Programmed death ligand 1 expression in hepatocellular carcinoma: A prognostic marker and therapeutic target for liver cancer?
Authors: Antonio Solinas, Diego F. Calvisi
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 2038 The recent advent of immune checkpoint inhibitors represents a breakthrough in cancer treatment. "Immune checkpoint" refers to the inhibitory pathways maintaining self‐tolerance and control of the immune response in peripheral tissues. Objective and durable clinical responses have been reported in several malignancies treated by immune checkpoint blockade, including melanoma, non‐small cell lung carcinoma, squamous head and neck cancer, renal cancer, ovarian cancer, colorectal cancer, and Hodgkin lymphoma.1 More than 700 clinical studies involving monoclonal antibodies aimed at blocking the programmed death 1 (PD‐1) axis or the cytotoxic T‐lymphocyte antigen 4 axis are currently registered at the ClinicalTrials.gov site. Cancer treatment based on immune checkpoint blockade stems from the observation that oncogenesis is associated with the expression of tumor‐associated antigens (TAAs). Since the discovery of the first melanoma‐associated antigen, more than 400 TAAs have been reported.2 The interaction of TAAs and the host immune system includes immune surveillance, immune editing, and immune tolerance. Accordingly, while tumor cells with high TAA expression are eliminated, tumor cells with low TAA expression can proliferate; and in established malignancies, the tumor microenvironment leads to immune suppression and promotes tumor progression.3 Mechanisms of tumor immune suppression include expression of the PD‐1 ligand (PD‐L1). This protein interacts with activated T and B lymphocytes, natural killer cells, dendritic cells, and macrophages that in turn express PD‐1, leading to regulatory T‐cell proliferation and exhaustion of effector cells. Documented TAAs in hepatocellular carcinoma (HCC) are alpha‐fetoprotein, glypican‐3, melanoma‐associated antigen A1, and the New York esophageal squamous cell carcinoma‐1. More than 50% of cluster of differentiation 8‐positive (CD8+) T cells from patients with HCC recognize the overlapping peptides derived from these TAAs at a rate that is inversely correlated with HCC stage. However, despite the high rate of TAA recognition, the effector cell function evaluated by interferon‐γ secretion and cytolytic activity is impaired.4 At this point, it remains unclear whether, and to what extent, PD‐1 blockade restores antitumor activity in patients with HCC. In this issue of hepatology, Calderaro et al.5 report new data on the expression of PD‐L1 in tumor cells and PD‐1 in the tumor infiltrate in a large series of HCC. The authors observed that PD‐L1 was expressed in 17% of tumors and that, among positive cases, the rate of PD‐L1‐positive tumor cells ranged from 1% to 30%. PD‐L1 expression in tumor cells was associated with HCC progenitor subtype, characterized by poor clinical outcome. In keeping with this finding, PD‐L1 expression was associated with common markers of aggressiveness and, above all, macrovascular invasion. PD‐L1 expression on tumor cells results from constitutive signaling pathway alterations (innate immune resistance) or, alternatively, is an adaptation to the tumor‐specific immune response (adaptive immune resistance). The negative finding of PD‐L1 expression in other HCC subgroups suggests that the progenitor subtype of HCC is constitutively resistant to the immune response. Interestingly, there was no association between Wnt/β catenin pathway activation, a major oncogenic cascade in HCC, and PD‐L1 expression in tumor cells. The relatively low rate of PD‐L1‐positive HCC observed by Calderaro et al. contrasts with the findings reported in a study from China.6 In a series of 128 primary HCCs, PD‐L1 was expressed in 96 of 112 hepatitis B virus‐positive tumor samples and associated with p53 and apurinic/apyrimidinic endonuclease 1 expression. The discrepant results of PD‐L1 expression in HCC from patients of different geographical areas suggest that distinct mutations drive HCC oncogenesis and tumor immune escape. Molecular classification of HCC includes two major subtypes: the proliferation class, characterized by activation of signaling pathways including AKT/mammalian target of rapamycin, RAS/mitogen‐activated protein kinase, transforming growth factor beta, insulin‐like growth factor, and the nonproliferation class, characterized by high expression of the Wnt/β‐catenin pathway. The HCC proliferation class is associated with hepatitis B virus infection, high levels of alpha‐fetoprotein, and poor clinical outcome. The nonproliferative class of HCC is characterized instead by low levels of alpha‐fetoprotein, low vascular invasion, and better outcome.7 Taken together, these findings support the hypothesis that PD‐L1 is mainly expressed within the proliferation class of HCC. These data have important clinical implications. The negative finding of PD‐L1 expression in 83% of HCCs from Caucasian patients and the scanty expression in some of the PD‐L1‐positive cases raise the question of which patients will benefit from PD‐1 blockade. In a phase 1/2 trial aimed at evaluating the safety and tumor activity of nivolumab in 51 patients with advanced HCC, the overall response rate was 7% and the overall survival rate at 18 months was 48%. These results were substantially confirmed in the interim analysis of the dose‐expansion cohorts. The latter analysis included 91 patients with a follow‐up ≥18 weeks and an overall response rate of 9%. Of note, responses were observed in patients with and without quantifiable PD‐L1, as assessed by immunohistochemistry, and unrelated to the etiology of the liver disease.8 The controversial role of PD‐L1 expression in tumor cells as a biomarker of PD‐1 blockade is possibly related to the characteristics of the tumor infiltrating cells. The immune microenvironment within HCC is heterogeneous. A proinflammatory microenvironment is characterized by high expression of innate immune genes, such as tumor necrosis factor, interleukin‐6, and chemokine (C‐C motif) ligand 2, and high activity of chemokine (C‐X‐C motif) ligand 10 and chemokine (C‐C motif) ligand 5 chemokines, which are related to T helper 1 and natural killer cell recruitment. By contrast, the high rates of regulatory T cells and myeloid‐derived suppressor cells are associated with immune tolerance.9 The expression of PD‐1 and PD‐L1 in different subpopulations leads to substantially different effects. Costaining of PD‐1 and lymphocyte subsets in tissue samples of nasopharyngeal carcinoma and control specimens diagnosed as lymphoid hyperplasia showed that PD‐1 was expressed by CD4 cells only in controls and by CD4 and CD8 lymphocytes in tumor infiltrating cells. In addition, the rate of PD‐1 positivity strongly correlated with a locoregional relapse. It is also known that interaction of PD‐L1 with PD‐1 constitutively expressed on regulatory T lymphocytes regulates their development and function and that in HCC PD‐L1 expression in activated monocytes increased with disease progression. At variance with the relatively low expression in tumor cells, when Calderaro et al. evaluated the tumor infiltrate, PD‐L1‐positive cells were found in 76% of cases, with a median number of 6/20 fields. PD‐L1 expression was associated with poor cell differentiation and high lymphocytic PD‐1 expression. These findings suggest that in HCC PD‐L1 is expressed by heterogeneous subsets of tumor infiltrating cells. In this respect, it is of relevance that PD‐L1 expression was high in 12/13 cases in the lymphoepithelioma‐like subtype of HCC. This finding is inconsistent with the natural history of lymphoepithelioma‐like HCC. In fact, patients with this subtype of HCC have a better outcome compared to patients with classical HCC. A possible explanation of this apparently contradictory finding is that tumor infiltrating lymphoepithelioma‐like HCCs mostly consist of T cells, the majority of T cells are CD8‐positive, and the ratio of CD8 to forkhead box p3‐positive regulatory T cells is 25.9.10 By contrast, the ratio of CD8 to forkhead box p3‐positive regulatory T cells in classical HCC is 2.9. Mechanisms of recruitment and maintenance of function of tumor infiltrating cells are of the utmost importance in order to tailor immunotherapy of HCC patients. Mechanisms of T‐cell homing have been extensively studied in colorectal cancer. It was shown by integrating gene expression, phenome mapping, tissue microarrays, and T‐cell repertoire that specific chemokines and adhesion molecules correlated with different subsets of immune cells and, ultimately, with prolonged disease‐free survival. The corresponding integrated setting in HCC has been clarified only in part. Blockade of the PD1/PD‐L1 axis may represent a therapeutic option in a subgroup of HCC patients (Fig. 1), provided that the mechanisms linking tumor progression and immunosuppressive microenvironment are clarified. The work by Calderaro et al. represents a step forward in this direction.Figure 1: Scheme summarizing the findings by Calderaro et al. and the possible therapeutic stratification of the patients based on PD‐L1 expression in the HCC specimens. Abbreviation: AFP, alpha‐fetoprotein.