FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy

Abstract

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.

Figure 1. Primers used for multiplex amplification of HRAS, KRAS, NRAS, PIK3CA, and FGFR3.

Figure 2. Probes for the detection of HRAS, KRAS, NRAS, PIK3CA and FGFR3 mutations.

Figure 3. RAS-BC mutation assay.

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

Figure 4. Frequencies of FGFR3, PIK3CA and RAS mutations in primary bladder tumors of 257 patients.

Frequencies in all primary bladder tumors (A) (n = 257) and in specific tumor stages: pTa/T1-G1/G2 (B) (n = 194), pTa/T1G3 (C) (n = 29) and muscle-invasive tumors (D) (n = 34).

Figure 5. Relations between mutations in primary bladder tumors of 257 patients.

Figure 6. Frequencies of FGFR3, RAS, PIK3CA mutations and p53 overexpression according to stage and grade.

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

Figure 7. Detailed overview of the mutation status of 54 primary and 184 recurrent tumors.

A: mutant primary tumors and their recurrences; B: wild-type primary tumors and their recurrences. The first column indicates the primary tumor. The successive boxes indicate temporally sequential recurrences removed in different transurethral resections (indicated by a sequence number on top). Multifocal tumors removed at the same transurethral resection are positioned underneath each other. Stage and grade of the tumors, mutation status (indicated by a color) and patient ID of the 54 patients is indicated.

Figure 8. Frequency of mutations in recurrence events of patients with a mutant pTa/T1G1/2 primary bladder tumor.

Frequency of FGFR3, RAS, and PIK3CA mutations is indicated.