- Top of page
- Materials and Methods
- Conflict of Interest
While fibroblast growth factor receptor 3 (FGFR3) is frequently mutated or overexpressed in nonmuscle-invasive urothelial carcinoma (UC), the prevalence of FGFR3 protein expression and mutation remains unknown in muscle-invasive disease. FGFR3 protein and mRNA expression, mutational status, and copy number variation were retrospectively analyzed in 231 patients with formalin-fixed paraffin-embedded primary UCs, 33 metastases, and 14 paired primary and metastatic tumors using the following methods: immunohistochemistry, NanoString nCounterTM, OncoMap or Affymetrix OncoScanTM array, and Gain and Loss of Analysis of DNA and Genomic Identification of Significant Targets in Cancer software. FGFR3 immunohistochemistry staining was present in 29% of primary UCs and 49% of metastases and did not impact overall survival (P = 0.89, primary tumors; P = 0.78, metastases). FGFR3 mutations were observed in 2% of primary tumors and 9% of metastases. Mutant tumors expressed higher levels of FGFR3 mRNA than wild-type tumors (P < 0.001). FGFR3 copy number gain and loss were rare events in primary and metastatic tumors (0.8% each; 3.0% and 12.3%, respectively). FGFR3 immunohistochemistry staining is present in one third of primary muscle-invasive UCs and half of metastases, while FGFR3 mutations and copy number changes are relatively uncommon.
- Top of page
- Materials and Methods
- Conflict of Interest
Our results demonstrate that some FGFR3 IHC staining is present in nearly one third of primary muscle-invasive UC tumors and half of metastases. While the sample size was relatively small, over half of metastases from paired primary tumors with FGFR3 expression retain FGFR3 protein expression, albeit at low levels in most specimens. Tissue cores were obtained from pathologist-reviewed H&E tumor specimens and incorporated into a TMA. Since the whole tissue section was not available to be stained for FGFR3, we do not know if FGFR3 expression is homogenous throughout the tumor. The presence or absence of FGFR3 IHC staining does not appear to have an impact on OS in patients with metastatic disease who are treated with platinum-based chemotherapy. On the basis of our findings, we believe that the importance of FGFR3 as a driver of tumor growth and progression in metastatic UC is unclear, and further functional evidence is required to support this hypothesis.
The frequency of FGFR3 mutation in our cohorts is low, with 2% in primary tumors and 9% in metastases, although the sample size was small and the confidence intervals are wide. A recent study by Gust et al. reported FGFR3 mutations in 11% (10/95) of cases of high-grade invasive UC . Ross et al. also recently published their finding of FGFR3 mutations in 6% (2/35) cases of high-grade metastatic muscle-invasive disease, both of which were detected in primary tumors . In contrast, previous reports have found FGFR3 mutations in up to 20% of muscle-invasive UC using mass spectrometry-based genotyping assay and 15% using sequencing [16, 3]. While Al-Ahmadie et al. used frozen tumors, the majority of which were from cystectomy cases for their mutation analysis, Tomlinson et al. performed their studies on FFPE tissue, although they did not specify whether these were TURBT or cystectomy samples. Thus, the use of FFPE tissue in our study is unlikely to be the reason for the lower mutation rate among our samples. Unlike Al-Ahmadie et al., we did not perform Sanger sequencing of all FGFR3 coding exons. However, the mutations identified in their study and in the work by Tomlinson et al. were included in our OncoMap and MIP platforms. The majority of our samples came from patients in Greece and Spain, raising the possibility that differences in exposures between these populations and those studied by Al-Ahmadie et al. and Tomlinson et al. may account for the differences in the mutation profile of these cohorts.
The most frequently identified FGFR3 mutation in our study was Y373C, followed by R248C. Other studies have reported S249C as the most commonly identified FGFR3 mutation, with lower frequencies of Y375C and R248C (10–20%) [3, 17-19]. The majority of patients in these studies had low-grade, nonmuscle-invasive disease in contrast to our muscle-invasive cohort. Thus, changes in tumor biology reflected by differences in grade or stage may account for some of this discrepancy. Although we only identified four FGFR3 mutants among 161 primary UC tumors and three mutants among 33 metastases, tumors with FGFR3 mutations in our study showed a statistically significant increase in FGFR3 mRNA levels and a trend toward increased FGFR3 protein expression by IHC, similar to what has been observed in nonmuscle-invasive disease [20, 3]. This preliminary finding of an association between FGFR3 mutation and protein expression requires confirmation in a larger cohort of tumors with FGFR3 mutations.
There are several limitations of this study. First, mutation analysis of FGFR3 was performed by hotspot sequencing using two different technologies, OncoMap and MIP array, rather than whole exon sequencing based on when the work was performed and technology available at that time; older samples were tested using Oncomap, and OncoScan was used when more recent samples became available. Among the eight tumors tested for mutations using both platforms, one tumor had a discordant result with Y373C mutation detected using OncoMap (iPLEX and hME) but the mutation, while detected in OncoScan, did not reach the frequency threshold for positivity. This discordance may reflect low tumor purity, heterogeneity, or subclonality within the tumor specimen. Since representative tissue cores were obtained from pathologist-reviewed tumor specimens and incorporated into a TMA, our results do not rule out heterogeneous staining and/or mutation profiles within tumors. In addition, no external validation of our results was possible, as no other clinically annotated cohort of patients with primary or metastatic UC was available for analysis.
It is important to note is that no established cutoffs have been developed for FGFR3 IHC staining. We reported any staining as “positive” based on a previous report , although many of our specimens showed weak staining. In contrast, other studies required positive FGFR3 staining in at least 5% of cells for a specimen to be considered positive [17, 21]. Despite using the same threshold for positivity, though, these groups reported different percentages of positive staining tumors ranging from 15% of pT2 and 2% of pT3 UC (all of which were moderately or poorly differentiated)  to 49.2% of high-grade pT2 UC .
Thus, despite a more inclusive threshold for positive FGFR3 staining, our observation that 29% of the primary tumors in our cohort had positive FGFR3 staining is reasonable based on the published literature. Finally, the majority of patients in our cohorts received platinum-based combination chemotherapy and thus their tumors may not be representative of the biology of UC in patients unable to receive chemotherapy due to impaired performance status.
Several lines of preclinical data suggest that FGFRs hold promise as therapeutic targets in UC. FGFR3 and FGFR1 are either mutated or overexpressed in the majority of nonmuscle-invasive UC , and in a percentage of muscle-invasive UC [17, 23]. Furthermore, inhibition of FGF signaling in both FGFR3 mutant and WT cell lines results in reduced proliferation, thus suggesting a role for FGFR3 “oncogene addiction” that is independent of mutation status [24, 25]. However, the clinical results to date have been disappointing. A phase II study of TKI258 (dovitinib), an oral inhibitor of FGFR3, VEGFR, and PDGFR, was recently terminated due to limited single agent activity in pretreated, advanced UC patients (NCT00790426), including in the cohort with a presumed FGFR3 mutation, despite encouraging in vitro inhibition of tumor proliferation . Other agents currently in development that may demonstrate increased clinical activity include pan-FGF inhibitors, dual FGFR, and VEGFR inhibitors and FGFR-specific antibodies conjugated to the gelonin toxin . The discrepancy between promising in vitro studies and lack of clinical benefit may reflect either lack of target specificity of the drug, that is, the wrong drug for the right target, or a lack of understanding of how FGFRs contribute to UC, which in turn prevents identification of a sensitive subset of tumors. Recent work demonstrates that FGFR3 fusion with transforming acid coiled coil 3 (TACC3) via 4p16.3 and t(4:7) rearrangements result in receptor activation independent of FGFR3 mutation or overexpression and confer sensitivity to FGFR-selective agents . This work suggests that chromosomal translocations, rather than mutational status or expression alone, could be predictive of response to FGFR inhibition. Furthermore, these fusions were identified in less than 10% of lines studied (4 of 43 and 2 of 32, respectively) . FGFR3 fusions (FGFR3-TACC3 and FGFR3-JAKMIP1) were recently identified in 5.7% (2/35) cases of high-grade metastatic muscle-invasive disease . Thus, small unselected phase II studies are unlikely to include patients whose tumors may be driven by these translocations.
FGFR3 IHC staining does not appear to have prognostic or predictive value in patients with metastatic UC. This finding, however, does not eliminate FGFR3 as a potential therapeutic target in metastatic disease. While FGFR3 staining is rarely intense, the presence of detectable protein in primary and metastatic tumors suggests that FGFR3 might be targeted by an antibody-mediated approach and result in successful growth inhibition, as recently demonstrated by Gust et al., provided we have predictive biomarkers with which to identify tumors that are dependent on FGFR3 signaling .
In summary, FGFR3 expression is present in both primary and metastatic UC, and the majority of metastases retain IHC expression of FGFR3 if it was expressed in the primary tumor. While FGFR3 mutation or copy number gain is a rare event, studies of these tumors may shed light on how FGFR3 signaling contributes to UC growth and proliferation. Furthermore, based on preclinical studies that suggest the FGFR pathway, and specifically FGFR3, may be a valid therapeutic target, we believe this area of investigation warrants further exploration.