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Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and its incidence is rising in many countries. Like most types of cancer, hepatocarcinogenesis is a multistep process involving a number of different genetic alterations that ultimately lead to malignant transformation of the hepatocyte. Large geographic differences in the incidence of HCC suggest that environmental factors likely contribute to its development. However, the relative contribution of different factors and their molecular interactions are still poorly understood.
Epidemiological studies indicated that chronic viral infection is the most important oncogenetic agent for HCC development and hepatitis B (HBV)-associated carcinogenesis is currently construed as a multifactorial process comprising both direct and indirect mechanisms that likely act synergistically.1 In the Western world, 70–90% of HCC develop in a cirrhotic liver. Cirrhosis has been considered as a pre-neoplastic condition, as it leads to a hypermutagenic status that affects cell cycle genes and promotes deregulated proliferation.2 The strong association between cirrhosis and HCC suggests that the common pathway for hepatocarcinogenesis may be the increased liver cell turnover induced by chronic liver injury and regeneration, which induces or selects a malignant clone. However, while HBV-related HCC mostly develops in patients with underlying cirrhosis in the Caucasian population, the tumour occurs more frequently in the absence of cirrhosis in Asian patients. Thus, although it is clear that HBV-induced cirrhosis likely represents a major factor involved in liver carcinogenesis, evidence for a direct effect of HBV is rapidly growing.3
HBV-induced HCC probably results from a combination of different interacting effects; for example, the integration of HBV DNA and the transactivating activity of viral proteins, such as the enigmatic HBx and the truncated PreS2/S. Integration of HBV sequences into the host cell genome is not essential for viral replication, but it allows the viral genome to persist and represents the early step of clonal tumour expansion. This potentially occurs through rearrangements and/or partial deletions in the host chromosome at the integration site, resulting in genetic alterations such as activation of cellular oncogenes and the inactivation of tumour suppressor genes.2 4 The transactivating potential of the regulatory proteins is a further important oncogenetic effect of integration. HBx acts as a powerful transactivator for transcription of a wide range of cellular and viral genes, including promoters, enhancers, proto-oncogenes (c-myc, c-fos), and cytokine-encoding genes (TNF-α, TGF-β), which modulate apoptosis, cell proliferation and the response to the DNA repair mechanism.5
Over the past decade, increasing attention has been paid to the question of whether the risk of developing HCC is influenced by patients’ viral status, such as HBV genotypes, viral load and emergence of genomic mutations arising during the course of chronic infection. There is some evidence that these factors play an important role in the outcome of chronic liver disease, but the mechanisms underlying this process remain unknown.6–8
Phylogenetic analyses of HBV isolates have identified eight different genotypes (A–H) varying in geographic prevalence and strongly associated with ethnicity. Among the major genotypes, HBV type A is common in North America and western Europe, genotype B and C are prevalent in Asia, and genotype D is most commonly found in the Mediterranean countries.9 How the various HBV genotypes influence HCC evolution remains a matter of heated debate. Much information on the clinical and biological relevance of the genotypic diversity has recently emerged from the scientific literature, namely the correlation between the infecting HBV lineage and different clinical outcome. Although available data are often controversial, comparative studies showed that genotype D is associated with a more severe disease and the highest risk for HBV persistence and recurrence with respect to genotype A, and may predict the occurrence of HCC in younger patients.10 The pathogenic mechanisms underlying the different outcome of chronic hepatitis caused by genotype A and non-A (B, C, D and E) is related to the ability of non-A genotypes to harbour the most common point mutation, that is, the G to A change at position 1896 in the precore region. This creates a translational stop codon leading to abrogation of “e” antigen (HBeAg) production and forms a base pairs with the nucleotide (nt) at position 1858, located in the stem of the epsilon encapsidation signal of the pre-genomic RNA.11 Thus, the precore stop codon mutation prevails in patients infected with genotype D, whereas it is less prevalent in genotypes B and C. The persistence of high viraemia in patients with precore mutants could be the mechanism involved in the progression of liver disease to cirrhosis and HCC.12
HBV genotypes B and C are prevalent in southern Asia, and the natural history of patients with genotype B differs from those with genotype C. The evaluation of clinical and virological differences between HCC patients infected by genotype B or C in Japan, Taiwan and China yielded contrasting results.7 13 14 In Japan and China, there is strong evidence that HBV genotype C is associated with a more active and rapidly progressive liver disease – and, consequently, with the risk of developing liver cirrhosis and HCC – with respect to genotype B, which has a relatively good prognosis.15 16 In fact, the patients infected by genotype B were older, had a higher rate of hepatitis HBeAg seroconversion, lower viral load, and rarely developed HCC.17 By contrast, a case–control study suggests that up to 90% of young patients with HBV-related HCC in Taiwan were infected by genotype B, whereas genotype C was predominant in patients with cirrhosis without HCC.7 This observation suggests that type B strains in Taiwan are somewhat different from those in Japan and China. Contradictory results also emerged from two large-scale cross-sectional studies, which found that the distribution of HBV genotypes B and C were similar in patients with and without HCC.17 18 Therefore, the role of HBV genotypes in the development of HCC remains uncertain.
Because of the spontaneous error rate of viral reverse transcriptase, HBV infection is associated with the emergence of mutations that randomly occur along the viral genome, and generate different viral populations. These mutations arise during active viral replication, and mutant strains can become dominant if they offer an advantage to the fitness of the virus. Selective mutations in core promoter and precore regions enhance HBV virulence playing a potential role in liver carcinogenesis.19
In addition to the more common precore stop codon mutation, a double mutation in the basal core promoter region (BCP: nt 1742–1849) at nt T1762/A1764 has often been found in patients with advanced liver disease and HCC.19 The prevalence of both precore stop codon and BCP mutations seems be related to the ethnicity, HBV genotypes and disease severity. The effects of these mutations have been extensively studied by several groups, and found to down-regulate the precore mRNA production that results in a reduction of HBeAg expression.20 21 In addition, an increase in pre-genomic RNA transcription influences viral genome regulation.22 Experimental evidence suggests that BCP mutant strain may enhance HBV virulence by up-regulating viral replication. High HBV viraemia, in addition to viral genotype and core promoter heterogeneity, could favour progression of liver disease and HCC development.23 A case–control study from Taiwan showed that the risk of HCC was independently associated with BCP mutations and higher serum viral load even in patients without cirrhosis.24
Other potential biomarkers of HCC development have recently been identified in the mutations in the core promoter region (T1753C/A/G) and enhancer II (C1653T),25 26 which had been previously found to be associated with fulminant hepatitis or acute exacerbation of chronic hepatitis.27 Both BCP and C1653T mutations are functionally related to decreased or abolished expression of HBeAg, confirming the important role of e antigen in the pathogenesis of HBV disease both in acute and chronic conditions.28 In addition, because the core promoter region overlaps with HBV X sequence, it can be understood why these mutations not only induce a functional alteration of the x protein, but also to affect its transactivating ability. Although the actual implication of these findings is not clear, the active intracellular replication of the HBV variants might be implicated in the development of HCC.
Yuen et al29 (see page 98) evaluated the role of HBV infecting genotype, BCP (A1762T–G1764A), pre-core (G1896A), C1653T, T1753C/A/G mutations (fig. 1), viral load and cirrhosis, taken individually and in combination, as risk factors for the development of HCC in a large Chinese population with HBV-related HCC and in a control group without HCC. The study confirmed that BCP and C1653T mutations, high viral load and cirrhosis are independent risk factors for HCC development, and, interestingly, quantified the increased risk of HCC according to the single factor or their combination: BCP mutations and cirrhosis versus BCP wild-type and without cirrhosis: 22.2-fold; BCP mutations and HBV DNA levels of ⩾4 log10 versus BCP wild-type and HBV DNA <4 log10: 7.2-fold; CBP and C1653T mutations versus both wild-type: 9.9-fold. However, contrary to their previous report,25 the authors failed to confirm the HBV genotype C as an independent factor for HCC development at multivariate analysis. The strong association between genotype C and BCP mutant may be due to the high prevalence of this mutant in HCC patients. Thus, rather than a genotype-specific mutation, the BCP variation could be seen as a factor potentially involved in hepatocarcinogenesis by diverse HBV genotypes and could serve as a molecular marker for predicting the clinical outcome of patients with chronic HBV infection.
These results were extended and completed by the paper by Chou et al30 (see page 91), who analysed HBV genome heterogeneity in precore and core promoter (enhancer II and BCP) regions during a 14 years follow-up of HBV carriers. The aim of the study was to investigate the temporal relationship between HBV sequence evolution and the risk of HCC development. Six single-nt polymorphisms in the core promoter region (at positions 1703, 1719, 1726, 1727, 1730, 1799), highly correlated with genotype C and representing a class of mutations defining a specific group of HBV strains, were identified and found to be associated with the subsequent development of HCC. Longitudinal analysis indicated that the increased HCC risk for the at-risk sequence variants were attributable to the persistence of nt changes. These findings suggest that HBV evolves genetically not only among different genotypes, but also within the same type. As a result, HBV strains of the same genotype may differ in their oncogenetic potential. The most interesting finding of this extraordinarily long study is that in the carriers of either HBV genotype C or HBV harbouring the BCP double variants a substantial excessive risk could already be detected for 9 or more years before the diagnosis of HCC. Moreover, the detection of the BCP variant at both baseline and another time point during the follow-up was more consistently associated with an increased HCC risk than the detection at a single time point, suggesting that such a risk is strongly related to the persistence and number of these HBV variants.
In conclusion, the most important novel finding of this study is the identification of mutational patterns that could represent an “early” predictor of hepatocyte transformation. Such patterns should be devoid of potentially confounding factors that affect other features such as HBV infecting genotypes, strongly related to ethnicity also in the Asian countries, viral load, expressing the activity of liver disease, and cirrhosis, a well known, but late-onset pre-neoplastic factor.
However, before a given viral sequence profile can be deemed a diagnostic marker or pre-emptive target of hepatocarcinogenesis, the functional analysis of mutated HBV strains is essential to better understand the cellular events involved in the progression of liver disease to HCC.
Competing interests: None to declare.
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