{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T04:58:21Z","timestamp":1785560301884,"version":"3.56.0"},"reference-count":55,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T00:00:00Z","timestamp":1785369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003196","name":"Italian Ministry of Health","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003196","id-type":"DOI","asserted-by":"crossref"}]},{"name":"AstraZeneca"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background: Sensitive detection of EGFR 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 EGFR coverage, co-mutation evaluation and high sensitivity. Methods: This study evaluated the amplicon-based NGS Plasma-SeqSensei\u2122 Solid Cancer In Vitro Diagnostics (IVD) Kit (Sysmex) against the real-time PCR-based cobas\u00ae 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 EGFR 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. Results: Among 129 evaluable samples, the NGS assay demonstrated a marginally higher EGFR mutation detection rate, identifying mutations in 31\/129 cases (24.0%, 95% CI: 17\u201333), versus 28\/129 (21.7%, 95% CI: 15\u201330) by cobas, specifically for variants covered by both assays. Overall concordance was almost perfect (Cohen\u2019s Kappa = 0.89, 95% CI: 0.80\u20130.98), 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 \u2265 20 ng, p = 0.006). Conclusions: 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 EGFR variants and actionable mutations in other genes.<\/jats:p>","DOI":"10.3390\/diagnostics16152392","type":"journal-article","created":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T08:09:42Z","timestamp":1785398982000},"page":"2392","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparing Real-Time PCR and Amplicon-Based NGS for Routine EGFR Liquid Biopsy Profiling"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9614-0814","authenticated-orcid":false,"given":"Andrea","family":"Boscolo Bragadin","sequence":"first","affiliation":[{"name":"Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Tosello","sequence":"additional","affiliation":[{"name":"Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3079-8780","authenticated-orcid":false,"given":"Stefano","family":"Longo","sequence":"additional","affiliation":[{"name":"Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessia","family":"Padovan","sequence":"additional","affiliation":[{"name":"Department of Surgery, Oncology and Gastroenterology, University of Padua, 35124 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Baldissin","sequence":"additional","affiliation":[{"name":"Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6597-0265","authenticated-orcid":false,"given":"Paola","family":"Del Bianco","sequence":"additional","affiliation":[{"name":"Clinical Research Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-9279-5612","authenticated-orcid":false,"given":"Elisa","family":"Masetto","sequence":"additional","affiliation":[{"name":"Clinical Research Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-9594-5178","authenticated-orcid":false,"given":"Francesco","family":"Callegarin","sequence":"additional","affiliation":[{"name":"Clinical Research Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5218-4970","authenticated-orcid":false,"given":"Laura","family":"Bonanno","sequence":"additional","affiliation":[{"name":"Department of Surgery, Oncology and Gastroenterology, University of Padua, 35124 Padua, Italy"},{"name":"Medical Oncology 2, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia","family":"Pasello","sequence":"additional","affiliation":[{"name":"Department of Surgery, Oncology and Gastroenterology, University of Padua, 35124 Padua, Italy"},{"name":"Medical Oncology 2, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina","family":"Guarneri","sequence":"additional","affiliation":[{"name":"Department of Surgery, Oncology and Gastroenterology, University of Padua, 35124 Padua, Italy"},{"name":"Medical Oncology 2, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4810-7136","authenticated-orcid":false,"given":"Stefano","family":"Indraccolo","sequence":"additional","affiliation":[{"name":"Basic and Translational Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy"},{"name":"Department of Surgery, Oncology and Gastroenterology, University of Padua, 35124 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1002\/ijc.35278","article-title":"The GLOBOCAN 2022 Cancer Estimates: Data Sources, Methods, and a Snapshot of the Cancer Burden Worldwide","volume":"156","author":"Filho","year":"2025","journal-title":"Int. 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