Journal of Cancer 2020, Vol. 11 5440 Ivyspring International Publisher Journal of Cancer 2020; 11(18): 5440-5448. doi: 10.7150/jca.46158 Research Paper Correlation of PD-L1 and SOCS3 Co-expression with the Prognosis of Hepatocellular Carcinoma Patients Liuxi Chen1,2*, Xingxing Huang1,2*, Wenzheng Zhang1,2, Ying Liu1,2, Bi Chen1,2,3, Yu Xiang2, Ruonan Zhang1,2,3, Mingming Zhang2, Jiao Feng2, Shuiping Liu2, Ting Duan2, Xiaying Chen2, Wengang Wang1,2, Ting Pan2, Lili Yan2, Ting Jin2, Guohua Li2, Yongqiang Li1, Tian Xie1,2,3 and Xinbing Sui1,2,3 1. Department of Medical Oncology, the Affiliated Hospital of Hangzhou Normal University, College of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang, China. 2. Key Laboratory of Elemene Class Anti-cancer Chinese Medicine of Zhejiang Province, Hangzhou Normal University, Hangzhou, Zhejiang, China. 3. State Key Laboratory of Quality Research in Chinese Medicines, Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, P.R. China. *These authors contributed equally to this work. Corresponding authors: Xinbing Sui, E-mail: [email protected]; Tian Xie, E-mail: [email protected] or Yongqiang Li, E-mail: [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.03.19; Accepted: 2020.06.14; Published: 2020.07.11 Abstract Purpose: To investigate the correlation between the expression of PD-L1, SOCS3 and immune-related biomarkers CD276, CD4, CD8 in hepatocellular carcinoma (HCC) and further determine the relationship with clinicopathologic characteristics and the prognostic value of their co-expression in HCC patients. Methods: We assessed the expression of PD-L1, CD276, SOCS3, CD4 and CD8 by immunohistochemistry in tumor tissue from 74 HCC patients who underwent curative hepatectomy. Results: High expression of PD-L1 was significantly associated with high Edmondson grade (p<0.01) and elevated enzyme (p=0.037); high expression of CD276 was significantly correlated with high Edmondson grade (p=0.021); high expression of SOCS3 was significantly associated with age (p=0.026) and tumor size (p=0.041), while PD-L1 showed no significant correlation. The expression of PD-L1, CD276, SOCS3 protein and other clinicopathological factors (sex, vascular invasion, tumor number, tumor capsule, pT stage, liver cirrhosis, HBsAg, TBiL, AFP) showed no significant correlation (p>0.05). High expression of CD8 was respectively significantly associated with worse overall survival (OS) (p=0.002). There was no significantly difference between CD4 and CD8 high-expression and overall survival (OS) (p=0.100). Both high expression of PD-L1 (p=0.003) and low expression of SOCS3 (p=0.015) was significantly associated with worse overall survival (OS). But CD276 only had a trendency (p=0.166). Additionally, multivariate Cox regression models implied that PD-L1, SOCS3, as well as both CD4 and CD8 was an independent prognostic factor for OS (p<0.05). Furthermore, HCC patients with PD-L1 low-expression and SOCS3 high-expression had a better prognostic according to the different pT stages (p<0.05). Conclusions: We for the first time demonstrated that PD-L1 and SOCS3 were independent prognostic factor for HCC patients. Co-expression of low PD-L1 and high SOCS3 could be a better predictive marker for HCC patients. Key words: PD-L1, SOCS3, CD276, CD4, CD8, hepatocellular carcinoma, prognostic value Introduction Hepatocellular carcinoma (HCC) remains one of and the sixth highest in females, due to the lack of the most common and aggressive malignancies effective diagnostic options and the late treatment [1, worldwide [1]. It is reported that hepatocellular 2]. Incidence rates showed higher numbers in China carcinoma has the fifth highest mortality rate in males mainly associated with chronic hepatitis B virus http://www.jcancer.org Journal of Cancer 2020, Vol. 11 5441 (HBV) infection [3, 4]. Recurrence, metastasis and Suppressor of cytokine signaling 3 (SOCS3) is a frequent drug resistance are the main dilemma to the tumor suppressor gene and its inactivation may play integrative cancer treatment of HCC [5]. Recently, a role in HCC initiation and development [17]. The immunotherapy has been attempted to further deletion of the SOCS3 is an important mechanism for improve prognosis with advanced HCC [6]. By the activation of the STAT3 and this mechanism analyzing inflammatory gene including expression vitally act in the occurrence and progression of profiles, infiltrates, and regulatory molecules, an hepatitis-related liver cancer [18]. Zhang et al. found ‘immune class’ accounting for 30% of HCCs was that overexpression of SOCS3 strongly inhibited characterized by immune cell infiltration, STAT1 phosphorylation and PD-L1 up-regulation programmed cell death protein 1 (PD-1) and/or [19]. programmed cell death 1 ligand 1 (PD-L1) expression, To our knowledge, PD-L1 is respectively related IFN-γ signaling activation and markers of cytolytic to CD4, CD8, CD276, SOCS3, but the relationship activity [7]. between those co-expression and hepatocellular Programmed cell death protein 1 (PD-1) is a carcinoma has not been reported. Hence, we receptor mainly expressed by T cells, which retrospectively selected 74 hepatocellular carcinoma negatively regulates the effector phase of T cell cases and performed immunohistochemical analysis responses. To date, monoclonal antibodies to either for PD-L1, CD4, CD8, CD276 and SOCS3 for each PD-L1 (atezolizumab, avelumab, and durvalumab) or paraffin-embedded block. In addition, we also PD-1 (nivolumab and pembrolizumab) are approved investigated the correlation of the expression of CD4, for the cancer treatment [8]. PD-L1, as an independent CD8, PD-L1, CD276 and SOCS3 with clinico- predictive biomarker, is demonstrated low prediction pathological features and clinical outcomes. We are accuracy attributed to the technical obstacles (variable committed to find potential biomarkers in detection antibodies, differing IHC cutoffs, staining of combination with PD-L1 to predict hepatocellular tumor versus immune cells) and the complexity of carcinoma progression and prognosis. biological mechanisms (unevenly distribution, unstable expression) [9]. Therefore, we tried to Materials and Methods explore potential biomarkers in combination to verify Public Gene Expression Omnibus dataset what prognostic clinical utility they may hold. analysis The PD-1/PD-L1 interactions suppress CD8 cytotoxic T lymphocyte (CTL) survival, proliferation Two publicly available Gene Expression and effector function and can also induce the Omnibus (GEO) datasets (GSE102079 and GSE121248) apoptosis of tumor-infiltrating T cells [10]. PD-1/ containing gene expression information from PD-L1 interactions lead the differentiation of CD4 T paracancerous tissues and cancerous tissues were cells to Foxp3 regulatory T cells (Tregs) [11], which are downloaded from Gene Expression Omnibus known to further inhibit the immune system and (http://www.ncbi.nlm.nih.gov/geo/). The processed promote peripheral immune tolerance [12]. Tumor- data dealed with normalization procedures were infiltrating lymphocytes (TILs) are a group of directly obtained from the corresponding websites, lymphocytes located around tumor cells. The and no other transformations were performed. presence of TILs is associated with tumor regression Patient population and tumor samples and prognosis in HCC, which is often regarded as an All patient information and clinical data were "immunogenic tumor" [13]. obtained from National Human Genetic Resources B7-H3 (also known as CD276), a co-stimulator Sharing Service Platform. A total of 74 patients with molecule of the cell surface B7 protein superfamily, is formalin-fixed, paraffin-embedded tumors (FFPE) mainly expressed on dendritic cells along with T were selected retrospectively, who had undergone lymphocytes, B monocytes and NK cells [14]. It was surgery without neoadjuvant treatment between June reported that CD276 had a co-stimulating effect on the 2007 and March 2008. Patients were proliferation of both CD4 and CD8 T cells. histopathologically diagnosed as hepatocellular Combinatorial blockade of anti-CD276 and anti-PD-1 carcinoma. Tumor staging and grading was carried has approved that this approach can be mutually out according to the American Joint Committee on enhancing treatment effect of established tumors [15]. Cancer (AJCC), TNM Staging System for Urethral Kang et al. have shown that down regulation of Carcinoma (8th ed., 2017). When the patient died, the CD276 can significantly reduce cell migration and cause of death was determined by the attending invasion in HCC, which may be referred to epithelial- physician or by death certificate. to-mesenchymal transition through the JAK2/ STAT3/Slug pathway [16]. http://www.jcancer.org Journal of Cancer 2020, Vol. 11 5442 Immunohistochemistry covariates (p value <0.11) according to the univariate PD-L1, CD4, CD8, CD276 and SOCS3 analysis. Overall survival (OS) was from the date of immunohistochemistry (IHC) were available in 74 curative hepatectomy to death. Kaplan-Meier analysis cases. Each case was performed PD-L1, CD4, CD8, was used the log-rank test to compare differences. CD276 and SOCS3 immunohistochemical staining Two-tailed p value <0.05 was considered as using the same paraffin-embedded blocks. The statistically significant in all cases.