Elsevier

Journal of Hepatology

Volume 39, Issue 2, August 2003, Pages 253-261
Journal of Hepatology

Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection

https://doi.org/10.1016/S0168-8278(03)00217-4Get rights and content

Abstract

Background/Aims: The analysis of hepatitis B virus (HBV) X protein genetic variability and is correlation with liver disease severity have only been addressed, so far, on whole liver extracts. We have studied, therefore, the HBV X protein (HBx) gene sequence in morphologically well-characterised tumour and non-tumour liver cells from patients with HBV-related hepatocellular carcinoma.

Methods: Using laser capture microdissection (LCM), we picked up six to eight groups of tumour and non-tumour hepatocytes in serial frozen sections from six patients. After global DNA preamplification followed by HBx-specific polymerase chain reaction, the HBx gene was sequenced in each group of microdissected cells. We also validated the quantification of HBV-DNA in microdissected hepatocytes using HBV Amplicor®.

Results: Heterogeneous mutations in HBx gene were found in distinct cirrhotic nodules and tumour areas from the same patient. Mutations at aa 127, 130 and 131 were frequently detected but there was no distinct point mutation profile between tumour and non-tumour samples. In contrast, deletions in HBx gene, which were found in five/six patients, were more frequent in tumour-derived sequences (6/18) than in non-tumour-derived sequences (1/20).

Conclusions: We have shown that LCM provides a direct insight of intrahepatic HBV infection. Using this technique, we demonstrated the persistence of distinct HBx encoding sequences in clonally expanding cells, thus supporting the hypothesis that HBx deletions may be implicated in liver carcinogenesis.

Introduction

Chronic hepatitis B virus (HBV) infection is one of the main causes of hepatocellular carcinoma (HCC) worldwide. In addition to induce chronic inflammation and cirrhosis, HBV is suspected to have a direct effect in the process of liver carcinogenesis. Integration of HBV-DNA in host genome can cause chromosomal instability and insertional mutagenesis. There is also evidence for a direct impact of some viral proteins, such as HBV X protein (HBx), on cell proliferation and viability [1], [2]. HBx is a promiscuous transactivator that acts through protein-protein interaction [3], [4] and it has been shown to provoke liver tumours in certain strains of transgenic mice [5]. The role of HBx in human carcinogenesis is however far from being established. In particular, results of in vitro and in vivo experiments have shown that HBx may either accelerate cell cycle progression and inhibit apoptosis [6], [7] or, conversely, induce cell cycle arrest and apoptosis [8], [9], [10], [11], depending on experimental conditions.

It is important to take account of the fact that, during the natural history of infection, mutations tend to accumulate in the HBV genome. Two major types of HBx gene changes have been detected preferentially in patients with advanced liver disease. Firstly, some point mutations in HBx gene, in particular the double substitution K130M and V131I, are more frequent in the sera of patients with cirrhosis and/or HCC than in patients with mild liver disease [12], [13], [14], [15]. Secondly, disruptions in the HBx gene leading to the synthesis of C-terminally truncated proteins have been more frequently detected in tumour than in non-tumour liver [16], [17], [18], [19]. These results are consistent with the clonal expansion of cells carrying such deletions and mutations during liver carcinogenesis.

Our group has further studied the biological properties of the HBx mutants present in HCC. A double substitution in 130/131 alone did not affect biological effects of HBx but C-terminally truncated HBx had lost their anti-proliferative effect and cooperated with ras and myc oncogenes in cell transformation [11], [19]. However it is still necessary to ascertain whether their biological properties established in vitro have a real impact in vivo. Thus, a major issue is to characterise the HBx sequences present in infected liver cells but classical approaches based on macroscopic dissection are insufficient to accurately correlate the molecular and pathological findings.

Therefore, our goal was to study the HBx mutants present in small amounts of morphologically well-characterised hepatocytes from tumour and non-tumour cells of patients with HBV-related HCC. We developed an original approach combining laser capture microdissection (LCM) and polymerase chain reaction (PCR)-derived methods to sequence the HBx gene. The frequencies of deletions and point mutations were assessed and their distribution between tumour and non-tumour cells analysed. We also tested the feasibility of quantifying HBV-DNA in microdissected hepatocytes.

Section snippets

Patients and tissues

We have studied eight patients infected with HBV who had received surgical treatment for HCC. The principal clinical characteristics of the patients are given in Table 1. All but one of the patients were hepatitis B surface antigen (HBsAg)-positive in serum (patient III had been followed for several years for chronic HBsAg-positive hepatitis B, but was HBs-antibody positive at the time of surgery). Frozen tumour and non-tumour samples were included in OCT™ and 7 μm sections were serially cut

Intrahepatic HBV-DNA quantification and immunohistochemistry (Table 1)

HBcAg and HBsAg expression could be detected by IHC in 2/8 and 6/8 non-tumour sections, respectively. The only non-tumour tissue (patient I) which showed a strong HBcAg expression, exhibited the higher intrahepatic viral load. In addition, the viral load was lower in non-tumour tissues with no or only a weak HBsAg expression (patients III, VI, VII and VIII). In tumours, HBcAg and HBsAg were detected in 0/8 and 1/8 sections, respectively. The viral load was significantly lower in tumour than in

Discussion

We have developed an original approach to the study of HBV-induced hepatocarcinogenesis in vivo combining laser capture microdissection and PCR-derived techniques. We thus demonstrated the feasibility of quantifying viral load in microdissected hepatocytes. Moreover, we also provided evidence for selection of C-terminally deleted HBx encoding sequences, but not for point mutations, in clonally expanding cells.

The usefulness of microdissection to achieve an analysis of well-characterised cells

Acknowledgements

This work was supported by INSERM and LNC. JBT was supported by fellowships from AP-HP and ARC. The authors are indebted to Dr Frédéric Eberlé and Roche Diagnostics (Meylan, France) for having provided the Amplicor kits. They would also like to thank Drs Anne Marie Roque, Vincent Thibaut, Jean-Paul Duong, Gilles Deléage, Yves Chrétien and Alexandre Dos Santos for helpful discussions.

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    Both authors contributed equally to this work.

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