Prevalence of hepatocarcinoma-related hepatitis B virus mutants in patients in grey zone of treatment

Abstract

Background: Antiviral treatment of patients with chronic hepatitis B (CHB) in the grey zone of treatment comands risk management in order to optimize the health outcome. In this sense, the identification of HBV mutants related with an increased risk of hepatocellular carcinoma (HCC) could be useful to identify subpopulations with potential indication of antiviral treatment.

Aim: To analyze the prevalence/persistence of hepatitis B virus (HBV) preS and basal core promoter (BCP)/precore/core variants associated to HCC development in CHB patients in the grey zone.

Methods: Work was designed as a longitudinal retrospective study, including 106 plasma samples from 31 patients with CHB in the grey zone of treatment: Hepatitis B e antigen negative, HBV-DNA levels between 12-20000 IU/mL, normal or discordant transaminase levels during follow up and mild/moderate necro-inflammatory activity in liver biopsy or Fibroscan (up to 9.5 kPa). Serum HBV-DNA was tested using the Abbott Real Time HBV Assay and the BCP/precore/core and the hepatitis B surface antigen (HBsAg) coding regions were analyzed in positive samples by PCR/bulk-sequencing to identify the HCC-related HBV mutants.

Results: High-risk HCC related mutants were detected in 24 (77%) patients: 19 (61%) in the BCP/precore/core, and 7 (23%) in the HBsAg coding region (2 preS1 and 5 preS2 deletions). The prevalence of preS deletions was genotype-dependent: 3/5 (60%) patients with preS2 deletions and 1/2 with preS1 deletions were infected with the HBV-E genotype. Since HBV-E was the most prevalent in sub-Saharan patients, a correlation between preS deletions and ethnicity was also found: 6/8 (75%) sub-Saharan vs 1/19 (5%) Caucasian patients had preS deletions (P = 0.00016). Remarkably, this correlation was maintained in those patients infected with HBV-A, a minor genotype in sub-Saharan patients: 2/2 patients infected with HBV-A from West Africa vs 0/6 of Caucasian origin had preS deletions. The HCC related variants were the major strains and persisted over time (up to 48 mo). Patients with preS deletions had a significant higher prevalence of F2 fibrosis stage than the negatives (57% vs 10%, P = 0.0078).

Conclusion: HBV genetic analysis of selected populations, like sub-Saharans infected with HBV-E/A genotypes, will allow identification of subpopulations with risk of HCC development due to accumulation of high-risk HBV variants, thus commanding their increased clinical surveillance.

Keywords: Grey zone; Hepatitis B virus; Hepatitis B virus treatment; Hepatocellular carcinoma; PreS deletions.

Figure 1

Features of samples analyzed. A: Number of patients with HBV-DNA amplification positive results in the different genomic regions analysed; B: Ethnic group distribution among the different HBV genotypes. The 4 patients in whom were confirmed sequential changes of the genotyping results during the follow-up were included in both genotype categories. BCP: Basal core promoter; HBV: Hepatitis B virus.

Figure 2

Analysis of basal core promoter /precore/core mutants related with the e-antigen negative status. A: Prevalence of individual mutations of the in the 3 most frequent HBV genotypes analyzed; B: Frequency of coexistence of more than one BCP/precore/core mutants; and C: Frequency of the most common combination of mutations in the different genotypes. The frequency calculation was performed including only the 23 patients with precore/core sequences data, not in on intention to treat basis. BCP: Basal core promoter; HBV: Hepatitis B virus; A: HBV-A; D: HBV-D; E: HBV-E.

Figure 3

Analysis of basal core promoter/precore/core region mutants related with the hepatocellular carcinoma risk. A: Frequency distribution of BCP/precore/core and HBsAg mutations (Black, grey and white bars show the viral variants with the highest, median and lowest evidences of association with HCC, respectively); B: Sequences of the 7 patients with preS deletions. BCP: Basal core promoter; HCC: Hepatocellular carcinoma.

Figure 4

Analysis of preS deletion mutations related with the hepatocellular carcinoma risk. A: Frequency of coexistence of hepatocellular carcinoma related mutations in both BCP/precore and preS regions (Dark bars indicate the patients with theoretical highest risk of hepatocellular carcinoma development, based in the coexistence of mutations in the HBsAg and precore region, or the simultaneous detection of 3 or more mutation irrespective of the genomic region); B: Prevalence of preS deletions in different genotypes and ethnic groups. Only a Caucasian patient infected with HBV-H genotype had a preS1 deletion. BCP: Basal core promoter.

Figure 5

Persistence over time of the high-risk mutations as major population in the patients analyzed.

Figure 6

Differences between patients with or without preS deletion mutants. A: Differences between patients with or without preS deletion mutants at fibrosis level; B: Differences between patients with or without preS deletion mutants at evolution of transaminase levels during follow-up. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.