Morphological heterogeneity in beta-catenin–mutated hepatocellular carcinomas: implications for tumor molecular classification

Summary

Beta-catenin (CTNNB1) is commonly mutated in hepatocellular carcinoma (HCC). CTNNB1-mutated HCC has important clinical correlates, such as being immune cold and less likely to respond to immune checkpoint inhibitor therapies. It remains unclear, however, if they are a morphologically homogenous group of tumors. To better understand the association between the morphology, CTNNB1 mutations, and other molecular features, a detailed study of 338 The Cancer Genome Atlas cases was performed. A characteristic histological morphology was strongly associated with CTNNB1 mutations but was present in only 58% of CTNNB1-mutated HCCs. Tumors with APC mutations tended to have the classic morphology; those with AXIN mutations did not. Pseudoglands are a key feature of the classic morphology, and they were associated with CTNNB1 mutations, male gender, specific CTNNB1 mutation site, and lack of TP53 mutations. Differential gene expression analysis stratified by the presence/absence of pseudoglands identified 60 differentially expressed genes (FDR <5%); clustering according to these differentially expressed genes revealed three groups of tumors, one with pseudoglands and a strong association with genes regulated by Wnt signaling; within this group, TP53 mutations were associated with a loss of the typical morphology of CTNNB1-mutated HCCs. When stratified by gender, further differential gene expression showed Wnt-regulated genes were associated with pseudoglands in men but not women. These findings indicate HCC with CTNNB1 mutations are morphologically heterogeneous, with gene penetrance for morphology dependent in part on gender, specific CTNNB1 mutations, and co-occurring TP53 mutations. This heterogeneity has important implications for the classification of HCC.

Introduction

Hepatocellular carcinomas (HCCs) have heterogeneous molecular aberrations [1]. Beta-catenin (CTNNB1) mutations are among the most common mutations in HCC, present in up to 35% of cases [2]. Beta-catenin is a key protein of the Wnt signaling pathway, with activating mutations leading to enhanced signaling of the Wnt pathway. Additional key proteins are encoded by AXIN and APC; these proteins normally suppress Wnt pathway activation, and inactivating mutations can lead to increased Wnt pathway signaling. HCCs with CTNNB1 mutations are more likely to be well-differentiated, have a thin trabecular growth pattern (synonym is microtrabecular), have pseudoglands (synonym is microacini), and produce bile [[3], [4], [5]], histological findings that together create a composite morphological pattern for CTNNB1-mutated HCCs. Recent studies have identified important clinical correlates for CTNNB1-mutated tumors, with a subset that has less inflammation by gene expression analysis (“immune cold”) [6] and a subset that appears less likely to respond to checkpoint inhibitor therapies [7]. Clinical outcome studies have been inconsistent, with studies showing less aggressive [4] and more aggressive behavior [2] for CTNNB1-mutated HCCs.

An important question for morphological/molecular tumor classification is whether CTNNB1-mutated HCCs represent a homogenous entity. For example, the reported correlations between CTNNB1 mutations and the classic morphology are statistically strong, but a significant proportion of HCCs with CTNNB1 mutations lack the classic morphology [[3], [4], [5]]. On the other hand, anecdotal experience has shown that HCCs can sometimes have the classic morphology but lack CTNNB1 mutations. To better understand the morphology of CTNNB1-mutated HCCs and to refine the morphological-molecular classification, detailed studies were undertaken of the clinical-histological-molecular correlations for CTNNB1-mutated HCCs.