Inter-Site Comparison Of Performance Of An Ultra-Sensitive Sequencing Technology For Circulating Tumor Dna

Abstract The combination of liquid biopsies and ultra-sensitive next-generation sequencing (NGS) holds tremendous promise in oncology for the detection and surveillance of somatic mutations that correlate with diagnosis, prognosis, response prediction, resistance monitoring and tumor burden. We have developed a capture-based NGS workflow that provides ultra-sensitive detection of 4 mutation classes - single nucleotide variants (SNVs), insertions/deletions, copy number amplifications (CNAs) and fusions - from low input cell-free DNA isolated from plasma. Sensitivity and specificity of 2 different AVENIO ctDNA Analysis Kits (for Research Use Only) across 3 sites for 48 samples that included contrived DNA blends with known mutations validated by digital PCR and normal reference plasma samples were assessed. The AVENIO ctDNA Targeted Kit (Targeted) was designed to detect mutations in 17 genes, across 80kb, included in NCCN Guidelines for solid tumors plus additional variants relevant for therapy selection. The AVENIO ctDNA Expanded Kit (Expanded) was designed to detect mutations in 77 genes, across 200kb, associated with solid tumor including variants relevant for clinical trial research. Testing sites achieved 100% sensitivity for SNVs and fusions at 0.5% and 1% allelic frequency, respectively, from 30 ng of input DNA for both kits. Median coverage of 4,200-6,400X was greater with the Targeted kit relative to that of 2,700-3,800X with the Expanded kit. Specificity was 100% for the Targeted kit either by sample or variant. The Expanded kit had a specificity of 91.7-100% by sample and 99.7-100% by variant. At an expected 5X or 8X copy number amplification for multiple genes, both the Targeted and Expanded kits were 100% sensitive and 93.1-98.4% specific per gene. The specificity for fusion detection was 100% for both kits down to 1% mutant allele frequency. The AVENIO ctDNA Analysis Kits for plasma and NGS applications provide exquisite sensitivity and specificity for the simultaneous detection of 4 different mutation classes. Citation Format: Stephanie Yaung, Alex Lovejoy, Dan Klass, Fergal Casey, Aruna Arcot, Melissa Loyzer, William Chiu, Nabil Azhar, Sophie Beckert, Maria Lange, Sandra Siemann, Sebastian Froehler, Shelly Gunn, John F. Palma. Inter-site comparison of performance of an ultra-sensitive sequencing technology for circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2017-2744