Abstract
BACKGROUND: Liquid biopsy is an important non-invasive method of sampling the molecular profile of tumors for patients to access personalized oncology therapeutics but can be challenging. NGS-based methods require high sample quality, high sequencing depth and associated cost, with complex workflows, while PCR assays are limited in variant coverage. Aspyre Clinical Test for Lung® (Blood) is a simplified genomic profiling assay for NSCLC that targets 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, gene fusions and exon skipping events from plasma-derived cfDNA and cfRNA simultaneously. METHOD: Sensitivity, specificity, analytical accuracy and analytical precision at standard input levels (20 ng cfDNA and 42 ng cfRNA) were tested using a combination of contrived samples and extracts from clinical samples taken from both healthy volunteers and patients with NSCLC. The effects of potential interfering substances on assay performance were tested. Assay sensitivity and specificity were also assessed at lower sample input levels (5 ng cfDNA and 6 ng cfRNA). RESULTS: At standard input levels, median limits of detection were ≤0.25 % variant allele fraction for single nucleotide variants, ≤0.4 % variant allele fraction for insertions or deletions, ≤6 copies for gene fusions, and ≤100 copies MET exon 14 skipping events. The specificity from variant-free samples was 100 %. Tests of analytical accuracy yielded 100 % NPA and 94 % PPA between Aspyre Clinical Test for Lung (Blood) and either results from orthogonal NGS testing or expected outcomes of contrived samples. Results were 100 % replicable across multiple operators, reagent lots, days and equipment. At low input levels, median limits of detection were ≤0.8 % for single nucleotide variants and insertions/deletions, 6 copies for gene fusions and 100 copies for MET exon 14 skipping, with a false-positive rate of 0 %. CONCLUSIONS: We present validation studies of Aspyre Clinical Test for Lung (Blood) using contrived and clinical samples. The technology is simple and fast, yet highly sensitive, specific, robust and reproducible with a turnaround time of two days. Aspyre Clinical Test for Lung (Blood) facilitates access to cost-effective, rapid, actionable molecular profiling of plasma for patients with NSCLC.