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Comparing Real-Time PCR and Amplicon-Based NGS for Routine EGFR Liquid Biopsy Profiling
Andrea Boscolo Bragadin1, Valeria Tosello1, Stefano Longo1
1Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy.
Diagnostics (Basel, Switzerland)
|August 13, 2026
Summary
Next-Generation Sequencing (NGS) offers a sensitive and robust method for detecting EGFR mutations in advanced non-small-cell lung cancer (aNSCLC) liquid biopsies, showing high concordance with standard PCR methods.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Sensitive detection of Epidermal Growth Factor Receptor (EGFR) mutations in liquid biopsies is crucial for targeted therapy in advanced non-small-cell lung cancer (aNSCLC).
- Real-time PCR provides rapid results but has limited sensitivity, whereas Next-Generation Sequencing (NGS) offers broader coverage and higher sensitivity for EGFR mutations and co-mutations.
Purpose of the Study:
- To evaluate the diagnostic concordance between an amplicon-based NGS assay (Plasma-SeqSensei™) and a standard real-time PCR assay (cobas® EGFR Mutation Test v2) for EGFR profiling in aNSCLC patients.
- To assess the performance of NGS in detecting EGFR mutations in liquid biopsies compared to the established PCR method.
Main Methods:
- A study enrolled 130 patients with aNSCLC, analyzing liquid biopsies using both Sysmex's amplicon-based NGS kit and Roche's cobas® real-time PCR test.
- NGS libraries were sequenced on an Illumina NextSeq 550 platform.
- Diagnostic concordance for EGFR mutations was the primary objective, with a secondary analysis of cfDNA input requirements.
Main Results:
- The NGS assay showed a marginally higher EGFR mutation detection rate (24.0%) compared to cobas (21.7%) among 129 evaluable samples.
- Near-perfect overall concordance (Cohen's Kappa = 0.89) was observed between the NGS and PCR methods.
- A minimum cfDNA input of 20 ng was identified as critical for optimal assay sensitivity and reliable mutation detection (OR = 4.41, p=0.006).
Conclusions:
- The Sysmex NGS assay is a robust, highly sensitive, and reliable method for EGFR mutation detection in aNSCLC liquid biopsies.
- NGS performance is comparable to standard RT-PCR, with the added clinical benefit of detecting a wider range of EGFR variants and other actionable mutations.
- The study highlights the importance of sufficient cfDNA input for accurate mutation detection using NGS in liquid biopsies.
