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 coamplify 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.

<i>FGFR3, TERT, ТР53</i> mutations and the <i>FGFR3</i> gene expression in bladder cancer as prognostic markers

“…Рак мочевого пузыря в тест-системе Urodiag ® PCR Kit. С ее помощью определяют мутации FGFR3 методом модифицированной технологии аллель-специфичной ПЦР (MASO-PCR, Mutated Allele Specific Oligonucleotide-PCR) для диагностики рецидива НМИРМП по анализу мочи [34]. Применяют и аналогичные тест-системы с использованием ПЦР для определения в осадке мочи мутаций TERT при диагностике РМП и его рецидивов [25].…”

Urinary markers for bladder cancer diagnosis: A review of current status and future challenges

“…UroDiag is a commercially available multiplex qPCR-based assay that combines the analysis of four FGFR3 somatic mutations (G372C, R248C, S249C, and Y375C) and three epigenetic markers (methylation of HS3ST2, SEPTIN9, and SLIT2) in urinary-exfoliated cells. 65 The strategy of this assay involves utilizing FGFR3 mutations, which are observed in over 50% of patients with NMIBCa and represent urinary markers for low-grade BCa, to compensate for the low sensitivity of epigenetic markers for low-grade BCa. 66 UroDiag showed a sensitivity of 97.6% (94% for low-grade tumors, 100% for high-grade tumors), and a specificity of 84.8% in the initial diagnosis of 272 patients (167 BCa patients and 105 controls), with a sensitivity of 94.5% (93.6% for lowgrade tumors, 96% for high-grade tumors) and a specificity of 75.9% in a surveillance setting of 158 patients.…”

“…UroDiag is a commercially available multiplex qPCR‐based assay that combines the analysis of four FGFR3 somatic mutations (G372C, R248C, S249C, and Y375C) and three epigenetic markers (methylation of HS3ST2, SEPTIN9 , and SLIT2 ) in urinary‐exfoliated cells 65 . The strategy of this assay involves utilizing FGFR3 mutations, which are observed in over 50% of patients with NMIBCa and represent urinary markers for low‐grade BCa, to compensate for the low sensitivity of epigenetic markers for low‐grade BCa 66 .…”

Liquid Biopsy Biomarkers in Urine: A Route towards Molecular Diagnosis and Personalized Medicine of Bladder Cancer

“…Since it has been demonstrated that high-risk tumors in general have high amount of hypermethylated genes than low-risk tumors [ 33 ], Roperch and Hennion [ 34 ] proposed a model combining a panel of FGFR3 mutations and a set of epigenetic markers (hypermethylation of the HS3ST2 , SEPTIN9 and SLIT2 ). Such a model in a study population including 263 subjects showed a very high diagnostic accuracy for the monitoring of BC patients with sensitivity/specificity/Negative predictive value (NPV)higher than 95%/76%/99%, respectively [ 35 ].…”