Abstract 3622: Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms

Abstract

Abstract Research detecting of somatic mutations in circulating cell-free DNA (cfDNA) using research blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. However, most of the existing mutation detection methods show insufficient sensitivity to detect cfDNA mutations since only small amount of mutant gene fragments, derived from tumor cells, is present in a large amount of normal circulating DNA background. We demonstrated a complete workflow that includes blood collection, cfDNA isolation, library preparation, sequencing, and data analysis to enable detection of rare DNA variants in blood plasma samples. Blood samples were collected using Streck™ DNA tubes followed by plasma preparation and cfDNA isolation using MagMAX™ Cell-Free DNA Isolation Kit. Library preparation was performed using Oncomine™ lung cfDNA kit. Barcoded libraries were pooled and sequenced on Ion Torrent™ Next Generation Sequencing Platforms. Sequencing data was analyzed in Torrent Suite™ using variantCaller-cfDNA plugin. ∼150 biomarkers relevant to non-small cell lung cancer were interrogated in one sequencing run. We demonstrated detection sensitivity at 0.1% frequency using engineered mutants that were spiked into control DNA samples. The workflow was tested on a set of research samples from matched tumor FFPE and blood plasma collected from research subjects with non-small cell lung cancer (NSCLC). About 1 mL of plasma was processed using the workflow described above. RecoverAll™ Multi-Sample RNA/DNA Isolation Workflow was used to isolate DNA from FFPE samples, followed by library preparation, sequencing and data analysis using the same workflow described above. Summary of variant calls from matched cfDNA and FFPE tumor samples are presented here. Results indicate high sensitivity of the workflow and expected levels of concordance between variants detected in the two types of research samples. In this study, we developed a highly sensitive and reliable research workflow to detect rare somatic mutations in circulating cfDNA samples. Significant overlapping of mutations discovered in FFPE tumor and cfDNA samples suggests that this workflow may be used to monitor tumor dynamics in NSCLC and potentially other tumors in the future. Disclaimer: For research use only. Not for use in diagnostic procedures. Citation Format: Jian Gu, Dumitru Brinza, Ann Mongan, Richard Chien, Dalia Dhingra, Fiona Hyland, Kelli Bramlett. Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms. [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 3622.