A simple and fast method for the simultaneous detection of nine fibroblast growth factor receptor 3 mutations in bladder cancer and voided urine
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Purpose:Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors.An FGFR3 mutationis associatedwith goodprognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases.These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detectionof recurrences inurine is animportant clinical issue. Inthis paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations. Experimental Design: The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide.The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of amutation. Results: The assay was found to be more sensitive than single-strand conformation polymorphism analysis.Mutations could still be detected with an input of only 1ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detectionwas 62%. Conclusions:We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.