Related Experiment Video For Allele-specific qPCR
Updated: Oct 28, 2025
A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction
Published on: April 5, 2018
Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world
Adam Szpechcinski1, Maciej Bryl2, Piotr Wojcik3
1Department of Genetics and Clinical Immunology, National Tuberculosis and Lung Diseases Institute, Warsaw, Poland.
Purpose:
The detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations.
Methods:
We analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I-IV) before treatment (n = 60) and after clinical progression on EGFR tyrosine kinase inhibitors (n = 30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH).
Results:
There was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ = 0.80; p < 0.0001) and 73.33% with therascreen (κ = 0.49; p = 0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively.
Conclusions:
Cobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.
