Prospective Evaluation of Hepatitis B 1762^T/1764^A Mutations on Hepatocellular Carcinoma Development in Shanghai, China

Chronic infection with the hepatitis B virus (HBV) is the most important risk factor for hepatocellular carcinoma (HCC) in humans. Given that only a fraction of persons chronically infected with HBV will eventually develop HCC, viral characteristics may contribute to the HBV-induced hepatocarcinogenesis. A double mutation in the HBV genome, an adenine-to-thymine transversion at nucleotide 1762 and a guanine-to-adenine transition at nucleotide 1764 (1762^T/1764^A), has been found in liver tumors (1-3). Recently, we have found that a specific HBV double 1762^T/1764^A mutation was not only detectable in plasma samples at the time of HCC diagnosis but also can be measured in some individuals at least 5 years before cancer diagnosis (4). HBV carriers with these HBV double mutations experienced an earlier age at death from HCC than HBV carriers without the mutations (5). In addition, these HBV double mutations were associated with an ∼70% increased risk of HCC among individuals with total HBV DNA level of ≥10,000 copies/mL plasma at baseline (6). Here, we evaluated the role of HBV 1762^T/1764^A double mutations in the development of HCC in a prospective cohort of 18,244 men in Shanghai, China.

The design of the Shanghai Cohort Study has been described in detail elsewhere (7, 8). Briefly, 18,244 men (∼80% of eligible subjects) between 45 and 64 y of age were enrolled in the study between January 1986 and September 1989. In addition to in-person interviews soliciting information on use of tobacco and alcohol and medical history, we collected a 10-mL blood sample from each participant at baseline. The Institutional Review Boards at the University of Minnesota and Shanghai Cancer Institute have approved this study.

Identification of incident cancer cases and deaths among cohort participants has been accomplished through record linkage of our cohort data set with those from Shanghai Cancer Registry and Shanghai Municipal Vital Statistics Office and by annual recontact of all surviving cohort members. Cumulatively, 422 (2.3%) cohort participants had been lost to follow-up. As of December 31, 2001, 214 cohort participants developed HCC. In our original study (i.e., the parent study of the present one), we randomly chose 5 to 10 control subjects for each case among all cohort members, who were individually matched to the index cases by date of birth (±2 y), month and year of blood collection, and neighborhood of residence at recruitment. We tested serum samples of all 214 cases and the 1,100 matched controls for the presence of hepatitis B surface antigen (HBsAg) using a standard RIA (AUSRIA, Abbott Laboratories), and 131 cases and 106 controls were positive for HBsAg.

For the present study, we randomly chose 50 cases from the 131 HBsAg-positive HCC patients to preserve precious prediagnostic serum samples for other ongoing research projects. All 106 identified control subjects with seropositive HBsAg were included in the present study given the relatively low prevalence of HBV double mutations in other Asian populations (6, 9). We included an additional 20 HBsAg-negative serum samples (10 from HCC cases and 10 from controls) as negative controls in the laboratory measurements. The determination of HBV 1762^T/1764^A double mutations was done with some modification as previously described (4, 5). Briefly, DNA was isolated from serum. PCR was done on this reaction mix using the following primers: HBVx-7F, 5-TTTGTTTAAAGACTGGGAGGACTGGAGGGAGGAGATTAGGTTA-3; HBVx7R, 5-TGGTGCGCAGACCAATTTATGCTGGAGGCCTCCTAGTACAA-3. The thermocycling conditions were 95°C for 2 min, then 40 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by a final extension of 72°C for 2 min. Negative controls (no DNA added) were included for each set of PCR reactions. PCR product was purified by ethanol precipitation and digested with 8 units of BpmI (New England Biolabs) overnight at 37°C in a volume of 50 μL to release 8-bp internal fragments. A phenol-chloroform extraction followed by an ethanol precipitation in the presence of SeeDNA (Amersham Pharmacia) was done to purify samples for analysis.

The copy numbers of HBV DNA with the 1762^T/1764^A double mutations were quantified from 200 μL serum and determined by quantitative real-time PCR as described previously (5). The real-time PCR was done using an ABI 7300 Real-Time PCR System using wild-type and mutant-specific fluorogenic probes (Applied Biosystems). Real-time PCR was done on 2 μL of serum-isolated template DNA. Real-time PCR was done on 5 ng template DNA from cell pellet samples and 100 pg template DNA from media samples. Primer pair and wild-type and mutant-specific probe sequences for HBV 1762^T/1764^A are 5′-CCGACCTTGAGGCATACTTCA-3′ for the forward primer and 5′-CCAATTTATGCCTACAGCCTCCTA-3′ for the reverse primer. The probes were VIC-AGGTTAAAGGTCTTTGTAC and 6FAM-AGGTTAATGATCTTTGTAC for the wild-type and mutants, respectively. Real-time PCR was done in a total volume of 25 μL consisting of 1× TaqMan Universal Master Mix (Applied Biosystems), 900 nmol/L of each primer, 200 nmol/L of each probe, and template DNA. Thermal cycler conditions were as follows: 50°C for 2 min, then 95°C for 5 min, followed by 45 cycles of 95°C for 30 s and 60°C for 1 min. Each sample was run in duplicate. Serial dilutions of HBV nts1762/1764 wild-type and mutant plasmid DNA standards were included in every 96-well plate analyzed. In addition, HepG2 cell DNA (an HBV-negative control) and no-DNA-added controls were included on each 96-well plate analyzed. All laboratory staff were blinded to case status during the analysis and interpretation of mutation data.

All 20 HBsAg-negative serum samples tested were absent for HBV 1762^T/1764^A mutation. Serum samples from one case and nine controls were insufficient for the assay. Thus, the present study included 49 cases and 97 control subjects.

Chi-square test was used to examine the difference in distribution by year of blood draw, neighborhood of residence at enrollment, smoking, alcohol drinking, and serum retinol levels in tertile, and t test was used for the difference in mean age between cases and controls. Although we used an individually matched case-control study design in the parent study, we broke the matched case-control sets to maximize the sample size for the present statistical analysis because HBsAg serologic status was not a matching factor. We used standard statistical methods for analyses of unmatched case-control data (10). Unconditional logistic regression models were used to calculate odds ratios (OR) for HCC and their corresponding 95% confidence intervals (95% CI) and P values associated with the presence of HBV 1762^T/1764^A double mutations. In addition to original matching factors (age, year of blood draw, and neighborhood of residence at recruitment), cigarette smoking (never, ever), heavy alcohol consumption (nondrinkers, <4 drinks per day, or ≥4 drinks per day), and serum concentration of retinol in tertile were included in all unconditional logistic regression models as covariates.

Statistical computing was conducted using the SAS version 9.1 (SAS Institute, Inc.) and Epilog windows version 1.0 (Epicenter Software) statistical software packages. All P values quoted are two-sided. Two-sided P < 0.05 was considered statistically significant.

The mean age (±SD) of cases (n = 49) at HCC diagnosis was 58.0 years (±4.2 years). The average time interval between blood draw and cancer diagnosis among cases was 3.1 (±1.8) years (range, 3.4 months to 6.5 years).

Cases were comparable with controls in terms of mean baseline age and body mass index (weight in kilograms divided by height in square meters) and frequencies by year of blood draw and neighborhood of residence at recruitment (Table 1). The prevalence of cigarette smoking and heavy alcohol consumption (≥4 drinks per day) was higher in cases than controls. As reported previously (11), individuals who developed HCC during follow-up had significantly lower level of serum retinol at baseline than control subjects who remained free of HCC (Table 1). All 49 cases and 97 controls with seropositive HBsAg reported a history of physician-diagnosed hepatitis and/or cirrhosis during the in-person interviews at baseline.

Twenty-two of 49 (44.9%) HCC cases and 21 of 97 (21.6%) controls tested positive for HBV 1762^T/1764^A double mutations, yielding an OR of 2.47 (95% CI, 1.04-5.85). The OR increased with increasing copies of the 1762^T/1764^A mutation (P_trend = 0.004). Men with ≥500 copies/μL serum had an OR of 14.57 (95% CI, 2.41-87.98; Table 2). We repeated the statistical analysis after excluding 6 cases of HCC that were diagnosed within 1 year of the blood draw. The results became slightly stronger. Compared with those negative for the HBV double mutations, men with the double mutations had an OR of 2.66 (95% CI, 1.08-6.59). The OR was 16.31 (95% CI, 2.35-113.00) for men with ≥500 copies/μL serum (data not shown).

To evaluate the potential differential effect of HBV mutation on risk of HCC among individuals with different lengths of follow-up, we examined the association between the HBV double 1762^T/1764^A mutation and HCC risk stratified by the time interval (years) between baseline sample collection and date of cancer diagnosis among HCC cases. The HBV mutation-HCC risk association for cases with short-term follow-up was similar to those with relative long-term follow-up. OR was 2.35 (95% CI, 0.83-6.66) in cases diagnosed within 3 years post-enrollment and 2.81 (95% CI, 0.87-9.06) in cases with longer follow-up.

Although the majority (61%) of HCC cases in this cohort of 18,244 middle-aged and older Chinese men tested positive for HBsAg, HCC is not an inevitable consequence of chronic infection with HBV. Only a fraction (∼15%) of HBV carriers develop HCC over their lifetime, suggesting that other factors may modify the risk of developing HCC among HBV carriers. Previously, we have shown significant modifying effects of dietary aflatoxin and retinol exposures on HBV-related HCC risk in this study population (7, 11, 12). We also showed that genetic mutations in the cytokine genes played an important role in determining an individual's susceptibility to HBV-related HCC (13).

The role of HBV DNA mutation in hepatocarcinogenesis continues to be of great interest. The prevalence of HBV 1762^T/1764^A double mutations in this middle-aged male population in China was 21.6%, consistent with studies in other Asian populations (6, 9). A recent Taiwan study reported that 28.6% of HBV carriers possessing at least 10,000 copies/mL plasma of total HBV DNA tested positive for these HBV double mutations (6). Our present finding of a statistically significant, 2.5-fold increased risk of HCC associated with the 1762^T/1764^A double mutations after adjustment for potential confounders also is consistent with the 1.7-fold increased relative risk noted in the Taiwan study (6). A novel finding of the present study is a strong, dose-response relationship between the mutant copy numbers of the HBV 1762^T/1764^A double mutations and HCC risk. HBV carriers with ≥500 mutant copies/μL serum experienced an ∼15-fold increased risk of HCC relative to HBV carriers without the double mutations.

The HBV genome encodes its essential genes with overlapping open reading frames; therefore, the 1762^T/1764^A double mutation in the HBV genome can alter the expression of multiple proteins. The molecular basis for the formation of the double mutations and the temporal relation for HBV infection is still a source of active investigation. Thus, whether these mutational changes can be acquired and transmitted from person to person is not completely understood.

Several mechanisms of hepatocarcinogenesis relating to the HBV 1762^T/1764^A double mutations have been hypothesized. The 1762^T/1764^A double mutation may enhance HBV virulence by increasing the host immune response (14, 15), increasing viral replication (16-18), or altering the coding region for the X antigen (3, 19). The 1762^T/1764^A double mutation seems to enhance the efficiency of viral replication either by modulating the relative levels of the precore and core RNAs or by creating a hepatocyte nuclear factor-1 transcription factor binding site (20). In cellular studies, the 1762^T/1764^A double mutation increased the replication of the viral genome two times; in the case of some of the rarer triple mutations, an 8-fold increase in genome replication was found (21, 22). The X antigen of HBV has potential transactivation activity for both viral and host genes, and it has been shown to interact directly with p53 and the DNA repair enzyme XAP-1 (23). Mutations in the basal core promoter region, which overlaps the coding sequence for the X antigen of HBV, may result in amino acid changes in the X antigen. The HBV 1762^T/1764^A double mutation also affects the amino acid sequence of the HBV X gene because it resides in codons 130 and 131, thereby inducing lysine-to-methionine and valine-to-isoleucine alterations, respectively (24). These amino acid changes may interfere with cell growth control and DNA repair and may cause HCC (23, 25).

A strength of this study is that the HBV 1762^T/1764^A double mutations were determined in serum collected before the development of HCC from participants who were not treated with any anti-HBV drugs, thus ruling out the possibility that the HBV double mutations were caused by the malignant transformation of hepatocytes or clinical treatment for HCC or hepatitis B. The present study has some limitations. Our analysis of HBV mutants was based on a single serum sample obtained at study entry; thus, the changes in HBV mutation status over time could not be assessed. In the present study, we used, for the first time, real-time PCR assay for 1762^T/1764^A. The assay did not allow for the quantification of total HBV DNA levels. An earlier report based on a cohort study in Taiwan showed a statistically significant association between HBV DNA level and HCC risk (26). However, this positive association became statistically nonsignificant after adjustment for the HBV 1762^T/1764^A double mutants and other potential confounding factors, whereas the association between the HBV DNA double mutations and HCC risk remained statistically significant after adjustment for HBV DNA load (6). These data indicated that the role of HBV DNA double mutations in the development of HCC is independent of and more important than total HBV DNA level.

In summary, the present study showed that HBV 1762^T/1764^A mutation is a codeterminant of HCC risk among HBV carriers. This genetic feature of HBV, in addition to other environmental and host factors, may lead to better identification of HBV carriers at very high risk of HCC and targeting of aggressive antiviral therapy in this subgroup of infected individuals.

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Xue-Li Wang, Yue-Lan Zhang, and Jia-Rong Cheng of the Shanghai Cancer Institute for their assistance in data collection and management, and the staff of the Shanghai Cancer Registry for their assistance in verifying cancer diagnoses in study participants.