Supplementary Material for: Association between Genetic and Immunological Background of Hepatocellular Carcinoma and Expression of Programmed Cell Death-1

<b><i>Background and Aim:</i></b> Immune checkpoint inhibitors are promising agents for the treatment of hepatocellular carcinomas (HCC) refractory to conventional therapies. To enhance the efficacy of this treatment, immunological and molecular characteristics of HCC with programmed cell death ligand 1 (PD-L1) should be explored. <b><i>Methods:</i></b> Clinical backgrounds, PD-L1 expression, and the amount of CD8+ tumor-infiltrating mononuclear cells (TIMCs) were analyzed in 154 HCCs. The expression of 3 stem cell markers and co-inhibitory receptors on tumor cells and TIMCs, respectively, were examined by immunohistochemical analysis. Somatic mutations in the 409 cancer-associated genes and TERT promoter were determined; HCCs were classified based on the presence of gene alterations affecting the 8 oncogenic pathways. The results were validated using the dataset from the Cancer Genome Atlas. <b><i>Results:</i></b> The expression of PD-L1 in the HCCs was positively correlated with progressive tumor features, the presence of cytokeratin 19 (CK19), Sal-like protein 4 (SALL4), and the mutations of genes involving the phosphatidyl inositol 3-kinase (PI3K)-Akt pathway. Although CD8+ cells were densely infiltrated in PD-L1-positive tumors, these TIMCs frequently expressed multiple co-inhibitory receptors. However, a subset of PD-L1-positive tumors characterized by activating mutations of the PI3K-Akt pathway showed a low degree of TIMCs. Conversely, PD-L1-negative HCCs were associated with mutations in the β-catenin pathway and a small number of TIMCs, although the expression of co-inhibitory receptors was rare. <b><i>Conclusions:</i></b> PD-L1-positive HCCs frequently showed an inflamed phenotype with stem cell features; a subset of PD-L1-positive HCCs with mutations in the PI3K-Akt pathway showed a non-inflamed phenotype. In HCCs with dense infiltration of TIMCs, CD8+ cells expressed multiple co-inhibitory receptors, suggesting T cell exhaustion. On the other hand, PD-L1-negative HCCs showed mutations leading to β-catenin activation and exhibited a non-inflamed background. These characteristics should be taken into consideration for developing novel combination therapies using immune checkpoint inhibitors.