Development and Broad Application of a Double Drop-Off ddPCR Assay for Simultaneous Detection of Four FGFR3 Mutations in Tissue and Liquid Biopsy Samples

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.