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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Carcinoma, Non-Small-Cell Lung*/genetics ; Carcinoma, Non-Small-Cell Lung*/drug therapy ; ErbB Receptors*/genetics ; Lung Neoplasms*/genetics ; Lung Neoplasms*/diagnosis ; Lung Neoplasms*/drug therapy ; Lung Neoplasms*/pathology ; High-Throughput Nucleotide Sequencing*/methods ; Mutation* ; Real-Time Polymerase Chain Reaction*/methods; Humans ; Female ; Male ; Middle Aged ; Aged ; Sensitivity and Specificity ; Exons/genetics

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 EGFR 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 EGFR 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 EGFR 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 EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR 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 EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR -positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.

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not applicable As the invited paper, a 100% discount of APC (2900 CHF) was offered to this submission.

Keywords: epidermal growth factor receptor; molecular diagnostics; mutations; non-small-cell lung cancer; targeted next-generation sequencing

EC 2.7.10.1 (ErbB Receptors)
EC 2.7.10.1 (EGFR protein, human)

Date Created: 20240727 Date Completed: 20240727 Latest Revision: 20240903

20240903

PMC11277480

10.3390/ijms25147908

39063150