Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies, accounting for over 1 million deaths annually worldwide. There is a clear, causative relationship between HCC and hepatitis B virus (HBV) infection. The highest incidence of disease (> 10 to 20 cases per 100,000) is found in central and Southeast Asia, and in tropical Africa [1]. South Korea is also a high incidence area at 24.5 per 100,000. Moreover, according to the Korean Central Cancer Registry of 2010, HCC is the second and fourth most common cause of cancer death in males and females, respectively, in South Korea. Compounded by underlying cirrhosis, the majority of patients with HCC can be treated with palliative therapy using transarterial chemoembolization or sorafenib [2]. However, sorafenib is limited by its high cost and side effects, which include diarrhea, alopecia, fatigue, cutaneous desquamation, and hypertension. Additionally, sorafenib cannot induce tumor necrosis, although it can inhibit the growth of HCC [3]. After strategies to minimize tumor growth with all of the above therapeutic modalities have failed, only supportive care can be provided. Thus, new therapeutic modalities are needed.

Programmed cell death 1 (PD-1) is an inhibitory receptor of T cells and activated B cells. This receptor is a member of the CD28 family that interacts with the B7 family ligands, including PD-1 ligand 1 (PD-L1, B7-H1) and PD-L2 (B7-DC). These ligands are key check-point molecules of T cells. While PD-L1 is generally expressed on both hematopoietic cells and non-hematopoietic cells, such as T cells, B cells, endothelial cells, and epithelial cells, PD-L2 expression is limited to antigen presenting cells and macro-phages [4]. The interaction of PD-1 and PD-Ls (PD-L1 and PD-L2) is generally correlated with immune tolerance and evasion by tumor cells. The prognostic significance of PD-Ls and the anti-tumor effects of anti–PD-1 antibody have also been reported in many cancers, including breast, lung, ovarian, and melanoma. However, the roles of PD-Ls in antitumor immunity and their clinical significance have not been fully investigated in HCC. Therefore, in the present study, we examined the expression of PD-Ls in different HCC tissues by immunohistochemical staining and investigated the correlation of overexpression of PD-Ls and prognosis in HCC.

Many studies have reported that PD-1 inhibits T-cells proliferation, survival and function in in vitro and in vivo. Some studies of chronic inflammation and autoimmune disease have shown that PD-1 lymphocyte levels tend to be elevated in many autoimmune diseases, such as rheumatoid arthritis and Sjogren’s syndrome. Moreover, antiviral effects have been reported in chronic lymphocytic choriomeningitis infection and human immunodeficiency virus infection [5]. In malignancies, several studies have demonstrated that PD-L1 was associated with poor prognosis in other cancers, including melanoma, non-small cell lung carcinoma, esophageal carcinoma, gastric carcinoma, pancreatic carcinoma, renal cell carcinoma, and ovarian carcinoma [4,6-11].

In the present study, PD-L1 and PD-L2 were overexpressed in up to 27.1% and 23.5% of HCC specimens. These rates were not higher than those found for other malignancies, including cancers of the lung (50%), esophagus (44%), stomach (42%), breast (23%), and kidney (37%) [12]. We did find that PD-L1 was significantly correlated with tumor size, recurrence and PIVKA-II levels, and that PD-L2 levels were correlated with histological differentiation. Moreover, we found that patients with low expression of PD-Ls had significantly better survival than those with high expression in each group. In particular, simultaneous overexpression of both PD-Ls was more strongly correlated with poorer survival and postoperative recurrence compared to low expression of both PD-Ls. Multivariate analyses supported the finding that the expression of PD-Ls and tumor size were significantly associated with poorer prognosis independent of other potentially confounding factors, such as histological differentiation, presence of microvascular invasion, and tumor stage, which was also examined in univariate analyses. Although the overexpression of PD-L2 was not related to the postoperative recurrences in multivariate analyses, other factors, including overexpression of PD-L1 and tumor size were significantly associated with tumor recurrences. These results were similar to those of a previous study by Gao et al. [13]. Although the proportion of cirrhosis and HBV positive were higher when compared with our study (88% vs. 99.4%), their study also revealed a relationship between PD-L2 and clinical prognosis. These investigators not only looked into PD-Ls expression, but also granzyme B⁺, FoxP3⁺ regulatory T-cells infiltration on tissue microarrays of 240 patients with HCC. They reported that overexpression of PD-L2 and PD-L1 were significantly related to poorer survival, but that the difference in recurrence was not statistically significant [13]. These findings indicate that HBV infection had less of an effect on PD-L1 and PD-L2 expression levels in HCC, and that PD-1 blockade is more important than single PD-L1 or PD-L2 knockdown in targeting cancer. Another study that lacked clinical or prognostic data also showed that the expression of PD-L1 in HBV and HCC patients was higher at earlier stages of HCC during tumor progression, and that when the stage increased, there was a lower level of PD-L1 expression. Moreover, HBV infection had no significant influence on PD-L1 or PD-L2 expression in HCC [14]. However, a different study revealed that circulating PD-1/PD-L1 expression was associated with poor prognosis in HBV-related HCC patients following cryoablation [15]. Some preclinical studies in animal models reported that tumor cells in a PD-1 overexpression group grew much slower in a murine hepatocarcinoma model. Moreover, macrophages and cytotoxic T cells were increased in the soluble PD-1-CH50 peptide group because of their ability to inhibit the interaction of PD-1 and PD-L1 [16].

PD-L2 is principally induced through Th-2-associated cytokines. PD-L2 is mainly expressed by antigen-presenting cells, including macrophages, dendritic cells, mast cells, and some B cells, in response to interleukin (IL) 4 and interferon. In contrast, PD-L1 is expressed by a wide variety of immune cells and non-immune cells, as well as most normal tissue cells. The expression of PD-L2 is generally lower than that of PD-L1 [17]. The regulatory pathways of PD-L2 are different from those of PD-L1. The two major pathways that have been reported are the nuclear factor kB pathway and the signal transducer and activator of transcription 6 pathways [18]. Ohaegbulam et al. [4] revealed that PD-L1 and PD-1 binding requires more complex conformational changes than direct binding of PD-L2 to PD-1. Generally, it is known that the binding strength of PD-L2 to PD-1 is three times greater than that of PD-L1. In addition, the simultaneous binding of PD-L1 and L2 to PD-1 has been disproven, implying that the two ligands compete with each other to bind to the receptor [4]. The relationship between PD-L2 and disease prognosis remains controversial. Rozali et al. [17] reported that PD-L2 positive patients had a worse prognosis than negative patients with esophageal carcinoma. However, no correlation with disease free survival was found in many other cancers, including pancreatic, ovarian, and HCC [17]. In our investigation, the overexpression of PD-L2 was found to be correlated with overall survival significantly, but the difference was not statistically significant for DFS. Our results sustained the hypothesis that the PD-L2 in the tumor immune escape differ depending on the status of organ's environments, for example, our cases had very high proportion of HBV and HCV.

Recently, cancer immunotherapies have been highlighted in other malignancies, such as melanoma [8]. Previously, this category was primarily composed of cell-based immunotherapies, such as dendritic, natural killer, and genetically modified T cells, as well as non-cell–based immunotherapy with cytokines, oncolytic viruses, and T-cell checkpoint inhibitors. To date, T-cell checkpoint inhibitors are actively in development for enhancing antitumor immunity [3]. Two papers reporting that the blockade of cytotoxic T lymphocyte antigen-4 (CTLA-4) improved overall survival in metastatic melanoma were recently published [19,20]. In addition, three different checkpoint inhibitors, ipilimumab (anti-CTLA4), pembrolizumab (anti–PD-1), and nivolumab (anti–PD-1), have been approved by the U.S. Food and Drug Administration for melanoma. The drugs have demonstrated great clinical effects on melanoma and non-small cell lung cancer [21]. However, the mechanisms of hepatic carcinogenesis are complex, including genetic, angiogenic, and immunologic factors [3]. Moreover, the liver maintains self-tolerance because it contains specialized cells, such as hepatocytes and dendritic, liver sinusoidal endothelial, Kupffer, hepatic stellate cells, which enhance the secretion of anti-inflammatory cytokines, including IL-10, and transforming growth factor β. These cells induce immunosuppression via the release of such cytokines and by elevating the expression of CTLA-4 and PD-L1 [22]. Although this immunosuppression may contribute to difficulty in targeting HCC, lower toxicity and immunological responses for clinical outcomes have been shown in clinical trials of patients with HCC following treatment with tremelimumab (anti–CTLA-4) [23]. In addition, combination treatments with tumor ablation and nivolumab (anti–PD-1) are ongoing (NCT01658878).

In conclusion, our study demonstrated that the overexpression of PD-Ls in patients with HCC was strongly correlated with overall survival and tumor recurrence. Moreover, PD-Ls are independent prognostic factors for worse overall survival; thus, their overexpression may be a therapeutic target for HCC. However, further multicenter investigations and functional studies are needed to fortify these findings. Accordingly, additional research is warranted to investigate the use PD-Ls as predictive biomarkers for HCC. Moreover, further studies to target PD-1 and PD-Ls in HCC are urgently needed for HCC immunotherapy.