Clinicopathologic significance of genetic alterations in hepatocellular carcinoma

Abstract

Hepatocarcinogenesis may involve multiple mutations with distinctive pathogenetic and clinicopathologic significance. To test this hypothesis, 68 cases of hepatocellular carcinoma (HCC) were studied prospectively for genetic–clinicopathologic correlation. Ten pathologic characteristics were evaluated. TP53 (alias p53) gene mutation was studied by a polymerase chain reaction (PCR)–single-strand conformation polymorphism–sequencing; CDKN2B (alias p15) and CDKN2A (alias p16) gene methylation by methylation-specific PCR; and genetic imbalances by comparative genomic hybridization (CGH). TP53 gene mutations occurred in 25% of cases, more than half being codon 249 G to T transversion. Methylation of CDKN2A was frequent (61.7%); of CDKN2B, rare (5.9%). The CGH analysis showed a median of nine aberrations per case, with amplifications more frequent than deletions. Isochromosomes might be involved in about 25% of cases. Amplifications of 1q and 8q were most frequent. Clinicopathologic correlations showed that CDKN2A methylation was significantly associated with tumors arising in cirrhotic livers; amplifications of 17q was significant in multiple parameters of tumor invasiveness (size, venous invasion, poor cellular differentiation, microsatellite formation); other amplifications (1q, 6p, 10p, and 20p) were also significant in tumor invasion; and deletions (at 1p, 11q, 4q, and 14q) were significant in tumor growth. Consistent patterns of genetic alterations were defined in HCC, which might represent distinctive pathways in hepatocarcinogenesis.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world [1], with a marked geographic variation in incidence, being very prevalent in sub-Saharan Africa and Asia, but uncommon in the West [1]. This is due to different risk factors. Chronic hepatitis B virus (HBV) infection [2] and exposure to the carcinogen aflatoxin [3] are important risk factors for African and Asian populations. Chronic HBV carriers have a relative risk of nearly 200:1 for developing HCC [2], which may be due to cirrhosis and the oncogenic potential of the HBV genome [4]. The carcinogenic potential of aflatoxin relates to its capacity to induce G:C to T:A transversion in the TP53 gene, with specific clustering at the third base of codon 249 [5]. It also acts synergistically with HBV infection to increase the risk of HCC [6]. Chronic hepatitis C virus (HCV) is a risk factor in the West [7], although the underlying pathogenetic mechanisms remain undefined. Hepatocellular carcinoma has a poor prognosis, with a 5-year survival of less than 3% in inoperable cases.

Genetic mutations also play significant roles in hepatocarcinogenesis. The TP53 gene is pivotal in maintaining genomic stability, and mutations of the gene have been implicated in HCC formation. In addition to the clustering of mutations to codon 249 in aflatoxin prevalent areas [3], [5], mutations also occur in other exons in sporadic cases in most parts of the world [8], [9], [10].

CDKN2A (alias p16, MTS1) is another candidate gene involved in hepatocarcinogenesis [10]. It is a potent cyclin-dependent kinase inhibitor [11] that halts cell-cycle progression at the G1–S phase boundary. Loss of CDKN2A function may lead to unregulated cellular proliferation [11]. The localization of the CDKN2A gene to 9p21, which is often deleted in HCC [12], suggests that it may be a tumor-suppressor gene. Interestingly, aberrations of CDKN2A appear to show some heterogeneity in different localities. Several studies in Japan, Australia, and Taiwan failed to show any homozygous deletion, mutation, or methylation inactivation of the CDKN2A gene in HCC [12], [13], [14]. On the other hand, studies from Hong Kong showed that CDKN2A was often inactivated in HCC, predominantly through methylation of the 5′ CpG island in the promoter region [15]. The reason for this difference is unclear. CDKN2B (alias p15, MTS2) is another inhibitor of the cyclin-dependent kinases CDK4 and CDK6 [16]. Methylation inactivation of CDKN2B occurred predominantly in hematologic malignancies [16]. It also is localized to 9p21 and so may also be a candidate gene in HCC.

Hepatocarcinogenesis is likely to involve multiple steps of genetic mutations. To test the hypotheses that the different genetic alterations involved in this multistep process may have distinctive pathogenetic roles; that these aberrations may be interrelated; and that they may be of clinicopathologic importance, we conducted a prospective study of HCC patients for TP53 gene mutation, CDKN2B and CDKN2A gene methylation, and genomic aberrations with comparative genomic hybridization (CGH). We further examined if these genetic lesions were interrelated, and defined their significance with a comprehensive panel of clinicopathologic parameters.