Abstract 490: High-throughput mutational analysis in cell-free DNA by targeted next-generation sequencing

Abstract

Abstract Circulating cell-free DNA (cfDNA) in plasma offers a non-invasive approach to monitor tumor molecular profiling in real-time at multiple time-points, detection of emerging genomic alterations associated with drug resistance and clarifying cancer prognosis and diagnosis of cancer recurrence or progression. We developed a robust, multiplexed and PCR-based targeted next-generation sequencing approach to detect actionable mutations in cfDNA with low DNA input (1-5 ng). We employed the latest Ion TorrentTM S5/XL bench-top sequencer with Ion ChefTM Automation System and successfully automated the entire 2-day workflow from libraries preparation to semi-conducting chip sequencing. The hands-on time has been reduced to only 30 min. We will present data showing validation of this NGS platform using commercially available DNA control, archival FFPE tissue and cfDNA matched samples and demonstrate robust sensitivity and specificity by using the off-the-shelf Ion AmpliSeq Cancer Hotspot Panel v2 that covers 50 oncogenes. We also compared this approach to other orthogonal technologies, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR) and achieved 100% concordance across these platforms. NGS can detect mutations down to 1% allelic frequency with 100% sensitivity and specificity. This robust and automated NGS platform is being routinely used in our lab to analyze cell-free DNA samples from multiple clinical trials. These efforts enable the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient's tumor evolution in real time, and aid in the development of personalized therapies for cancer patients. Citation Format: Nga wan Rachel Tam, David Shames, Walter Darbonne. High-throughput mutational analysis in cell-free DNA by targeted next-generation sequencing. [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 490.