Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels

Abstract Liquid biopsy tests are an integral part of the molecular diagnostic workup of patients with multiple cancer types. We have extended validation studies of a new liquid biopsy test panel that uses an amplicon-based targeted next-generation sequencing (NGS) panel with a semiconductor-based system, the Ion GeneStudio S5 Prime. The workflow requires very low input (20 ng) of cell-free nucleic acids extracted from human plasma and has been demonstrated to yield results in less than 72 hours from sample receipt at the laboratory. This report details the analytic performance of the test panel for detecting copy number variations (CNVs), fusions and exon skipping variants in plasma. These data add to previously reported validation data for the detection of somatic nucleotide variants (SNV) and indels [97.7% - 100% concordant with orthogonal droplet digital PCR (ddPCR) tests]. The updated study sample sets included consented clinical and normal healthy, donor specimens, and reference cell lines to detect fusions and exon skipping [HCC78 (ROS1), CRL-5935 (EML4-ALK), KM12 (NTRK1) and HTB-178 (MET exon 14 skipping)], and CNVs [CRL-5928 (HER2 gain), SK-BR-3 (HER2, CDK6, EGFR and MYC gains), HCC-827 (EGFR gain) and CRL-5909 (MET gain)]. The limit of detection (LOD) for fusion/skipping was initially established at a molecular coverage of 42 copies and was then verified by detection of three additional fusion/skipping variants. The LOD for CNVs was initially established at 1.40 fold-change and was then verified with the detection of three additional CNVs. Verification was conducted with specimens near the initially established LOD. In the precision studies, all expected fusion/skipping variants were detected in all runs and all days of testing (n=18/18) generating a hit rate of 100%. An average CV of 20.5% (range 8.7% to 34.8%) for repeatability and 20.8% (range 15.7% to 30.5%) for reproducibility was established. Twenty eight of 29 CNVs were detected, generating a hit rate of 96.6%. An average CV of 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for reproducibility was established for detected CNVs. We conclude that the validated test panel meets the criteria for being rapid, highly sensitive and specific, and has utility for the reporting of clinically relevant variants of interest in multiple cancer types. Citation Format: Audrey Audetat, Cherie Tschida, Sarah Kreston, Leisa Jackson, Hestia Mellert, Adam Stephen, Gary A. Pestano. Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5323.