Analytical performance and concordance with next-generation sequencing of a rapid multiplexed dPCR panel for the detection of actionable DNA and RNA biomarkers in non-small cell lung cancer

Abstract

AbstractBackgroundOver the last ten years, the discovery and FDA approval of targeted therapies for lung cancer has significantly improved patient survival rates. However, despite these improved survival rates, only 68% of patients receive molecular testing that results in assignment of targeted therapy1,2. Barriers to timely access to biomarker information include no testing ordered3,high nucleic acid input requirements, and problematic turnaround time (TAT) by NGS (> 14 days)4.Here we report the analytical performance and concordance with next-generation sequencing (NGS) of a highly-multiplexed research use only (RUO) panel using digital PCR (dPCR). The HDPCR NSCLC panel reports the status for variants (SNV, indels, and fusions) in eight actionable genes using amplitude modulation and multi-spectral encoding in dPCR5.MethodsThe panel’s analytical sensitivity and reactivity were determined using DNA and RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue spiked with plasmid DNA or in-vitro transcribed RNA. Concordance was established on 106 FFPE samples previously characterized using the Oncomine Precision Assay® or pathology results. Discordant resolution was resolved with Archer Fusionplex® and Variantplex® panels.ResultsThe analytical sensitivity, reported as estimated mutant allele fraction (MAF), for DNA targets (EGFRexon 19 deletions,EGFRexon 20 insertions,EGFRS768I,EGFRL858R,EGFRT790M,EGFRL861Q,BRAFV600E,EGFRG719X,ERBB2exon 20 insertions andKRASG12C) ranged from 0.8% – 4.9% with 40 ng of DNA input, and 2.4% to 10.9% with 15 ng of DNA input. For RNA fusion targets (ALK, RET, ROS, NTRK1/2/3, andMETexon 14 skipping), the analytical sensitivity ranged from 24 - 150 copies with 5 ng of total RNA input. The population prevalence-based coverage ranged from 89.2% to 100.0% across targets and >99.0% in aggregate. The accuracy of the assay was >97% with respect to the comparator method.