Abstract

BackgroundOver 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 therapy 1,2. 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 panels 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(R) or pathology results. Discordant resolution was resolved with Archer Fusionplex(R) and Variantplex(R) panels. ResultsThe analytical sensitivity, reported as estimated mutant allele fraction (MAF), for DNA targets (EGFR exon 19 deletions, EGFR exon 20 insertions, EGFR S768I, EGFR L858R, EGFR T790M, EGFR L861Q, BRAF V600E, EGFR G719X, ERBB2 exon 20 insertions and KRAS G12C) 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, NTRK 1/2/3, and MET exon 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.