Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection.
OBJECTIVE
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.
Iavarone
Massimo
M
Centre National de Référence des Hépatites Virales, Institut Pasteur/CHU Necker, 156 rue de Vaugirard, 75730, Paris Cedex 15, France.
Trabut
Jean-Baptiste
JB
Delpuech
Oona
O
Carnot
Françoise
F
Colombo
Massimo
M
Kremsdorf
Dina
D
Bréchot
Christian
C
Thiers
Valérie
V
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D017353:Gene Deletion; D006509:Hepatitis B / Q000473:pathology / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology*; D008297:Male; D042282:Microdissection; D008875:Middle Aged; D008969:Molecular Sequence Data; D017354:Point Mutation; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein