The recent success of selective kinases inhibitors in a subset of cancer types has generated hope that targeted inhibitors can be developed for a broader range of human cancer types. Thus far, this paradigm has been most effective with inhibitors of kinases that are mutationally activated. Examples include imatinib in patients with chronic myelogenous leukemia (abl
) and gastro-intestinal stromal tumor (c-kit
), erlotinib in non-small cell lung cancer (EGFR
) and most recently PLX4032 in melanoma (BRAF
] In the case of imatinib, abl
translocations are pathognomonic for chronic myelogenous leukemia and thus pre-treatment patient selection beyond classical cytological characterization is not required in order to identify the population of patients most likely to respond. In contrast, it is now apparent that solid tumors as traditionally classified by tissue of origin harbor a diversity of mutations that confer overlapping selective advantage.
For example, EGFR
mutations are all non-overlapping in non-small cell lung cancer.[34
] While the mutual exclusivity of these alterations suggests that they all regulate overlapping downstream effectors of transformation, the specific oncogene mutated is highly predictive of response to targeted inhibitors of the pathway.[35
] Such observations suggest that the response of patients to targeted kinase inhibitors will depend strongly upon the complement of mutations within an individual patient’s tumor and that such predictors (both positive and negative) can be identified. The experience with EGFR targeted inhibitors in lung cancer further suggests that it is preferable to know the tumor genotype of patients prospectively in order to use this information to select the appropriate patients for trial.[12
] This is particularly important if the frequency of a given mutation is low.
FGFR-3 is one of four members of the fibroblast growth factor receptor family of receptor tyrosine kinases that promote cell growth and proliferation.[37
] Activation of this receptor family is mediated by receptor dimerization promoted by bivalent ligand binding facilitated by the co-association of an accessory molecule such as heparin.[37
] Several prior studies have reported that activating mutations of FGFR-3 are particularly common in low-grade urothelial carcinoma.[20
] Notably, the specific alleles found somatically mutated in bladder cancers when identified in the germline are associated with a variety of autosomal dominant skeletal disorders including severe achondroplasia with developmental delay and acanthosis nigricans as well as thanatophoric dysplasia.[17
] Most of these FGFR3
mutations occur in the extracellular domain of FGFR-3 and promote constitutive receptor dimerization and activation. These findings along with additional preclinical studies using human cancer cell lines expressing mutant FGFR3
suggest that tumors expressing an activating FGFR3
mutant allele may be selectively sensitive to inhibitors of this kinase.[40
As the bulk of the morbidity and mortality of bladder cancer is attributable to the development of muscularis propria-invasive high-grade disease, we sought to determine the frequency of FGFR3 mutations in high-grade bladder cancer and their prognostic relevance in this population. Although the frequency of FGFR3 mutation was lower in high grade (17%) versus low-grade (84%) urothelial cancers, in patients with HGUC, FGFR3 mutation was associated with an equally aggressive disease course as that of wild type tumors, with a high rate of recurrence and progression to muscularis propria-invasive disease. The poor outcome overall of patients with FGFR3 mutant HGUC justifies the testing of selective inhibitors of this kinase in patients with this disease. The high prevalence of FGFR3 mutations in low grade UC may, likewise, represent and attractive target for inhibition in this patient group.
The low frequency of FGFR3 mutation in the high-grade cohort, however, raises a number of logistical hurdles to the accrual of sufficient patients with advanced bladder cancer to trials of selective FGFR-3 inhibitors. With the goal of facilitating the identification of high-grade tumors harboring FGFR3 mutations, we compared in detail the morphologic appearance of FGFR3 mutant and wild-type tumors. Our retrospective review of 133 high-grade tumors found that FGFR3 mutation was associated with specific, easily recognizable histopathologic features that were characterized by the presence of a prominent exophytic, papillary component lined with polygonal cells possessing koilocytoid nuclei.
Our prospectively identified case control test set of 24 tumors exhibiting the predictive morphologic features and the 25 tumors lacking these features, further supports the importance of careful histopathologic review, as FGFR3 mutations were identified in 54% of the cases in the former group whereas all 25 control cases of the latter group were FGFR3 wild type. These results suggest that detailed histological evaluation of HGUC can aid in the identification of patients with FGFR3 mutations and thus facilitate the recruitment of such patients to trials of selective FGFR-3 inhibitors. Interestingly, the suggestive morphology of FGFR3 mutation accurately predicted the presence of such mutations in primary tumors of the upper tract, including renal pelvis and ureter, in addition to urinary bladder tumors. This observation may help to expand the applicability of FGFR3 targeting approaches to include tumors of the upper urinary tract as well.
Notably, 11 of 24 (46%) of the tumors in the prospective set that displayed some or all the features that predict for the presence of FGFR3 mutations were wild-type for the hotspot mutations included with our Sequenom assay. As the prospective set consisted entirely of FFPE samples, it is possible that in some of these cases, our failure to detect FGFR3 mutations may have been attributable to the lower sensitivity of this assay in FFPE as compared to frozen tissue. Alternatively, such cases may have harbored non-hotspot FGFR3 mutants not included within our Sequenom panel or genomic alterations that phenocopy FGFR3 mutation such as receptor amplification or FGF ligand overexpression. In support of this latter possibility, two FGFR3 mutant tumors from the 133 frozen samples in which all FGFR3 coding exons were sequenced contained non-hotspot mutations (D468N and S131L), which have not been reported in the Catalogue of Somatic Mutations in Human Cancer but similarly possessed an extensive exophytic papillary component indistinguishable from the tumors harboring the commonly recurrent hotspot mutations.
The presence of an FGFR3 mutation in HGUC does not ensure that these tumors remain dependent upon this kinase. Prior studies of urothelial cancer have shown that significant genetic heterogeneity is present in bladder tumors resected from individual patients as well as within regions of the same tumor. It is possible that FGFR3 mutations are key drivers of tumor growth and survival in low-grade tumors but that additional genetic events relegate FGFR3 mutations to passenger status in the high-grade tumors in which they are found. Twenty of the 31 FGFR3 mutant high-grade tumors in our series exhibited evidence of a low-grade component. Careful dissection and genetic analysis of the high- and low-grade components from three such tumors clearly demonstrated that the FGFR3 mutation was present in the high-grade component and confirmed that our finding of FGFR3 mutations in such tumors was not artifactual due to the presence and intermixing of a second low-grade component. Furthermore, given the concordance of the mutant alleles between high and low-grade components, our data suggest that in such tumors, the low-grade disease was in fact a precursor to the high-grade disease. These results also suggest that a subset of high and low grade tumors may possess overlap of activated signaling pathways. As for the FGFR3 patients for which no low-grade component could be detected, this result could be explained by either sampling error or the replacement of the low-grade tumor by overgrowth of the high-grade component. Alternatively, in some of these cases, no preceding low-grade component may have existed.
Finally, as FGFR3
mutations are predominantly found in superficial low grade UC, it was possible that the detected mutations within our high-grade set may have been present only in the exophytic, non-invasive regions of the tumors whereas the invasive component may have been FGFR3
wild-type. Using the Sequenom assay on FFPE material consisting of only the invasive component, however, we were able to confirm the presence of an FGFR3
mutation in the invasive component for all but one of the tumors for which sufficient tissue was available for analysis (). This is consistent with a previous report by Tomlinson et al.[27
] Moreover, in both FGFR3
mutant cases for which metastatic material from pelvic lymph nodes was available, the identifiable FGFR3
mutant was detectable in both the primary and metastatic lesion. These data provide further support to the contention that FGFR3
mutations are present in a subset of high-grade metastatic urothelial cancers. Future studies will be needed, however, to confirm that mutant FGFR3
remains biologically relevant in this context and thus represents a rational therapeutic target.