FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer

Abstract

FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) and represents a potential target for therapy. In multiple myeloma, both over-expression and mutation of FGFR3 contribute to tumour development. To define the population of UC patients who may benefit from FGFR-targeted therapy, we assessed both mutation and receptor over-expression in primary UCs from a population of new patients. Manual or laser capture microdissection was used to isolate pure tumour cell populations. Where present, non-invasive and invasive components in the same section were microdissected. A screen of the region of the highest tumour stage in each sample yielded a mutation frequency of 42%. Mutations comprised 61 single and five double mutations, all in hotspot codons previously identified in UC. There was a significant association of mutation with low tumour grade and stage. Subsequently, non-invasive areas from the 43 tumours with both non-invasive and invasive components were analysed separately; 18 of these had mutation in at least one region, including nine with mutation in all regions examined, eight with mutation in only the non-invasive component and one with different mutations in different regions. Of the eight with mutation in only the non-invasive component, six were predicted to represent a single tumour and two showed morphological dissimilarity of fragments within the block, indicating the possible presence of distinct tumour clones. Immunohistochemistry showed over-expression of FGFR3 protein in many tumours compared to normal bladder and ureteric controls. Increased expression was associated with mutation (85% of mutant tumours showed high-level expression). Overall, 42% of tumours with no detectable mutation showed over-expression, including many muscle-invasive tumours. This may represent a non-mutant subset of tumours in which FGFR3 signalling contributes to the transformed phenotype and which may benefit from FGFR-targeted therapies.

Figure 1

Distribution of FGFR3 mutations in UC tumours. A. FGFR3 mutation distribution according to tumour stage. B. FGFR3 mutation distribution according to histological grade.

Figure 2

Morphology and FGFR3 expression in tumour samples. A and B, Different, non-continuous regions from the same tumour section showing similar morphology. The non-invasive component (A) contained a Y375C mutation and the invasive component (B) was wildtype; C, Morphologically dissimilar regions of tumour that are physically merging. In this sample the low grade tumour (arrows) showed FGFR3 mutation (S249C+Y375C) and the high grade tumour (arrowheads) was wildtype; D and E, Distinct regions of the same sample showing different FGFR3 staining pattern. A, B, C, bars = 100μm; D, E, bars = 50μm.

Figure 3

Patterns of FGFR3 staining in normal urothelium and bladder tumours. Normal ureteric urothelium (A) and bladder tumour samples (B-D). A, staining pattern 1; B, staining pattern 0; C, staining pattern 2; D, staining pattern 3; bars = 100μm.

Figure 4

Distribution of FGFR3 staining intensity in UC tumours. The staining intensity of the FGFR3 antibody was classed as the same as or below normal (low) or above normal (high). A. FGFR3 staining intensity according to stage (black bars=low, grey bars=high). B. FGFR3 staining intensity according to grade (black bars=low, grey bars=high). C. Distribution of FGFR3 staining classification according to the presence (grey bar) or the absence (black bar) of FGFR3 mutation.

Figure 5

Comparison of FGFR3 staining intensity with mutation status. Tumours with a low (A and B) or high (C and D) level of FGFR3 staining were compared with stage (A and C) and grade (B and D). Frequency represents the percentage of tumours in a particular stage or grade. Black bars represent FGFR3 wildtype tumours and the grey bars represent FGFR3 mutant tumours.