A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

Abstract

Background We have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine.Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays.Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

Molecular and Immunohistochemical assessment of Fibroblast Growth Factor Receptor 3 (FGFR3) gene as a survival time predictor in bladder cancer patients

“…The development of reliable and affordable tools to detect BC is a challenge. Genetic and epigenetic alterations in DNA have been reported in the development and progression of BC [5].…”

“…Genetic analysis of BC has identified genetic abnormalities in a number of chromosomes and genes that have been known to play a significant role in development of BC pathogenesis and have emerged as a marker for non-aggressive disease and a promising therapeutic target [6]. In the mutational path, Since 2002 the fibroblast growth factor receptor gene 3 (FGFR3) appears to be the most frequently mutated gene in BC [5].…”