Companion diagnostics, which predict the efficacy of molecular targeted agents based on genetic information, are indispensable for the treatment of advanced non-small cell lung carcinoma. Recent increase in the number of molecular targeted agents and the corresponding target genes have led to the demand for the simultaneous testing of multiple genes. Although gene panels using next-generation sequencing (NGS panels) are ideal for this purpose, conventional panels require high tumor content, and biopsy samples often do not meet this requirement. We developed a new NGS panel called a compact panel, to accommodate biopsy samples without the restriction of tumor content. The compact panel is characterized by high sensitivity, with detection limits for mutations of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for EGFR exon 19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Mutation detection also has a high quantitative ability, with correlation coefficients ranging from 0.966 to 0.992. The panel detected fusions in samples whose tumor cell content was as low as 1%. The compact panel exhibited good concordance with approved tests as follows: EGFR positive, 100.0 (95% confidence interval 95.5-100); EGFR negative, 90.9 (82.2-96.3); ALK positive, 96.7 (83.8-99.9); ALK negative, 98.4 (97.2-99.2); ROS1 positive, 100 (66.4-100); ROS1 negative, 99.0 (97.1-99.2); MET positive, 98.0 (89.0-100); MET negative 100 (92.8-100). The analytical performance demonstrated that the compact panel can handle various types of biopsy samples obtained by routine clinical practice, without requiring strict pathological monitoring like in case of conventional NGS panels.
Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow
