Abstract 742: Development and evaluation of a non-invasive qPCR assay for sensitive detection of FGFR2/3 alterations in urine samples as a companion diagnostic for erdafitinib

Abstract Introduction: Fibroblast growth factor receptor (FGFR) alterations, including point mutations and gene fusions, play a pivotal role in various cancers, particularly urothelial carcinoma. Erdafitinib, an FDA-approved therapy for locally advanced or metastatic urothelial carcinoma with FGFR2/3 alterations, currently relies on tissue-based diagnostics, which are invasive and may not always be feasible. Non-invasive liquid biopsy approaches, such as qPCR assays using urine cell pellets (UCP), offer significant advantages in terms of patient convenience and safety while providing accurate detection of clinically relevant alterations. Methods: We developed and evaluated the Predicine FGFR2/3 qPCR Kit, a real-time multiplex PCR assay designed to detect four FGFR3 mutations (R248C, S249C, G370C, Y373C) and five FGFR2/3 fusions (FGFR3-TACC3 V1/V3, FGFR3-BAIAP2L1, FGFR2-BICC1, FGFR2-CASP7) from urine DNA and RNA. The assay integrates proprietary buffers to preserve nucleic acids, allowing simultaneous detection of DNA mutations and RNA fusions. Analytical performance, including sensitivity, specificity, and accuracy, was assessed using gradient dilutions, known positive controls, and clinical urine samples. Detection thresholds were benchmarked against NGS and ddPCR. Results: The assay demonstrated high sensitivity, detecting DNA variants at allele frequencies as low as 0.25% and RNA fusions at 6-7 copies. Specificity exceeded 98% across all tested alterations in healthy donor samples, and positive percentage agreement (PPA) exceeded 95% for FGFR3 mutations and FGFR2/3 fusions compared to NGS results. In a retrospective cohort of 60 clinical urine samples, mutation detection sensitivity reached 100% for most variants, except for FGFR3-S249C, while specificity ranged from 92% to 100%. UCP-derived nucleic acids enabled streamlined workflows with a single vial of urine. Mutation detection achieved a limit of 0.1% for DNA and 6.25 copies for RNA fusions. Conclusion: The Predicine FGFR2/3 qPCR Kit offers a rapid, robust, and non-invasive solution for detecting FGFR alterations in urine samples. By providing high sensitivity, specificity, and efficiency, it represents a promising alternative to invasive tissue-based diagnostics for identifying patients eligible for FGFR-targeted therapies such as erdafitinib. These findings highlight its potential utility as a companion diagnostic, with further clinical validation planned to support its integration into routine molecular diagnostics. Citation Format: Meirong Zhang, Mingzhu Li, Yue Zhang, Hang Dong, Haoran Tang, Feng Xie, Qing Xu. Development and evaluation of a non-invasive qPCR assay for sensitive detection of FGFR2/3 alterations in urine samples as a companion diagnostic for erdafitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 742.