The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more variants in both assays. Additionally, we observed to be a frequently co-mutated gene in -positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.