BackgroundFifty percent of patients with muscle–invasive bladder cancer (MI-BC) die from their disease and current chemotherapy treatment only marginally increases survival. Novel therapies targeting receptor tyrosine kinases or activated oncogenes may improve outcome. Hence, it is necessary to stratify patients based on mutations in relevant oncogenes. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival, however two-thirds develop recurrences. Tumor specific mutations can be used to detect recurrences in urine assays, presenting a more patient-friendly diagnostic procedure than cystoscopy.Methodology/Principal FindingsTo address these issues, we developed a mutation assay for the simultaneous detection of 19 possible mutations in the HRAS, KRAS, and NRAS genes. With this assay and mutation assays for the FGFR3 and PIK3CA oncogenes, we screened primary bladder tumors of 257 patients and 184 recurrences from 54 patients. Additionally, in primary tumors p53 expression was obtained by immunohistochemistry. Of primary tumors 64% were mutant for FGFR3, 11% for RAS, 24% for PIK3CA, and 26% for p53. FGFR3 mutations were mutually exclusive with RAS mutations (p = 0.001) and co-occurred with PIK3CA mutations (p = 0.016). P53 overexpression was mutually exclusive with PIK3CA and FGFR3 mutations (p≤0.029). Mutations in the RAS and PIK3CA genes were not predictors for recurrence-free, progression-free and disease-specific survival. In patients presenting with NMI-BC grade 3 and MI-BC, 33 and 36% of the primary tumors were mutant. In patients with low-grade NMI-BC, 88% of the primary tumors carried a mutation and 88% of the recurrences were mutant.Conclusions/SignificanceThe mutation assays present a companion diagnostic to define patients for targeted therapies. In addition, the assays are a potential biomarker to detect recurrences during surveillance. We showed that 88% of patients presenting with low-grade NMI-BC are eligible for such a follow-up. This may contribute to a reduction in the number of cystoscopical examinations.

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Purpose: Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, 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 detection of recurrences in urine is an important clinical issue. In this 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 a mutation. 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 detection was 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.Bladder cancer is the fifth most common cancer in the western world with an incidence of 20 new cases per year per 100,000 people in the U.S. (1). Unfortunately, these statistics do not include superficial pTa bladder cancer, which represents the most common type of bladder cancer. In the Netherlands, the incidence of both superficial and invasive bladder cancer is estimated as about 30 new cases per year per 100,000 people. This is in accordance with data from global cancer statistics for the western world (2). Superficial bladder tumors are removed by transurethral resection. However, up to 70% of these patients will develop one or more recurrences, and it has been estimated that 1 in 1,450 people is under surveillance for bladder cancer in the United Kingdom (3). Cystoscopy is an uncomfortable, invasive, and expensive procedure, but currently remains the gold standard for detection of recurrences. Because patients have to be monitored perpetually and have a long-term survival, bladder cancer is the most expensive cancer when calculated on a per patient basis (4).Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have been reported in >50% of primary bladder tumors (5, 6).1 Most of the somatic mutations found in bladder cancer are identical to germ line mutations responsible for skeletal disorders such as thanatophoric dysplasia and achondroplasia (7). It has been reported that FGFR3 mutations are very frequent ...