pauli Chantal , Robinson Brian , Mosquera Juan Miguel , Nanus David , Tagawa Scott T. , Rubin Mark A. 
Published: August 2015

Abstract
Background: Beyond first-line platinum based chemotherapy, there are currently no FDA-approved therapies for advanced platinum-resistant urothelial carcinoma (PRUC). Our objective was to generate the first detailed genomic profile of metastatic PRUC to identify molecular changes critical to the development of platinum-resistance and metastasis. Methods: Following informed consent, we collected 50 urothelial tumor samples, from 23 patients consented through our precision medicine protocol. Metastatic tumor samples were obtained from metastatic biopsies or surgical metastatectomy. Germline samples were prospectively collected for every patient and matched archival formalin-fixed paraffin-embedded primary tumors from the same patients were retrieved. Our cohort comprises 23 metastatic samples, 37 platinum resistant urothelial samples and 18 trios of matched primary, metastatic and germline samples including two rapid autopsy cases that yielded tumor samples from multiple primary and metastatic sites. We performed whole exome sequencing of all tumor and germline samples and RNA sequencing on 18 advanced PRUC tumors. We utilized our in-house computational pipeline for integrated analysis of somatic single nucleotide variants and somatic copy-number alterations and to analyze the clonal evolution of the primary and metastatic in each patient. We utilized the DAVID bioinformatic tool for pathway analysis. Results: Advanced PRUC samples were enriched for several molecular alterations, we identified 414 recurrently mutated genes (defined as genes mutated in 2 or more samples and having the highest number of mutations per nucleotide) including common genes such as TP53(45%) as well as actionable alterations in PIK3CA(11%) and TSC1(19%). Frequent copy number alterations included CDKN2A deletions (33%), E2F3(10%) and ERBB2(7%) amplifications. Pathway analysis showed an enrichment of mutations in genes involved in controlling apoptosis (p = 1.0E-7, FDR 1.7E-4) and cell cycle regulation (P = 1.6E-2, FDR 2.3E1) compared to the TCGA dataset of untreated urothelial carcinoma primary tumors.We reconstructed phylogenetic trees from matched primary, metastatic and germline trios revealing evidence of divergent clonal evolution between primary tumors, lymph node and visceral metastases. In some cases, shared mutations occurred with gradually increasing variant allele frequencies across primary tumors, lymph node metastasis and visceral metastases respectively, indicating clonal selection of mutations that were present in the primary tumor. We also identified unique acquired mutations in metastatic lesions that were not present in primary tumors. Conclusions: This study generates a detailed molecular profile of the genomic landscape of PRUC revealing evidence of extensive heterogeneity and clonal selection underlying platinum-resistance and metastatic spread.
Citation Format: Bishoy M. Faltas, Himisha Beltran, Kenneth Eng, Chantal Pauli, Brian Robinson, Juan Miguel Mosquera, David Nanus, Scott T. Tagawa, Mark A. Rubin. Next generation sequencing for discovery of somatic mutations and clonal relationships in platinum-resistant metastatic urothelial cancer. [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 3880. doi:10.1158/1538-7445.AM2015-3880