Abstract 797: Targeted next generation sequencing of FFPE lung cancer tissue.

Abstract

Abstract In view of a personalized medicine, the screening of somatic mutations in an individual tumor that predict therapeutic outcome has become an increasing need in the clinical and molecular diagnostics. In most laboratories, Sanger sequencing has become an accepted standard, but due to the limited screening throughput and the increasing demand for targeted sequencing, the application of next-generation sequencing in the molecular diagnostic has turned into focus. Whole-genome or whole-exome sequencing approaches provide a comprehensive view of an individual tumor mutation load, but the current high costs and the excess of information without a direct clinical implication limit the routine use of this technology. In this study, we evaluated a targeted resequencing approach that focuses on somatic hotspot cancer mutations based on semiconductor sequencing. We especially took attention on the reliability of the method concerning sample quality, mutation type and reproducibility. We used DNA extracted from formalin-fixed, paraffin embedded (FFPE) tumor tissues (biopsy and resection) of different tumor degree and overall quality that were previously screened for the presence of EGFR mutations (exon 18-21) by Sanger sequencing. 190 amplicons covering hotspot mutations in 46 genes were generated in a multiplex PCR reaction (AmpliSeqTM), barcoded and 8 samples were run on a single IonTorrent 318 chip. We were able to successfully sequence all samples with a mean coverage rate of 670.000 reads (AQ20:560.000) and an average read depth of 2947 AQ20 reads / amplicon. In all but one sample, variant calling identified the EGFR mutation, missing only one low level 9bp insertion in exon 20. Moreover, we identified additional 43 mutations in 17 genes and uncovered three previously unknown EGFR amplifications. Taken together, amplicon-based semiconductor sequencing is a powerful and cost-effective method working with low-quality DNA material, enabling routine diagnostic next-generation sequencing. Citation Format: Volker Endris, Albrecht Stenzinger, Roland Penzel, Arne Warth, Peter Schirmacher, Wilko Weichert. Targeted next generation sequencing of FFPE lung cancer tissue. [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 797. doi:10.1158/1538-7445.AM2013-797