Abstract 3148: Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy

Abstract

Abstract Next-generation sequencing (NGS) technology enables rapid analysis and turnaround of multiple genes for clinically actionable somatic variants. Genetic sequencing on tumor biopsy tissue to inform treatment decisions is the hallmark of precision medicine. However, a one-time, single-site tissue sample may not be a true representation of tumor dynamic profile. A liquid biopsy, based on circulating cell-free DNA (cfDNA), can capture the entire heterogeneity of the disease, and offer what tissue biopsies can’t, the opportunity to take serial samples in order to monitor tumor genomic evolution in real time. We have recently developed a proprietary cfDNA enrichment process that requires only droplet volumes of blood. Here, we presented clinical validation of the blood-drop liquid biopsy NGS assay interrogating 2855 mutation hotspots in 50 cancer-associated genes using the AmpliSeq cancer panel and Ion Torrent Proton sequencer. One nanogram of cfDNA prepared from 20 uL of plasma/serum was used as template to amplify 207 mutation hotspot amplicons. Deep sequencing of total 95 clinical specimens (≥ 1000× average coverage across the capture regions) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). The variants at AF of ≥ 10% in most samples (90/95), except 5 cases, were confirmed by another laboratory. Repeated sequencing of 15 samples, and progressively diluted samples from two reference standard DNAs with known mutations and AF demonstrated reliable detection sensitivity at 1% for most single nucleotide variants (SNV) with high within-run and between-run reproducibility. Validation also revealed sensitivity and specificity for detecting variants at 5% AF was 100% (95% CI = 95.7-100.0) using Tru-Q 4 NGS reference standard DNA, and NA19240 or NA12878 HapMap individual, respectively. Overall, our new-generation liquid biopsy NGS assay, with the capability to multiplex and simultaneously sequence multiple patient samples using finger-stick blood, is a robust and sensitive method for mutation analysis of clinical significance, further expedite treatment decision-making and identify targeted therapies for eligible patients in a time- and cost-efficient manner. Citation Format: Chen-Hsiung Yeh, Jonathan Spurgin, Andrew Ford, John Athanasuleas, Upender Manne. Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy. [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 3148.