Multiplex IF - QuPath Workflow for Simultaneous Assessment of FGFR3 Expression and T-cell Infiltrations in Upper Tract Urothelial Carcinoma

Background: Fibroblast growth factor receptor 3 (FGFR3) mutation is enriched in upper tract urothelial carcinoma (UTUC) and was shown to be associated with immune-cold tumour microenvironment. The T-cell depleted phenotype is a barrier to an effective treatment by immune checkpoint inhibitors. FGFR3 inhibition could be potentially useful in repopulating T cells in the tumour. However, heterogenous expression of FGFR3 in tumour tissues has hampered the identification of its immune regulatory effects. Purpose: To develop a tool for comprehensive analysis of spatial effects of FGFR3 on the tumour immune microenvironment in UTUC. Methods: Diagnostic FFPE specimens (n=24) were obtained from NHS GG& C Biorepository. FGFR3 hotspot mutations in exons 7, 10 and 15 was determined by PCR using GoTaq Green Master Mix (Promega) and Sanger sequencing. Serial Cloner was used for mutation analysis. Multiplex immunofluorescence (mIF) panel with FGFR3 (B9), CD3, CD8, PD-1, PanCK was performed using ImmPRESS Polymer reagents (Vector) with Opal-TSA (Akoya Biosciences). QuPath was used for quantitative image analysis. Results: Out of 24 cases, 17 (70.8%) showed the known FGFR3 hotspot mutations. FGFR3 mutation was not associated with FGFR3 expression level (H-score) or T cell infiltration. Quantitative image analysis by QuPath showed a high level of heterogeneity in both FGFR3 expression and T-cell infiltration. Overall 32.4% of T-cells were cytotoxic CD3+CD8+ and 9.0% were CD3+PD-1+. Two distinct subgroups of UT tumours were identified based on patterns of T-cell density along FGFR3 expression gradient, negative, low and high. A lower level of T cell infiltration was observed when FGFR3 expression was higher in the immune-cold subgroup and muscle-invasive cases. Conclusions: QuPath workflow was established to investigate the impact of FGFR3 expression on tissue immune contexture. The association between FGFR3 expression levels and T-cell infiltration may implicate an immune regulatory role of FGFR3 in a subgroup of UTUC cases.