Abstract NG06: Genomic dissection of the clonal evolution dynamics of chemotherapy-resistant urothelial carcinoma
Authors: Faltas Bishoy M. , Prandi Davide , Tagawa Scott T. , Molina Ana M. , Nanus David M. , Sternberg Cora , Rosenberg Jonathan , Mosquera Juan Miguel , Robinson Brian , Elemento Olivier , Sboner Andrea , Beltran Himisha , Demichelis Francesca , Rubin Mark A. 
Published: July 2017

Introduction: Chemotherapy-resistant urothelial carcinoma (UC) has no uniformly curative therapy. Understanding how selective pressure from chemotherapy directs UC’s evolution and shapes its clonal architecture is a central biologic question with important clinical implications.

Methods: To address this question, we performed whole-exome sequencing and clonality analysis of 72 UCs including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy.

Results: Our analysis provided several important insights. Our findings show that chemotherapy-treated UC is characterized by significant intrapatient mutational heterogeneity. The majority of mutations were not shared. On average, only 28.4% (range 0.2%-76.4%) of mutations were shared between pre- and postchemotherapy samples. This effect was consistent across primary-primary tumor pairs and primary-metastatic tumor pairs (p=0.17, Wilcoxon test). Surprisingly, even mutations in previously reported driver genes, including PIK3CA, KMT2D (MLL2), ATM, and TP53, were not consistently shared between matched prechemotherapy and postchemotherapy tumors. We confirmed these findings with targeted sequencing of 250 common driver genes achieving an average coverage of 400x and an excellent concordance with variant allele frequencies obtained from whole exome sequencing (Pearson correlation = 0.93, P&lt;10-171). Phylogenetic analysis revealed that both branching evolution and metastatic spread were very early events in the natural history of UC. At the copy number level, our analysis revealed limited intrapatient heterogeneity with respect to interpatient heterogeneity, suggesting that each patient’s cancer is relatively stable during evolution at the copy number level. We discovered two distinct copy-number-based clusters. Cluster A was defined by 9p21 (CDKN2A, CDKN2B, and MTAP) deletions in the setting of euploid copy number background. Cluster B was characterized by several enriched amplifications, including 1q21.1 (SETDB1 and MLLT11) amplifications (P=0.0002, Fisher's exact test) and 6p22.3 (E2F3) amplifications (P=0.001, Fisher's exact test). This cluster was also enriched with TP53 mutations (P=0.0001, Fisher's exact test). We validated the presence of these clusters in the TCGA dataset of untreated UC and confirmed that these copy-number alterations predate chemotherapy and are clonally static during the lifetime of UC tumors. On the other hand, the mutational landscape of UC tumors evolved significantly after chemotherapy. We observed a significant increase in the number of clonal mutations in the postchemotherapy samples across the study cohort (P=0.0134, Fisher’s exact test). Chemotherapy-treated UC was enriched with clonal mutations involving L1-cell adhesion molecule (L1CAM) and integrin-signaling pathways. To understand the mutational mechanisms driving the evolution of UC, we compared the patterns of single base transitions between chemotherapy-naïve and chemotherapy-treated tumors. We observed a significant increase in C&gt;A nucleotide substitutions in tumors treated with cisplatin-based chemotherapy consistent with previously described specific mutagenesis signature induced in C. elegans genome after cisplatin treatment. Further analysis of context motifs of various base substitutions showed enrichment in C&gt; T or G changes at the TCW motifs (where W = A or T), which is highly suggestive of APOBEC-induced mutagenesis. APOBEC induced-mutations were clonally enriched in chemotherapy-treated UC and continued to shape UC's evolution throughout its lifetime.

Conclusions: Our findings have several potential clinical implications: First, genomic divergence between untreated and treated clones suggests that clinically actionable molecular targets in metastatic chemotherapy-treated tumors will be potentially missed when relying only on biopsies of untreated primary tumors at the time of diagnosis, and that repeat metastatic biopsies during the course of clinical care would be needed to detect the most recent version of the rapidly changing molecular landscape of a given patient’s UC. Second, further study of the functional role of L1CAM and integrin signaling in mediating chemotherapy resistance in UC could lead to a potential strategy for reversing or preventing chemotherapy resistance by targeting these pathways. Third, despite its initial effectiveness in eliminating cancer cells, platinum-based chemotherapy is associated with unintended significant mutagenic editing of the genomic landscape of postchemotherapy tumors. Our insight into the nature of these edits is crucial towards a complete understanding of the basis of chemotherapy resistance in advanced UC, which lays the foundation for the development of rational therapeutic strategies for preventing the emergence or reversing the chemotherapy-resistant state of UC. In summary, our results demonstrate that advanced chemotherapy-treated UC undergoes extensive and dynamic clonal evolution throughout the lifetime of the tumor with significant genetic editing that continues during and after chemotherapy. Our findings lay the foundation for an evolutionary understanding of advanced chemotherapy-treated UC and present opportunities for advancing cancer precision medicine.

Citation Format: Bishoy M. Faltas, Davide Prandi, Scott T. Tagawa, Ana M. Molina, David M. Nanus, Cora Sternberg, Jonathan Rosenberg, Juan Miguel Mosquera, Brian Robinson, Olivier Elemento, Andrea Sboner, Himisha Beltran, Francesca Demichelis, Mark A. Rubin. Genomic dissection of the clonal evolution dynamics of chemotherapy-resistant urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG06. doi:10.1158/1538-7445.AM2017-NG06