Abstract 2379: Spatial and temporal clonal evolution during development of metastatic urothelial carcinoma
Authors: Thomsen Mathilde B.H. , Nordentoft Iver , Lamy Philippe , Hoyer Soren , Vang Soren , Hedegaard Jakob , Borre Michael , Jensen Jorgen B. , Orntoft Torben F. , Dyrskjot Lars 
Published: July 2016

Abstract
Background: To better treat metastatic bladder cancer, it is important to understand the clonal evolution in space and time and to identify aggressive subclones giving rise to metastatic spread. Patients with metastatic bladder cancer have a median survival of 3-6 months if untreated and 13-14 months median survival if they receive adjuvant chemotherapy. Targeted therapy using e.g. antibodies or small molecule cancer drugs has not yet shown convincing results - likely due to lack of genetic profiling prior inclusion to therapy. Here we performed a thorough characterization of the spatial and temporal tumor heterogeneity in three patients with metastatic bladder cancer by whole exome sequencing (WES) of multiple small cellular regions from primary and metastatic tumor biopsies.
Methods: Biopsies from primary tumors, lymph node metastases, and distant metastases from three patients were studied. DNA was extracted from small cellular regions procured by laser-microdissection or punctures along with matched germline DNA. WES was performed using either Nextera Rapid Exome Capture or TruSeq Nano DNA Library Prep combined with SeqCap EZ Exome Capture and sequenced on the Illumina HiSeq2000 or NextSeq500 platforms.
Results: Exome sequence information from 24 cellular regions and germline DNA was obtained. The average mean target coverage obtained for tumor samples was 68X(27-215X) and germline was 167X(95-301X). Mutation calling using MuTect identified 256, 265 and 378 somatic SNVs in tumors from the three patients. We observed low spatial intra-tumor heterogeneity but large inter-tumor (primary vs metastasis) heterogeneity. We further identified 6-12 SNVs in known disease driver genes per patient. Public (present in all sampled regions) disease driver mutations could be identified in all cases, however, over time regional driver mutations emerged, especially following metastatic spread. We observed low allele frequencies across all cellular regions, and based on this we hypothesize that the distribution of the individual clones is intermixed. Further, we observed a similar intermix of sub clones in the metastatic lesions, which may be explained by metastases seeding as cell clusters or in individual clones in parallel.
Conclusions: The three patients studied showed low spatial intra-tumor heterogeneity but large genetic diversity between primary tumor and metastatic lesions. The differences between primary tumors and metastatic lesions observed emphasize the challenges in designing rational targeted therapy solely based on the genetic profile of the primary tumor.
Citation Format: Mathilde B.H. Thomsen, Iver Nordentoft, Philippe Lamy, Soren Hoyer, Soren Vang, Jakob Hedegaard, Michael Borre, Jorgen B. Jensen, Torben F. Orntoft, Lars Dyrskjot. Spatial and temporal clonal evolution during development of metastatic urothelial carcinoma. [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 2379.