Related Experiment Video
Updated: Jun 16, 2026
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable EGFR Variants in
Adam Szpechcinski1, Joanna Moes-Sosnowska1, Paulina Skronska1
1Department of Genetics and Clinical Immunology, The Institute of Tuberculosis and Lung Diseases, 01-138 Warsaw, Poland.
Abstract:
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.
Insights
Next-generation sequencing (NGS) offers superior detection of epidermal growth factor receptor (EGFR) variants in non-small-cell lung cancer (NSCLC) compared to qPCR. NGS provides exact variant identification and precise allelic frequency, enhancing diagnostic accuracy for targeted therapies.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Targeted therapies for non-small-cell lung cancer (NSCLC) require accurate companion diagnostics for epidermal growth factor receptor (EGFR) variants.
- Next-generation sequencing (NGS) is increasingly replacing traditional methods like qPCR for detecting actionable EGFR mutations.
Purpose of the Study:
- To evaluate the diagnostic utility of targeted NGS for detecting druggable EGFR variants in clinical NSCLC samples.
- To compare the performance of NGS against a validated qPCR assay using clinical and reference DNA materials.
Main Methods:
- Analyzed 59 NSCLC tissue and cytology specimens using both targeted NGS ('TruSight Tumor 15') and qPCR ('cobas EGFR mutation test v2').
- Assessed NGS sensitivity, specificity, and repeatability using DNA reference materials with known EGFR variant allelic frequencies (VAF).
- Evaluated concordance between NGS and qPCR in clinical samples.
Main Results:
- NGS demonstrated high analytical sensitivity with a lower detection limit below 5% VAF in reference material, correctly identifying all EGFR variants.
- NGS showed excellent repeatability for VAF assessment (CV% 0.02-3.98).
- Overall concordance between NGS and qPCR in clinical samples was 76.14%, with discrepancies often due to qPCR false-positive EGFR exon 20 insertions. 15% of samples showed discordant results. TP53 co-mutation was frequently observed in EGFR-positive NSCLC.
Conclusions:
- Targeted NGS offers superior performance over qPCR for EGFR variant detection in NSCLC, including precise variant identification and VAF calculation.
- NGS exhibits high analytical sensitivity and repeatability, potentially improving diagnostic reports for targeted therapy selection.
- The study highlights NGS's advantages in accurately characterizing EGFR mutations and identifying co-mutations like TP53 in NSCLC.
Related Concept Videos
01:11Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase
01:29Pharmacogenomics: Identification of New Drug Targets