CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma

Abstract

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4⁺ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4⁺ tumor-infiltrating lymphocytes (TILs^{CTLA-4 High}) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILs^{CTLA-4 Low} (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3⁺ TILs (TILs^{FOXP3 High}) had poorer prognoses than patients with low FOXP3⁺ TILs (P = .021, P = .034, respectively), and the density of CTLA-4⁺ TILs was positively correlated with FOXP3⁺ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (Tumor^{PD-L1 High}) and/or TILs^{CTLA-4 High} presented worse OS and a higher recurrence rate than patients with TILs^{CTLA-4 Low}Tumor^{PD-L1 Low}. Moreover, multiple tumors, lymph node metastasis, and high Tumor^PD-L1/TILs^CTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. Tumor^PD-L1/TILs^CTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

Keywords: cytotoxic T-lymphocyte-associated antigen-4; hepatolithiasis; intrahepatic cholangiocarcinoma; prognosis; programmed death ligand-1.