Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine.

Abstract

271 Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary matrix metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. The LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. Results: To validate the new multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. [Table: see text]