Expression of Programmed Cell Death-Ligands in Hepatocellular Carcinoma: Correlation With Immune Microenvironment and Survival Outcomes

Haotian Liao; Wen Chen; Yunlu Dai; Joseph J Richardson; Junling Guo; Kefei Yuan; Yong Zeng; Kunlin Xie

doi:10.3389/fonc.2019.00883

Expression of Programmed Cell Death-Ligands in Hepatocellular Carcinoma: Correlation With Immune Microenvironment and Survival Outcomes

Front Oncol. 2019 Sep 11:9:883. doi: 10.3389/fonc.2019.00883. eCollection 2019.

Authors

Haotian Liao¹, Wen Chen², Yunlu Dai³, Joseph J Richardson⁴, Junling Guo⁵, Kefei Yuan⁶, Yong Zeng^{1

6}, Kunlin Xie¹

Affiliations

¹ Liver Transplantation Division, Department of Liver Surgery, West China Hospital, Sichuan University, Chengdu, China.
² Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States.
³ Faculty of Health Sciences, University of Macau, Macau, China.
⁴ Department of Chemical and Biomolecular Engineering, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of Melbourne, Parkville, VIC, Australia.
⁵ John A. Paulson School of Engineering and Applied Sciences, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, United States.
⁶ Laboratory of Liver Surgery, West China Hospital, Sichuan University, Chengdu, China.

Abstract

The quantity of programmed cell death-ligand 1 (PD-L1) is regarded as a predicting factor of clinical response to anti-PD-1 axis immunotherapy. However, the expression of PD-L1 and its prognostic value in hepatocellular carcinoma (HCC) patients remain debated. Meanwhile, the molecular features of PD-1's other ligand, namely PD-L2, as well as its correlation with clinicopathological parameters and HCC tumor microenvironment (TME), are still poorly understood. In this study, immunohistochemistry (IHC) data from 304 HCC patients were used to determine the clinicopathological features of PD-L1 and PD-L2 and their correlation with CD8⁺ T cells in HCC. Moreover, fresh clinical HCC samples were used to identify the immune cell subtypes expressing PD-L1 and PD-L2. By using The Cancer Genome Atlas (TCGA) dataset, we further assessed the correlation between mutation signature, copy number variation (CNV), number of neoepitopes, immune gene expression, immune/stromal cell infiltration to the expression of PD-L1 and PD-L2. While membrane expression of PD-L2 was observed in 19.1% of tumor samples, no obvious expression of PD-L1 was detected on tumor cell membranes. High expression of PD-L2 on tumor membranes and PD-L1 in immune stroma were both significantly associated with poorer overall survival (OS) and disease-free survival (DFS) outcomes. Flow cytometry analysis and immunofluorescence showed that macrophages were the main immune cell subtype expressing both PD-L1 and PD-L2. Moreover, positive expression of PD-Ls was correlated with higher CD8⁺ T cells infiltration in immune stroma. CNV analysis showed a similarity between PD-L1 and PD-L2 in affecting gene expression. In addition, higher levels of PD-Ls correlated with higher expression of immune related genes, enhanced cytolytic activity, and larger proportions of immune/stromal cell infiltration. Collectively, our study reveals the impact of both PD-L1 and PD-L2 on the HCC tumor microenvironment for the first time, providing insight for new therapeutic options.

Keywords: anti-PD-1 axis therapy; hepatocellular carcinoma; macrophage; programmed cell death-ligands; tumor immune stroma; tumor microenvironment.