Two Distinct Characteristics of Immune Microenvironment in Human Hepatocellular Carcinoma with Wnt/β-Catenin Mutations

<b><i>Introduction:</i></b> Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. <b><i>Methods:</i></b> This study includes 152 resected HCCs; mutations in the <i>catenin beta-1</i>, <i>adenomatous polyposis coli,</i> or <i>AXIN1</i>, or <i>AXIN2</i> genes were defined as Wnt/β-catenin mutations. With hierarchical cluster analyses, TME was classified into inflamed or non-inflamed classes based on the gene expressions associated with T-cell activation. Expression profiles of molecules related to cell differentiation and biliary-stem cell markers were compared between the TME classes to investigate whether differences in tumor traits were associated with TME. <b><i>Results:</i></b> Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of <i>AXIN2 and GLUL,</i> which are involved in the canonical Wnt/β-catenin signaling and hepatocyte differentiation, respectively. Non-inflamed tumors showed higher enhancement on the hepatobiliary-phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) compared to inflamed tumors. HCCs classified as inflamed class are revealed to have high numbers of CD3+, CD4+, and CD8+ tumor infiltrating lymphocytes on immunostaining. This class is associated with increased expression of anti-epithelial cell adhesion molecule and FOXM1 accompanied by upregulation of genes related to interferon-gamma signaling, dendritic cell migration, regulatory T cells, and myeloid-derived suppressor cell activation and recognized as low enhancement nodule on Gd-EOB-DTPA-enhanced MRI. <b><i>Conclusion:</i></b> Heterogeneity of tumor traits and TME was observed in HCC with Wnt/β-catenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the <i>HNF4A</i> but also by <i>FOXM1</i>, both of which are downstream transcription factor of the Wnt/β-catenin signaling pathway.