Abstract The ability to harness the power of next generation sequencing for characterizing the cancer genome would be extremely valuable to the cancer research community and ultimately improve diagnosis and individualized therapy. We present a targeted approach using massively parallel sequencing to simultaneously detect mutations, translocations and copy-number variations in archived clinical tumor specimen. Targeted sequencing was achieved by designing RNA baits to capture the exons of 504 genes with relevance to cancer. The bait set was augmented with specific intronic sequences to detect translocations often involved in cancer. The overall performance of the hybrid capture was improved by optimizing the tiling strategy of the baits. These improvements significantly shifted fragments with low or no-coverage closer to the overall mean, resulting in a more uniform coverage distribution. To validate the targeted gene panel we sequenced tumor samples harboring mutations, translocations and copy-number alterations that were previously identified by clinically approved assays. All the known alterations were confirmed with additional potentially actionable mutations. The ultimate goal of the targeted panel is to screen cancer patients quickly and economically allowing simultaneous detection of all common genomic alterations in the cancer genome. Citation Format: Paul Van Hummelen, Matthew Ducar, Robert T. Jones, Alina Raza, Ashwini Sunkavalli, Megan Hanna, Adri Mills, Ravali Adusumilli, Prateek Kumar, Laura Schubert, Marc Breneiser, Anna C. Cooley, Elizabeth Garcia, Lynette M. Scholl, Neal I. Lindeman, Nikhil Wagle, Levi Garraway, Kristian Cibulskis, Scott L. Carter, Michael Lawrence, Gad Getz, Matthew L. Meyerson, William C. Hahn, Laura E. MacConaill. Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2013-819

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Formalin-fixed paraffin embedded (FFPE) tissues are far more abundant in most tissue banks and pathology departments than fresh or fresh frozen (FF) samples, but typically yield varying degrees of degraded DNA as a result of the fixation process. The quality of FFPE tissues may vary based on a number of factors such as: age of the block, fixation time, storage and handling conditions. Being able to harness the power of next generation sequencing technologies to genomically characterize these abundant and diverse achieved samples would be tremendously valuable to the cancer research community and would enable the use of this material for clinical purposes. The goal of this study was to assess the performance of FFPE samples in next generation sequencing applications. We monitored close to 100 samples from tumor and normal tissues of FFPE, FF and blood origin. These samples underwent several processes for whole-exome or targeted sequencing, including DNA fragmentation, size selection, library preparation, and hybrid-capture enrichment. We observed variable performance across these samples at several of the above steps, which correlated predominantly with FFPE tissues and the age of block. Other quality metrics that showed relative lower performance of FFPE DNA were the cluster density, duplication rate or library complexity. Most FFPE samples still generated good quality sequence, however, older FFPE blocks, over 10 years, may need more input DNA or higher sequence depth to reach minimum coverage if somatic mutation analysis is the goal. There was no difference in performance between DNA from tumor and normal tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3178. doi:1538-7445.AM2012-3178

Formalin fixation followed by paraffin embedment (FFPE) is the most common method of preserving resected tissues. FFPEs can be easily stored, retrieved and processed for further analysis when compared to the logistical complications of processing fresh frozen (FF) material. Conversely, they may not be ideal for sequencing because of the nucleic acid fragmentation and artifacts introduced by fixation. Here we report our efforts in performing next-generation sequencing on more than 70 specimens comprising >40 FFPE samples, from low to high quality, and compared the performance of FFPE, FF and commercially available cell lines.