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).

Keywords: MASO-PCR; Mutation and methylation markers; NMIBC; Surveillance; Urine-based laboratory test; Urodiag® PCR kit.

Fig. 1

a–b Design of Mutation and Methylation PCR assays. The diagram 2a illustrates the Mutation assay with the position of the primers and fluorescent probes used for detection of human FGFR3 mutations (G372C, R248C, S249C, and Y375C) by MASO-PCR. The diagram 2b illustrates the Methylation assay with the position of the primers and fluorescent probes used in quantifying methylation degree of HS3ST2, SEPTIN9, and SLIT2 genes by QM-MSPCR. In both assays, the amplification curves are shown as examples, with Cts values above the established threshold as positive (Case 1) and below the threshold as negative (Case 2)

Fig. 2

DNA integrity assessed by PCR amplification of GLOBIN gene. DNA concentrations were determined by fluorometry. The GLOBIN gene was amplified with an amount of urine DNA comprised between 10 and 18 ng (4 μl of DNA sample) from each Filter (F). Amplification curves are shown from Pool 1 to Pool 4, respectively

Fig. 3

LoD for the Mutation assay. DNA from FGFR3 mutant plasmids was diluted into the wild-type DNA (standard human DNA). The proportion of mutant DNA was 50, 10, 5, and 1%, respectively. The representative amplification curves (a, c) and mean Ct values (b, d) are shown in the detection of the FGFR3 S249C/Y375C (a, b) and R248C/G372C (c, d) mutations by MASO-PCR

Fig. 4

LoQ for the Methylation assay. The limit of quantification (LoQ) for the QM-MSPCR1 (a) and QM-MSPCR2 (b) was determined by carrying out a series of dilutions with bisulfite converted DNA quantities of 10, 1, 0.1 and 0.01 ng