Molecular Drivers of the Non-T-cell-Inflamed Tumor Microenvironment in Urothelial Bladder Cancer

Abstract

Muscle-invasive urothelial bladder cancer is a common malignancy with poor outcomes for which immune checkpoint blockade is now showing promise. Despite clinical activity of PD-1/PD-L1-targeted therapy in this disease, most patients do not benefit and resistance mechanisms remain unknown. The non-T-cell-inflamed tumor microenvironment correlates with poor prognosis and resistance to immunotherapies. In this study, we determined tumor-oncogenic pathways correlating with T-cell exclusion. We first establish in this report that T-cell-inflamed bladder tumors can be identified by immune gene expression profiling with concordance with CD8(+) T-cell infiltration. Upregulation of genes encoding immune checkpoint proteins PD-L1, IDO, FOXP3, TIM3, and LAG3 was associated with T-cell-inflamed tumors, suggesting potential for sensitivity to checkpoint blockade. β-Catenin, PPAR-γ, and FGFR3 pathways were activated in non-T-cell-inflamed tumors. No difference was seen in overall somatic mutational density between groups. The three pathways identified represent targetable potential pathways of tumor-intrinsic immunotherapy resistance. Cancer Immunol Res; 4(7); 563-8. ©2016 AACR.

©2016 American Association for Cancer Research.

Figures

Figure 1

(A) T cell-inflamed and non-T cell-inflamed bladder tumors can be distinguished by immune gene expression profiling. Eighty-eight (33%) of tumors show minimal expression of T cell related immune genes (non-T cell-inflamed) while 95 (36%) show overexpression (T cell-inflamed). The remainders show a mixed expression pattern of T cell related genes. (B) Representative examples of CD8 immunohistochemical staining showing an absence of intratumoral T cells in a non-T cell-inflamed tumor (left) and marked T cell infiltration in a T-cell-inflamed tumor (right). (C) Immune subtypes identified by immune gene expression profiling show significant association with the presence of T cells by immunohistochemistry, defined as having >1 CD8+ T cell per 0.1 mm2 field (P = 0.01, Fisher's exact test, n = 19).

Figure 2

(A) Expression of PD-L1 is positively correlated with expression of CD8A (P < 0.0001, Pearson correlation). T cell-inflamed tumors (red) show higher expression of both genes compared with non-T cell-inflamed tumors (blue). (B) Additional immune inhibitory molecules, IDO1, FOXP3, TIM3, and LAG3 show a similar significant correlation (all P < 0.0001, Pearson correlation).

Figure 3

Cellular localization of molecules upregulated in the non-T cell-inflamed group and their relationship with (A) β-catenin and (B) PPARG. Arrows point from the regulator towards a molecule being regulated. Intensity of red color is proportional to level of upregulation. Dotted lines indicate indirect relationships. Lines highlighted in orange show predicted activation. (C) All bladder tumors showed cytomembranous staining for β-catenin (left), however a fraction of non-T cell-inflamed tumors showed nuclear staining for β-catenin (right). (D) Nuclear β-catenin staining was only found in tumors with an absence of CD8+ T cells (P = 0.036, Fischer's exact test, n = 19).