Supplementary Material for: Immunological Microenvironment Predicts the Survival of the Patients with Hepatocellular Carcinoma Treated with Anti-PD-1 Antibody

<b><i>Introduction:</i></b> Although immune checkpoint inhibitors (ICIs) have been considered as promising agents for the treatment of advanced hepatocellular carcinoma (HCC), previous clinical trials revealed that the response to anti-programmed cell death protein 1 (anti-PD-1) monotherapy was as low as 20%. Identifying subgroups that respond well to ICIs is clinically important. Here, we studied the prognostic factors for anti-PD-1 antibody treatment based on the molecular and immunological features of HCC. <b><i>Methods:</i></b> Patients who were administered anti-PD1 antibody for advanced HCC at Kindai University Hospital were included. Clinicopathological backgrounds and antitumor responses were examined in 34 cases where tumor tissues before treatment were available. Transcriptome analysis was performed using 40 HCC samples obtained from surgical resection, and immune status was compared between 20 HCCs with activating mutations in β-catenin and those without the mutations using transcriptome-based immunogram. <b><i>Results:</i></b> Univariate analysis showed that the disease control rate was significantly better in patients with α-fetoprotein &lt; 400 ng/mL, negative for β-catenin/glutamate synthetase (GS) staining, high combined positive score (CPS) of programmed death-ligand 1 (PD-L1), and increased infiltration of CD8⁺ cells in tumor tissues. Among them, negative staining of β-catenin/GS, CPS of PD-L1 ≥ 1, and high degree of CD8⁺ tumor-infiltrating lymphocytes (TILs) were significantly associated with longer survival in both progression-free survival (PFS) and overall survival (OS). The combination of these factors well stratified the survival of the patients on anti-PD-1 antibody in both PFS and OS (<i>p</i> &lt; 0.0001 and <i>p</i> = 0.0048 for PFS and OS, respectively). In addition, the immunogram revealed that tumor-carrying mutations in β-catenin showed downregulation of immune-related genes, especially in those related to priming and activation by dendritic cells, interferon-γ response, inhibitory molecules, and regulatory T cells. <b><i>Discussion/Conclusion:</i></b> The combined score including Wnt/β-catenin activation, CPS of PD-L1, and degree of CD8⁺ TILs in HCC is informative for predicting the response to ICI in HCC cases. Constitutive activation of β-catenin can induce an immune cold phenotype with downregulation of immune-related genes, and immunohistochemistry-based evaluation is beneficial for identifying the subgroup that shows a good response to ICI.