Improved detection of druggable EGFR variants in non-small cell lung cancer using targeted next-generation sequencing

<bold>Background:</bold> 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 <italic>EGFR</italic> variants in exons 18-21 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 <italic>EGFR</italic> variants testing in clinical NSCLC material previously analyzed by IVD-certified qPCR test with respect to DNA reference material. <bold>Methods:</bold> 59 NSCLC tissue specimens were tested for <italic>EGFR</italic> variants by the NGS ‘TruSight Tumor 15’ assay (Illumina) and the qPCR ‘Cobas EGFR mutation test’ (Roche Diagnostics). The sensitivity and specificity of NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of <italic>EGFR</italic> variants. <bold>Results:</bold> In DNA reference material, NGS demonstrated sufficient lower detection limit for diagnostic applications (VAF<5%); all <italic>EGFR</italic> variants were correctly identified. NGS showed high repeatibility of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 59%. The majority of discordant results concerned false-positive detection of <italic>EGFR</italic> exon 20 insertions by qPCR. <italic>TP53</italic> was frequently co-mutated gene in <italic>EGFR</italic>-positive NSCLC patients. <bold>Conclusion:</bold> NGS showed a number of superior features over qPCR in <italic>EGFR</italic> variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity) which might enhance the basic diagnostic report.