Diagnostics · Published 2026-07-30 · DOI 10.3390/diagnostics16152392
Andrea Boscolo Bragadin, Valeria Tosello, Stefano Longo, Alessia Padovan, Sara Baldissin, Paola Del Bianco, Elisa Masetto, Francesco Callegarin, Laura Bonanno, Giulia Pasello, Valentina Guarneri, Stefano Indraccolo
<b>Background</b><b>:</b> Sensitive detection of <i>EGFR</i> mutations in liquid biopsies of advanced non-small-cell lung cancer (aNSCLC) is vital for guiding targeted treatments. Real-time PCR offers a quick turnaround time but relatively low sensitivity while Next-Generation Sequencing (NGS) offers broader <i>EGFR</i> coverage, co-mutation evaluation and high sensitivity. <b>Methods:</b> This study evaluated the amplicon-based NGS Plasma-SeqSensei™ Solid Cancer In Vitro Diagnostics (IVD) Kit (Sysmex) against the real-time PCR-based cobas<sup>®</sup> EGFR Mutation Test v2 (Roche), the current routine standard at the Veneto Institute of Oncology IOV-IRCCS. We enrolled 130 patients with aNSCLC in the RARE Study between April 2022 and August 2025 who were referred to our institute. Liquid biopsies were taken at diagnosis or at progression and analyzed using two methods with the primary objective of assessing diagnostic concordance for <i>EGFR</i> profiling. Sysmex NGS libraries were sequenced on a NextSeq 550 sequencer (Illumina), with the NextSeq 500/550 Mid Output Kit v2.5 (150 Cycles) in single-end mode. <b>Results:</b> Among 129 evaluable samples, the NGS assay demonstrated a marginally higher <i>EGFR</i> mutation detection rate, identifying mutations in 31/129 cases (24.0%, 95% CI: 17–33), versus 28/129 (21.7%, 95% CI: 15–30) by cobas, specifically for variants covered by both assays. Overall concordance was almost perfect (Cohen’s Kappa = 0.89, 95% CI: 0.80–0.98), confirming the high reliability of both methods. Furthermore, we identified a cfDNA input threshold of at least 20 ng as critical for ensuring optimal assay sensitivity and reliable mutation detection (Odds Ratio = 4.41 for inputs ≥ 20 ng, <i>p</i> = 0.006). <b>Conclusions:</b> Ultimately, the Sysmex NGS proved to be a robust and highly sensitive assay. It delivers performance comparable to that of standard RT-PCR while providing the clinical advantage of concurrently detecting a broader spectrum of <i>EGFR</i> variants and actionable mutations in other genes.
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Bragadin, A., Tosello, V., Longo, S., et al. (2026). Comparing Real-Time PCR and Amplicon-Based NGS for Routine EGFR Liquid Biopsy Profiling. Diagnostics. https://doi.org/10.3390/diagnostics16152392