{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:25:27Z","timestamp":1787030727346,"version":"build-2736575974"},"reference-count":42,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background: Fibroblast Growth Factor Receptor 3 (FGFR3) mutations are common and clinically relevant alterations in bladder cancer, with implications for diagnosis, monitoring, and patient selection for targeted therapies. Tissue-based testing is often limited by sample availability, invasiveness, and the need for repeated sampling. This study aimed to develop and analytically validate a sensitive double drop-off droplet digital polymerase chain reaction (ddPCR) assay for simultaneous detection of four FGFR3 hotspot mutations in tissue and liquid biopsy samples. Methods: The assay targeted four FGFR3 mutations (S249C, R248C, Y373C, G370C) using reference probes that generate a constant fluorescence signal and wild-type-specific drop-off probes that lose binding when a mutation is present, thereby distinguishing wild-type double-positive droplets from mutant droplets with reduced drop-off fluorescence. Analytical validation was performed using synthetic mutant oligonucleotides, wild-type genomic DNA, and cell-free DNA (cfDNA) from healthy donors (HDs). Specificity, limit of blank (LOB), limit of detection (LOD), and assay precision were evaluated. Performance was compared with next-generation sequencing (NGS) in formalin-fixed paraffin-embedded (FFPE) tissue DNA. FGFR3 mutations were also assessed in matched plasma and urinary cfDNA from bladder cancer patients. Results: The assay demonstrated clear cluster separation, no cross-reactivity, and reliable detection of all mutations down to 0.2% mutant allele frequency (MAF). Strong agreement was observed with a mutation-specific singleplex ddPCR assay for S249C. Concordance with NGS in tissue DNA was 74.2%, with ddPCR identifying additional low-abundance mutations not reported by NGS. FGFR3 mutations were detected in plasma and urinary cfDNA, with complete concordance between matched plasma and urine samples. Conclusions: This ddPCR assay provides a rapid, sensitive, and cost-effective method for detecting clinically relevant FGFR3 mutations and may complement sequencing-based approaches for molecular monitoring using tissue, plasma, and urine specimens.<\/jats:p>","DOI":"10.3390\/cancers18162634","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T15:48:36Z","timestamp":1786722516000},"page":"2634","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Broad Application of a Double Drop-Off ddPCR Assay for Simultaneous Detection of Four FGFR3 Mutations in Tissue and Liquid Biopsy Samples"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-5555-3630","authenticated-orcid":false,"given":"Eleni","family":"Thanou","sequence":"first","affiliation":[{"name":"Analysis of Circulating Tumor Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikos","family":"Gavalas","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0834-9912","authenticated-orcid":false,"given":"Eleni","family":"Kabrani","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Pathology, Department of General and Anatomic Pathology, Aristotle University School of Medicine, 54124 Thessaloniki, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Foteini","family":"Grigoriou","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Konstantinou","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vasiliki","family":"Malamatini","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina","family":"Chourdaki Peristeri","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7796-5914","authenticated-orcid":false,"given":"Evi","family":"Lianidou","sequence":"additional","affiliation":[{"name":"Analysis of Circulating Tumor Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5996-7262","authenticated-orcid":false,"given":"Aristotelis","family":"Bamias","sequence":"additional","affiliation":[{"name":"2nd Propaedeutic Department of Internal Medicine, Attikon University Hospital, National & Kapodistrian University of Athens, Chaidari, 12462 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7760-9701","authenticated-orcid":false,"given":"Athina","family":"Markou","sequence":"additional","affiliation":[{"name":"Analysis of Circulating Tumor Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"7655","DOI":"10.1038\/s41598-025-92033-5","article-title":"Trends in the Global, Regional, and National Burden of Bladder Cancer from 1990 to 2021: An Observational Study from the Global Burden of Disease Study 2021","volume":"15","author":"Su","year":"2025","journal-title":"Sci. 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