Molecular Scoring of Hepatocellular Carcinoma for Predicting Metastatic Recurrence and Requirements of Systemic Chemotherapy

Abstract

Hepatocellular carcinoma (HCC) causes one of the most frequent cancer-related deaths; an HCC subset shows rapid progression that affects survival. We clarify molecular features of aggressive HCC, and establish a molecular scoring system that predicts metastasis after curative treatment. In total, 125 HCCs were examined for TP53, CTNNB1, and TERT promoter mutation, methylation of 8 tumor suppressor genes, and 3 repetitive DNA sequences to estimate promoter hypermethylation and global hypomethylation. A fractional allelic loss (FAL) was calculated to represent chromosomal instability through microsatellite analysis. Molecular subclasses were determined using corresponding and hierarchical clustering analyses. Next, twenty-five HCC patients who underwent liver transplantation were analyzed for associations between molecular characteristics and metastatic recurrence; survival analyses were validated using a publicly available dataset of 376 HCC cases from the Cancer Genome Atlas (TCGA). An HCC subtype characterized by TP53 mutation, high FAL, and global hypomethylation was associated with aggressive tumor characteristics, like vascular invasion; CTNNB1 mutation was a feature of the less-progressive phenotype. A number of molecular risk factors, including TP53 mutation, high FAL, significant global hypomethylation, and absence of CTNNB1 mutation, were noted to predict shorter recurrence-free survival in patients who underwent liver transplantation (p = 0.0090 by log-rank test). These findings were validated in a cohort of resected HCC cases from TCGA (p = 0.0076). We concluded that molecular risks determined by common genetic and epigenetic alterations could predict metastatic recurrence after curative treatments, and could be a marker for considering systemic therapy for HCC patients.

Keywords: chromosomal alteration; hepatocellular carcinoma; liver transplantation; methylation; molecular subclass; mutation; recurrence; systemic chemotherapy.

Figure 1

Molecular classification of HCC based on corresponding and hierarchical clustering analyses. Members of the A1-subclass are shown in blue, A2-subclass in green, B1-subclass in red, and B2-subclass in purple. (a) 125 HCCs were analyzed using the corresponding analysis based on the presence or absence or the CTNNB1, TP53, and TERT promoter mutations, methylation status on 8 TSG promoters (with or without hypermethylation), methylation status on the 3 kinds of rDNAs (with or without significant hypomethylation), and FAL score (<21% and ≥21%). (b) Hierarchical clustering analyses using x- and y-axis values of two-dimensional drawings of corresponding analysis shown in (a). Each subclass was determined based on the clusters.

Figure 2

Heat-map of the molecular alterations and clinical background of the cases in each subclass. The black rectangle represents positive, the white represents absence, and gray shows that information is missing. p-hyper denotes promoter hypermethylation determined by methylation levels of 8 tumor suppressor genes, and S-hypo denotes significant global hypomethylation determined by methylation levels of 3 kinds of repetitive DNA sequences. TERT-p mutation, TERT promoter mutation. FAL: fractional allelic loss (%) as a representative of the degree of chromosomal alterations. Mod-poorly: moderately-poorly differentiated.

Figure 3

Recurrence-free survival of HCC patients who underwent liver transplantation. The solid line represents the survival of cases with the aggressive molecular pattern (molecular risk factors ≥ 3), and the broken line represents the cases with mild molecular pattern (molecular risk factors ≤ 2). p = 0.0090 by log-rank test.

Figure 4

Recurrence-free survival (a) and overall survival (b) of HCC patients who underwent liver resection. The dataset included 376 HCCs referred from the Cancer Genome Atlas (TCGA). Among these, the results of whole exome sequencing, copy number values by Affymetrix SNP6, methylation analysis by HumanMethylation450 BeadChip, and clinical data, including the survival and curability of resection, are available for 168 HCC cases. These were subjected to Kaplan-Meier analysis. Since genome-wide methylation analysis was not applicable, the number of molecular risk factors ≥2 was considered as an aggressive molecular pattern, and those with 0–1 molecular risk factor was considered as showing a mild molecular pattern. The solid and the broken lines represent the survival of cases with aggressive and mild molecular patterns, respectively. (a) Kaplan–Meier curve for recurrence-free survival; p = 0.0076 by log-rank test. (b) Kaplan–Meier curve for overall survival; p = 0.1037 by log-rank test.