Abstract
BACKGROUND: Immunotherapy has demonstrated a limited clinical efficacy in approximately 5% of cholangiocarcinoma. The main challenges for an effective immunotherapy response in cholangiocarcinoma arise from the tumor microenvironment, which is poorly understood. METHODS: For a comprehensive analysis of the tumor microenvironment in cholangiocarcinoma, we performed multiplex immunohistochemistry with two 15-marker immune panels and Nanostring assays for a comprehensive analysis of 104 surgically resected cholangiocarcinomas including intrahepatic, hilar, and distal cholangiocarcinoma. We also validated some key findings with a batch integration analysis of published single cell RNA sequencing data. RESULTS: This study found that natural killer cells occupy the largest immune cell compartment in cholangiocarcinoma. Granzyme-B(+)CD8(+) effector T cells are significantly associated with better overall survival in both intrahepatic and distal cholangiocarcinoma. Above 85% of intrahepatic cholangiocarcinomas with higher density of PD-1(-)EOMES(-)CD8(+) effector T cells are associated with long-term survival. However, only the density of PD-1(-)EOMES(-)CD8(+) T cells in the tumor areas, but not in the peripheries of the tumors, is prognostic. In all three cholangiocarcinoma subtypes, T regulator cells are significantly associated with a poor prognosis; however, M1 and M2 tumor-associated macrophages or PD-L1(+) tumor-associated macrophage demonstrate different prognostic values. Combining PD-L1(+) M1 or M2, PD-L1(-) M1 or M2 tumor-associated macrophages, and T regulator cells to subgroup intrahepatic and distal cholangiocarcinoma, the prognosis is significantly better distinguished. Moreover, PD-L1(-) M2 tumor-associated macrophages is associated with a good prognosis in intrahepatic and distal cholangiocarcinoma, suggesting this subtype of M2 tumor-associated macrophages may be antitumoral. Interestingly, lower densities of various types of immunosuppressive cells are associated with decreased infiltration of effector T cells in distal and hilar cholangiocarcinoma, but not in intrahepatic cholangiocarcinoma. In intrahepatic cholangiocarcinoma, PD-L1(+) tumor-associated macrophages exert their immunosuppressive function likely through promoting T cell exhaustion. CONCLUSIONS: This study suggests that the densities of Granzyme-B(+)CD8(+) effector T cells and non-exhausted PD-1(-)EOMES(-)CD8(+) T cells and the PD-L1 status in the tumor-associated macrophages are prognostic makers in cholangiocarcinomas. The study also supports targeting PD-L1(+) tumor-associated macrophages as the immunotherapy for cholangiocarcinoma.