FGFR3, HRAS, KRAS, NRAS and PIK3CA Mutations in Bladder Cancer and Their Potential as Biomarkers for Surveillance and Therapy

Background: Fifty 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 Findings: To 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/Significance: The 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

Patients' perceived burden of cystoscopic and urinary surveillance of bladder cancer: a randomized comparison

OBJECTIVE: To compare, in patients with non-muscle-invasive low-grade (pTa/pT1, G1/G2) urothelial cell carcinoma of the urinary bladder, the perceived burden of flexible cystoscopy or surveillance by microsatellite analysis (MA) in voided urine, as such patients are normally recommended to adhere to regular cysto-urethroscopic surveillance (CUS). PATIENTS AND METHODS: In all, 220 participants of a randomized trial comparing CUS and surveillance by MA were asked to complete questionnaires 1 week after cystoscopy or urine sample collection. We assessed the discomfort and pain reported during CUS, experiences with MA, and physical symptoms, medical consumption and general functioning in the week after CUS/urine sampling. RESULTS: We analysed data from 732 questionnaires (197 patients) completed after CUS and 184 (67 patients) after collecting urine. The introduction of the cystoscope was reported to cause discomfort in 39% and pain in 35% of the responses to the questionnaires; the waiting time for the results of MA was reported as burdensome in 19%. Painful micturition was significantly more frequent in the week after CUS than after MA (30% and 12%, respectively). The frequency of fever (1% and 2%) and haematuria (7% and 6%) was similar in both groups. Older patients reported significantly less pain and discomfort from cystoscopy, and this was not related to having more previous cystoscopies. CONCLUSION: CUS caused pain and discomfort in about a third of patients. The burden of MA appeared fully attributable to the waiting time for the test result. The present results are a further motivation in the search for less invasive surveillance test

Combinations of mutations in primary bladder tumors of 257 patients.

<p>Combinations of mutations in primary bladder tumors of 257 patients.</p

RAS-BC mutation assay.

<p>Panel A: wild-type sample, panels B–D: samples with mutations. Position of the interrogated codons, nucleotides and genes is depicted at the bottom.</p

Frequencies of individual mutations in primary tumors of 257 patients.

<p>Frequencies of individual mutations in primary tumors of 257 patients.</p

Probes for the detection of <i>HRAS</i>, <i>KRAS</i>, <i>NRAS</i>, <i>PIK3CA</i> and <i>FGFR3</i> mutations.

<p>Probes for the detection of <i>HRAS</i>, <i>KRAS</i>, <i>NRAS</i>, <i>PIK3CA</i> and <i>FGFR3</i> mutations.</p

Frequency of mutations in recurrence events of patients with a mutant pTa/T1G1/2 primary bladder tumor.

<p>Frequency of <i>FGFR3</i>, <i>RAS</i>, and <i>PIK3CA</i> mutations is indicated.</p

Frequencies of <i>FGFR3</i>, <i>RAS</i>, <i>PIK3CA</i> mutations and p53 overexpression according to stage and grade.

<p>The correlation of these alterations in primary bladder tumors of 257 patients with stage (A) or grade (B) is indicated by p-values (χ²).</p

Relations between mutations in primary bladder tumors of 257 patients.

<p>Relations between mutations in primary bladder tumors of 257 patients.</p

Primers used for multiplex amplification of <i>HRAS</i>, <i>KRAS</i>, <i>NRAS, PIK3CA</i>, and <i>FGFR3</i>.

<p>Primers used for multiplex amplification of <i>HRAS</i>, <i>KRAS</i>, <i>NRAS, PIK3CA</i>, and <i>FGFR3</i>.</p