Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas

Abstract

Background and aims: Hepatitis B virus (HBV) DNA integration into or close to cellular genes is frequently detected in HBV positive hepatocellular carcinomas (HCC). We have previously shown that viral integration can lead to aberrant target gene transcription. In this study, we attempted to investigate common pathways to hepatocarcinogenesis.

Methods: By using a modified Alu-polymerase chain reaction approach, we analysed 50 HCCs along with 10 previously published cases.

Results: Sixty eight cellular flanking sequences (seven repetitive or unidentified sequences, 42 cellular genes, and 19 sequences potentially coding for unknown proteins) were obtained. Fifteen cancer related genes and 25 cellular genes were identified. HBV integration recurrently targeted the human telomerase reverse transcriptase gene (three cases) and genes belonging to distinct pathways: calcium signalling related genes, 60s ribosomal protein encoding genes, and platelet derived growth factor and mixed lineage leukaemia encoding genes. Two tumour suppressor genes and five genes involved in the control of apoptosis were also found at the integration site. The viral insertion site was distributed over all chromosomes except 13, X, and Y.

Conclusions: In 61/68 (89.7%) cases, HBV DNA was integrated into cellular genes potentially providing cell growth advantage. Identification of recurrent viral integration sites into genes of the same family allows recognition of common cell signalling pathways activated in hepatocarcinogenesis.

Figure 1

(A) Localisation on the hepatitis B virus (HBV) genome of the primers used in this study. Open circle represents the total HBV sequence, nucleotide 3200/1, DR1, and DR2 indicate numbering from the hypothetical EcoRI site (HBV subtype adw and accession number V00866 were used as reference sequences), direct repeat 1, and direct repeat 2, respectively. Arrows represent HB1 primers (HBV preS2/S (uPreS2), HBV X (pUTP), and HBV core region (uPre31), respectively). (B) Schematic protocol for amplification of the viral-host junction. Technical details of the procedure have been previously reported. (1) Small arrows represent primers. Primers represented as broken arrows are synthesised with dUTP instead of dTTP and can be denatured by uracil DNA glycosylase (UDG) treatment. The Alu specific primer has a 5′ tag sequence (Alu+tag). (2) After the tag introducing polymerase chain reaction (PCR), this PCR product is comprised of HBV sequence, cellular franking sequence, and newly synthesised complementary to the tag sequence (cTag), which is indicated by a rectangle. (3) The primers synthesised with dUTP are denatured by UDG and further amplification is performed by HB2 and tag primer, then a second PCR is carried out using HB3 and the Alu tag primer.

Figure 2

Hepatitis B virus (HBV) DNA integration in the human telomerase reverse transcriptase (hTERT) gene. The shaded box is the hTERT gene, the bold arrows indicate the position and orientation of the inserted viral sequence, and the thin arrows indicate the orientation of the hTERT gene. The distance between the ATG or stop codon and the HBV integration site is indicated (kb). The letters on the top of the bold arrow indicate the hepatocellular carcinoma code, as reported in table 1 ▶.

Figure 3

Distribution of the viral integration sites on the chromosome map. Red spots indicate viral integration sites. The names and chromosomal localisations of the genes are also indicated.