Abstract 5574: A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples
Authors: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes
Journal: Cancer Research (2018)

Abstract Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and &amp;gt;50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering &amp;gt;500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was &amp;gt;90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with &amp;gt;90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring. Citation Format: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes. A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5574.