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Identification of PDCD1 and PDCD1LG2 as Prognostic Biomarkers and Associated with Immune Infiltration in Hepatocellular Carcinoma

1Department of Hepatobiliary Oncology, Liver Cancer Center, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University, Tianjin, 300060, People’s Republic of China; 2NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300134, People’s Republic of China; 3Graduate School of Tianjin Medical University, Tianjin, 300070, People’s Republic of China; 4Tianjin First Central Hospital, Tianjin, 300192, People’s Republic of China; 5Department of Gastroenterology, Tianjin Haihe Hospital, Tianjin, 300350, People’s Republic of China


Introduction
Owing to its high prevalence and poor prognosis, hepatocellular carcinoma (HCC) has caused the second-highest cancer fatality rate globally. 1 HCC is associated with a number of known risk factors, such as metabolic liver disease (especially nonalcoholic fatty liver disease), alcoholism, chronic hepatitis B and C infection, and exposure to food toxins that include aristolochic and aflatoxins acid. Treatment options for HCC include thermal ablation (heat treatment), surgical resection, radiotherapy (radiation therapy), and liver transplantation (for individuals with early-stage disease). 2 Patients with middle-stage HCC are treated with catheter-based locoregional therapy. In addition, for patients with advanced-stage HCC, it has been established that kinase and immune checkpoint inhibitors are viable therapy alternatives. 3 Immune checkpoint molecules that include both stimulatory and inhibitory immune checkpoint molecules are considered ligand-receptor combinations that block or stimulate immune responses. A growing body of research evidence has illustrated that immune checkpoint molecules, regardless of stimulatory or inhibitory ones, are significantly expressed in high proportion in distinct types of tumors. Despite the fact that the primary role of immune checkpoint molecules associated with tumor cells is considered to be the facilitation of immune escape, it was recently demonstrated that these molecules perform essential functions in the maintenance of a variety of mechanisms, such as drug resistance, improved energy metabolisms, selfrenewal, metastasis, anti-apoptosis, angiogenesis, or even the epithelial-mesenchymal transition. 4 Moreover, another study suggested that loss of histone macroH2A1 could activate CD4 + CD25 + FoxP3 + regulatory T cells and improve paracrine-mediated chemoresistance in HCC. 5 Recently, inhibitory checkpoints, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), and also programmed cell death ligand 1 (PD-L1), have been discovered to attenuate antitumor immune responses in solid tumors. In addition, B7-1 and B7-2 are the co-stimulatory immune checkpoints in HCC that have attracted much attention. 6 However, there has been no thorough and systematic research on the prognostic significance of immune checkpoint inhibitors as well as their corresponding correlation with immune infiltration in HCC. 5 Immune checkpoints and their relative association with immune infiltration in human hepatocellular cancer (HCC) are investigated in the present using bioinformatics methods. This may provide new ideas for screening therapeutic targets for HCC.

Dataset and Preprocessing
The immune checkpoint gene expression profile was obtained from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/). To perform additional investigations, the TCGA HCC dataset (n=374) and related clinical features of HCC patients were acquired. The levels of immune checkpoints mRNA were analyzed and visualized by R (version 4.0.3) with packages ggplot2 and pheatmap.

Drug Sensitivity, Genetic Mutation, and Cancer-Related Pathway Analysis
Using the gene mutation data collected from the TCGA HCC dataset, we visualized the genetic mutation of eight immune checkpoints utilizing the "maftools" package in R. Using the Genomics of Drug Sensitivity in Cancer (GDSC), we obtained data on a sum of 265 small moles or drugs. Using Pearson correlation analysis, we then determine the correlation coefficient between immune checkpoints and these small compounds or drugs. The HCC cohort was classified into two groups with a medium level of immune checkpoints expression. in the case when PAS (immune checkpoint group High) > PAS (immune checkpoint group Low), we hypothesized that immune checkpoints may either activate or block a pathway. According to Ye et al, the approach of cancerrelated pathway analysis was conducted. 7

Prognosis Analysis and Predictive Nomogram
Kaplan-Meier survival analysis was utilized to delve into the prognostic significance of immune checkpoints in HCC. The p-values as well as a hazard ratio (HR) having 95% confidence intervals (CI)were analyzed using the Log rank test. The variables influencing the prognosis of HCC patients were found using univariate and multivariate cox regression analyses, and a prediction nomogram was developed on the basis of these factors. The "forestplot" R tool was utilized to depict the HR, P-value, and 95% CI of these components.

The Human Protein Altas
The Human Protein Altas (https://www.proteinatlas.org/) is a web-based database for performing extensive protein analyses in human malignancy. 8 The tissue atlas and pathology atlas modules were used in the present research to examine the protein levels of immune checkpoints in liver and HCC tissues. Infiltration of the Immune Checkpoints TIMER (https://cistrome.shinyapps.io/timer/), a web-based platform that allows for in-depth analyses of tumorinfiltrating immune cells, 9 was employed to evaluate the immune infiltration of immune checkpoints in HCC. The association between immune checkpoints and immune cell infiltration, as well as the expression of immune biomarkers, chemokines, and chemokines receptors, was examined by means of Pearson analysis, with a p-value of 0.05 serving as the cut-off value.

Functional Enrichment Analysis
HCC immune checkpoints were analyzed using LinkedOmics (http://www.linkedomics.org/adminphp), a web server that allows for extensive analyses. 9 With a false discovery rate (FDR) of 0.05, these immune checkpoints were submitted to the gene isolation service LinkedOmics for subsequent analysis. Subsequently, Gene Set Enrichment Analysis (GSEA) was utilized for the purpose of identifying immune checkpoint-related activities, such as analyses on gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. In the present research, the minimum number of genes (Size) was set at three whereas the maximum number of simulations was set at five hundred.

Results
The Expression of Immune Checkpoints in HCC Initial detection of immune checkpoint expression in HCC enabled us to better understand the function of immune checkpoints in this disease. According to the TCGA HCC cohort data, immune checkpoint mRNA expression demonstrated that the expression of SIGLEC15(p=2.39e-5), PDCD1LG2 (p=2.39e-13), LAG3 (p=1.94e-3), PDCD1 (p=5.64e-3), CTLA4 (p=1.52e-6) and PDCD1LG2 (p=4.54e-7) was elevated in HCC tissues in contrast with liver tissues ( Figure 1). Therefore, extensive alteration of immune checkpoints in HCC.

Analyses of Immune Checkpoints in HCC Using Cancer Hallmarks
Furthermore, we used cancer signature analysis to probe into the function of immune checkpoints in HCC. The genetic alteration immune checkpoints in TCGA HCC patients were comprised of missense mutations, and Del mutations ( Figure 2A). SNPs were the most prevalent variation type whereas C>A was the most prevalent SNV type, according to the data ( Figure 2A). Across all the immune checkpoints, LAG3 was the most common gene and about 15% of TCGA HCC cases experience LAG3 genetic mutation ( Figure 2B). The hallmark pathways of cancer, such as RTK, Cell Cycle, Hormone ER, PI3K/ AKT, Hormone AR, RAS/MAPK, DNA Damage Response, EMT, TSC/mTOR, as well as apoptosis pathways, perform a significant function in the onset and progression of tumors. According to a correlation study between immune checkpoints and cancer hallmark pathways, immune checkpoints in HCC participated in the activation of the apoptotic, Hormone ER, EMT, as well as the DNA damage inhibition response pathways ( Figure 2C). Furthermore, co-expression analysis indicated a strong association among every member of immune checkpoints except for SIGLEC15 ( Figure 2D). In general, one of the most important steps in the development of therapeutic drugs is to examine the relationship between the genes and the drug targets that already exist. A drug sensitivity analysis performed in the present research demonstrated that some immune checkpoints were susceptible to certain small molecules or medicines from the GDSC (negative association) ( Figure 2E).

Prognostic Significance of Immune Checkpoints in HCC
Previous study had revealed that some immune checkpoints might act as prognostic biomarker in many malignances. In our study, prognosis analysis indicated that PDCD1 and PDCD1LG2 were related to the progression free survival (PFS), disease-specific survival (DSS), overall survival (OS), and recurrence-free survival (RFS) of HCC patients (Tables 1 and 2 Figure 3B). Thus, PDCD1 and PDCD1LG2 may serve as prognostic biomarkers in the treatment of HCC.

The Development of a Prediction Nomogram Predicated on Clinicopathologic Characteristics and Immune Checkpoints
Univariate and multivariable analyses demonstrated that PDCD1, PDCD1LG2, and the stage pT were all independent variables influencing the prognosis of HCC patients ( Figure 4A and B). Based on clinicopathologic characteristics (pT stage) as well as potential prognostic biomarkers (PDCD1 and PDCD1LG2), we then used the cox regression technique to generate a predictive nomogram for OS over one, three, and five years in the discovery group. Furthermore, the calibration plots for OS over three and five years were anticipated more accurately in comparison with an ideal model in the TCGA HCC cohort ( Figure 4C and D).

Association Between Immune Checkpoints and Clinical Characteristics in HCC
To thoroughly examine the role of PDCD1 and PDCD1LG2 in HCC, we conducted a correlation analysis between PDCD1/PDCD1LG2 and the clinical characteristics of HCC. It was discovered that HCC patients with a strong tumor grade expressed more PDCD1 as opposed to individuals with a lower tumor grade (Supplementary Figure 1A, p=0.0017). As shown in Supplementary Figure 1B, patients with HCC who had distant tumor metastases exhibited a significantly elevated level of

Validation of PDCD1 and PDCD1LG2 Expression and Overall Survival in HCC
The expression of PDCD1 and PDCD1LG2 proteins in HCC and normal liver tissues were then investigated. As expected, PDCD1 was high staining in HCC tissues while it was low staining in normal liver tissues ( Figure 5A). PDCD1LG2 was low staining in HCC tissues while it was high staining in normal liver tissues ( Figure 5B). As shown in Figure 6A       (NK) cells, CD8+ T cells, Tregs, and exhausted T cells (Table 3). Moreover, we also analyzed the correlation between chemokines and PDCD1/PDCD1L as well as chemokines receptors, indicating that PDCD1/PDCD1LG2 had a positive association with the content of chemokines receptors and chemokines ( Figure 7E and F). These findings suggested a potential association between PDCD1/ PDCD1LG2 and immune infiltrates in HCC.

PDCD1/PDCD1LG2 Associated Functions
In order to explore PDCD1/PDCD1LG2 associated functions, we then detected the genes correlated with PDCD1/ PDCD1LG2 in HCC and the result was shown in Supplementary Figure 2A and B. GSEA was performed based on PDCD1/PDCD1LG2 and correlated genes. As a result, we found that PDCD1 in GO analysis was  involved in T cell activation, adaptive immune response, leukocyte proliferation, B cell activation, transcription factor activity, monooxygenase activity, cytokine receptor activity, and cytokine binding ( Figure 8A-C). PDCD1 in KEGG analysis was involved in Th17 cell differentiation, T cell receptor signaling pathway, Hematopoietic cell lineage, Drug metabolism, Chemical carcinogenesis, and Th1 and Th2 cell differentiation, ( Figure 8D). Moreover, we found that PDCD1LG2 in GO analysis was involved in cellular defense response, cytokine metabolic process, interferon-gamma production, leukocyte proliferation, adaptive immune response, coreceptor activity, cytokine binding, antigen binding, and cytokine receptor activity (Supplementary Figure 3A-C). PDCD1LG2 in KEGG analysis was implicated in TNF signaling pathway, Th1 and Th2 cell differentiation, NOD-like receptor signaling  Figure 3D).

Discussion
Immune checkpoints are essential in a variety of biological processes and have been shown to improve cancer patients' clinical outcomes. 13,14 In addition, they have also been reported in a growing body of research to be prognostic indicators in a variety of malignancies, including renal cell carcinoma and lung carcinoma. [15][16][17] Whether immune checkpoints might function as a prognostic biomarker in HCC and their relationship to immune infiltration were still unknown. Hence, the objective of the present research was to determine the significance of these immune checkpoints in both immune infiltration and prognosis of HCC patients. First, we explored the mRNA expression of these immune checkpoints in HCC, indicating that the mRNA levels of SIGLEC15, PDCD1LG2, LAG3, PDCD1, PDCD1LG2, and CTLA4 were increased in HCC tissues as opposed to liver tissues. Notably, the majority of these immune checkpoints were changed in HCC, suggesting that they may perform a critical function in the occurrence and progression of HCC. Furthermore, additional investigations demonstrated that immune checkpoints in HCC were related to the activation of the apoptotic signaling pathway. Apoptosis is an orderly and coordinated cellular process that occurs under physiological and pathological conditions. 18 Activation of apoptosis could inhibit the carcinogenesis and progression of cancer.
Prognosis analysis revealed that PDCD1 and PDCD1LG2 were prognostic biomarkers for HCC and high PDCD1 and PDCD1LG2 expression had a better RFS, OS, DSS, and PFS. A growing body of research evidence has demonstrated that some immune checkpoints may serve as markers for predicting the prognosis of cancers patients. PDCD1 was a prognosis biomarker in multiple cancers and the high PDCD1 expression was related to an unfavorable OS, uveal melanoma, and DSS in brain glioma. 19 PDCD1 was shown to be an immunotherapy target and a prognostic marker in non-small cell lung cancer. 20 HAVCR2 was reported to be a predictive biomarker for gastric cancer and was discovered to be adversely correlated with OS. 21 Our study also found that PDCD1/PDCD1LG2 showed a favorable relationship with CD8+ T cells, B cells, Macrophage, CD4+ T cells, Dendritic cells, and Neutrophils. These immune cells were a vital part of tumor microenvironments and exert a central role in tumor immune infiltration as well as biological mechanisms. Neutrophils could predict poor prognosis in HCC and promote tumor progression through p53 and STAT3 signaling pathways. 22 Moreover, tumor-associated Neutrophils recruit Macrophages that might facilitate the HCC progression and the formation of sorafenib resistance. 23 Positive associations were discovered between the expression of PDCD1/PDCD1LG2 and the expression of immune biomarkers, chemokines, and chemokine receptors. Actually, these immune biomarkers, chemokine, and chemokine receptors could serve as a prognostic biomarker and performs an essential function in tumor immune infiltration. CCL2 is a prognostic biomarker and blockade of CCL2/CCR2 signaling in HCC, could inhibit tumor growth by activating T cell anti-tumor immune response. 24 Furthermore, in the case of HCC, CCL14 is a predictive biomarker that has been shown to be associated with immune infiltrates. 25 Our study also has some limitations. First of all, most of the analysis of our study was performed on mRNA level, without protein level. Moreover, animal and in vitro studies should be performed to validate the function of PDCD1/PDCD1LG2 in the occurrence and progression of HCC, as well as its correlation with the infiltration of the immune cells into the microenvironment of the tumor.

Conclusion
To conclude, the present research performed a thorough investigation of the prognostic significance of immune checkpoints in HCC as well as their correlation with immune infiltration, which suggested that PDCD1 and PDCD1LG2 were prognostic biomarkers in HCC and related to immune infiltration.

Data Sharing Statement
The analyzed data sets generated during the study are available from the corresponding author on reasonable request. For personal use only.

Ethics Approval and Consent to Participate
Our study was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Informed consent was signed by all participating patients.

Disclosure
The authors declare that they have no competing interests.