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Carcinogenesis Advance Access originally published online on January 12, 2008
Carcinogenesis 2008 29(9):1685-1691; doi:10.1093/carcin/bgm301
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Role of hepatitis B virus genotype mixture, subgenotypes C2 and B2 on hepatocellular carcinoma: compared with chronic hepatitis B and asymptomatic carrier state in the same area

Jianhua Yin, Hongwei Zhang, Chengzhong Li1, Chunfang Gao2, Yongchao He, Yujia Zhai, Peng Zhang, Ling Xu, Xiaojie Tan, Jinsong Chen, Shuqun Cheng3, Stephan Schaefer4 and Guangwen Cao*

Department of Epidemiology
1 Department of Infectious Diseases, Changhai Hospital
2 Department of Experimental Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, People’s Republic of China
3 Department of Comprehensive Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, People’s Republic of China
4 Abteilung für Virologie, Universität Rostock, Germany

* To whom correspondence should be addressed. Email: gcao{at}smmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The role of genotype mixture and subgenotypes remains controversial in determining the clinical outcome of chronic hepatitis B virus (HBV) infection. We aimed to determine their role on the development and the recurrence of hepatocellular carcinoma (HCC). HBV genotypes, serum viral load and hepatitis B e antigen (HBeAg) seroconversion were determined in 462 HCC patients, 234 chronic hepatitis patients and 425 asymptomatic carriers born in Eastern China. In the 462 HCC patients, 62 (13.4%), 337 (72.9%) and 49 (10.6%) had HBV subgenotype B2, C2 and genotype mixture, respectively. Genotype mixture in HCC patients and hepatitis patients was associated with higher viral load than HBV C2 (P = 0.012, P = 0.000) and more frequent than asymptomatic carriers (P = 0.005, P = 0.000). HBV C2 was more prevalent in HCC patients compared with controls. Proportion of HBV B2 in HCC patients decreased consecutively from <30 to 50–59 years group (P = 0.024). Age-related changes of HBeAg seroconversion were not consistent with serum viral load in HCC patients with HBV B2 and genotype mixture, quite in contrast to hepatitis patients. By multivariate regression analysis, age ≥40 years and serum viral load (≥10 000 copies/ml) were independently associated with hepatocarcinogenesis, whereas age ≤50 years and HBV B2 were independently associated with HCC recurrence after surgical resection. In conclusion, HBV coinfections with two or three genotypes were associated with higher viral load and more severe course of the disease. HBV B2 infection was related to HCC recurrence. HBV C2 predominance in HCC patients was related to the high prevalence in Eastern China.

Abbreviations: AFP, alpha-fetoprotein; AntiHBe, antibody to hepatitis B e antigen; ASC, asymptomic HBsAg carrier; CH, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Hepatitis B virus (HBV) chronic infection affects >350 million people worldwide and >1 million die annually of HBV-related chronic liver diseases (1). In Mainland China, an endemic area with one-third of HBV carriers in the world and chronic carriers of hepatitis B surface antigen (HBsAg) are mostly infected during early childhood (2). Chronic HBV infection greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV infection is associated with up to 80–90% of HCC patients in China, India, Korea, Singapore and Vietnam (3).

The clinical course and outcome of HBV infection are affected by several factors including viral genotype. Eight HBV genotypes (A–H) have been identified by a sequence divergence >8% in the entire HBV genome. Genotypes are further separated into subgenotypes if the divergence in nucleotide sequence is between 4 and 8% (4, 5). The genotypes mostly prevalent in Asia are genotypes B and C (5–7). Subgenotype B2 (formerly Ba) is prevalent throughout Asia, including China, whereas the prevalence of subgenotype B1 (Bj) is restricted to Japan (5, 8). C1 (Cs) was described in Southern Asia, whereas C2 (Ce) was prevalent in the Far East (6, 9). Genotypes and subgenotypes have distinct geographical distributions and are associated with severity of liver diseases in different populations (10, 11). HBV genotypes affect the prevalence of mutations in the precore region and core promoter, thereby influencing the expression of hepatitis B e antigen (HBeAg) (12, 13).

Expression of HBeAg that usually indicates replication of HBV at higher rate is associated with an increased risk of HCC (14). HBV-infected patients seropositive for HBeAg have a significantly higher viral load than those seronegative for HBeAg. Elevated viral load (≥10 000 copies/ml) is a strong risk factor of HCC and often indicates poor prognosis of chronic HBV infection (15–17).

Perspective studies indicated that HBV genotype C, not HBeAg, was an independent risk factor for HCC (18, 19). HBV genotype C was shown to be associated with increased viral load and an increased risk of HCC compared with other genotypes in men (20). However, genotype B was found to be associated with HCC at younger age in a prospective study from Taiwan (10, 18). Despite remarkable differences in virological characteristics among HBV subgenotypes (5–7, 21–23), no clear clinical relevance of HBV subgenotypes has been elucidated yet. The impact of HBV genotype/subgenotype-related factors, such as serum viral load and HBeAg seroconversion on the course of infection, the development and prognosis of HCC, is not yet identified unequivocally.

The aims of the present study are to elucidate the role of HBV subgenotypes, genotype mixture, serum viral load and HBeAg seroconversion on the development of HCC in an area where HBV genotype C and B are highly prevalent and to determine how HBV subgenotypes affect serum viral load and HBeAg seroconversion in the patients with chronic hepatitis B (CH) or HCC.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
The study involved patients treated at the two affiliated hospitals of this University from April 2005 to June 2006 and asymptomic HBsAg carriers (ASCs) recruited in an epidemiological survey in September 2006. All participants had a positive marker of HBV, either HBsAg or HBV DNA. People with subclinical HBV persistent infection and normal aminotransferase level were termed as ASC. Patients with abnormal liver function and histological features, HBsAg seropositive for at least 6 months, positive HBeAg or negative HBeAg with detectable HBV DNA, were classified as having CH. Patients with liver cirrhosis were diagnosed on the basis of histological findings as diffuse fibrosis and formation of pseudolobuli, as well as ultrasonic features of liver cirrhosis plus evidence of hypersplenism. Patients with HCC were diagnosed on the basis of histological findings or an elevated serum alpha-fetoprotein (AFP) level (≥400 ng/ml) combined with at least one of the positive images on ultrasonography and computerized tomography. The patients and ASCs who were born in Eastern China including Shanghai, Jiangsu, Anhui and Zhejiang provinces were invited to participate in the study. Patients whose medical records showed a history of having received antiviral or immunosuppressive treatments were not involved in this study. Patients with acute hepatitis B, as confirmed by detection of high-titered antibody to hepatitis B core antigen of IgM class and antibody to HBsAg 2 weeks after the onset of the disease, were excluded. Patients who had other possible causes of hepatitis or liver cirrhosis, including autoimmune hepatitis, coinfection with hepatitis C virus or D virus, primary biliary cirrhosis, Wilson’s disease, hemochromatosis and other concurrent illness (e.g. alcoholism and uncontrolled diabetes), were also excluded. The study protocol conformed to the 1975 Declaration of Helsinki and was approved by the Ethics Committees of the Institutions, and an informed consent was obtained from each participant.

Before any treatment, 5 ml fasting blood was collected with a vacuum blood collection tube without anticoagulant. The serum was separated by centrifugation at 4°C and stored in a sterile tube at –80°C within 4 h of sample collection. Serological testing was performed as follows: HBV markers and antibody to hepatitis C virus by Architect-i2000, antibody to hepatitis D virus by ELISA kit (Abbott laboratories, North Chicago, IL); liver function tests by serum chemistry autoanalyzer (Model 7600, Hitachi, Tokyo, Japan) using commercial reagents (Wako, Japan); AFP by Bayer ACS-180 (Bayer, Germany) using commercial reagents (Bayer).

Serum HBV DNA load was measured in the LightCyclerTM (Roche, Germany) using Quantitative HBV PCR Fluorogence Diagnostic kit (PG Biotechnology, Shenzhen, China). The kit has a certified lower limit of detection of 500 copies/ml.

HBV genotype and subgenotype were determined by a multiplex polymerase chain reaction assay that had been previously developed in our laboratory (24). The specificity of the multiplex polymerase chain reaction was improved by raising the annealing temperature from 56 to 63°C and adding betaine (Sigma, St Louis, MO) up to a final concentration of 1 mol/l into the reaction.

All patients with HCC were evaluated for the feasibility of surgical resection. Patients with solitary tumors had a chance to receive surgical treatment. The patients were followed up for a year after surgical resection. Confirmation of HCC recurrence was based on the following criteria: positive lesion detection by at least two different imaging techniques (abdominal ultrasonography, computerized tomography or magnetic resonance imaging) or by the results of one imaging technique and a serum AFP level of 400 ng/ml or greater.

Statistical analysis
All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS12.0 for Windows, SPSS, Chicago, IL). Categorical variables, like positive rates of antibody to hepatitis B e antigen (AntiHBe), were tested by {chi}2 test. Continuous variables, like serum viral load with skewed distribution, were adjusted to normal distribution by transformation into logarithmic function and then tested by Student’s t-test. Non-parametric statistical method was also applied to compare the variables of skewed distribution. Analysis of variance and the Bonferroni procedure were used to determine differences among groups. EPI info (Centers for Disease Control and Prevention, Atlanta, GA) was used for the analysis of linear trend in proportion. Multivariate logistic regression analysis was used to identify factors independently associated with the development and the recurrence of HCC. A P value of <0.05 was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Distribution of HBV subgenotypes and genotype mixture
A total of 1121 HBV-infected persons, including 462 patients with HCC, 234 patients with CH and 425 ASCs, were investigated. Patients’ data were summarized in the supplementary Table I, available at Carcinogenesis Online. HBV genotypes A–D, subgenotype B2, C1 and C2 were found. Distribution of HBV subgenotypes B2, C2 and other genotypes was not significantly different in ASCs from Shanghai, Jiangsu, Anhui and Zhejiang provinces ({chi}2 = 12.12, P = 0.059). HBV genotype C (C2, 97.3%) and B (B2, 100%) were more prevalent than other genotypes (Table I). HBV subgenotype C2 was more frequent in the patients with HCC than in the patients with CH and ASCs (P = 0.022, P = 0.001). Eight different genotype mixtures, mainly genotype C-based mixture, were detected. Those included A/C, B/C, C/D, A/B/C, C/E, B/C/D, A/D and A/B. Genotype mixture was more frequent in the patients with HCC and those with CH than that in ASCs, indicating that HBV coinfections with two or three HBV genotypes play an important role in a more severe course of the disease.


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  		Table I. Distribution of HBV genotype, subgenotype and genotype mixture in the patients and ASC from Eastern China

 
Clinical relevance of HBV B2, C2 and genotype mixture
Median age of the patients with CH was 11 years, whereas median age of ASCs was 12 years, younger than that of the patients with HCC. Gender ratio of ASCs was significantly different from that of HCC patients and CH patients whose male gender prevailed significantly (P = 0.000 for each). In assessing the role of genotypes/subgenotypes on clinical outcome of chronic HBV infection, age and gender were considered as major confounding factors. We stratified the participants into six age groups and adjusted the age of the patients with CH and ASCs by adding 11 and 12 years, respectively. HBV subgenotype C2 was more frequent in the patients with HCC than in ASCs at 40–49 years age group (P = 0.007), whereas HBV subgenotype B2 was less frequent in HCC patients than in ASCs at 30–39 years (P = 0.033), 40–49 years (P = 0.003) and 50–59 years (P = 0.004) age groups. A linear trend in the proportion of HBV B2, rather than HBV C2, in the patients with HCC was significant at the age groups from <30 to 50–59 years ({chi}2 = 5.07, P = 0.024) (Table II). In male HBV infectants, HBV subgenotype B2 was less frequent in HCC patients than in ASCs only at 30–39 years age group (P = 0.023) (Table II). HBV genotype B2 was associated with early onset of HCC. Proportion of HBV C2 was stable in all age groups of HCC patients.


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  		Table II. Distribution of HBV B2, C2 and genotype mixture in the patients with HCC and controls after the adjustment for age and gender

 
Although significant differences in serum AFP, alanine aminotransferase, total bilirubin, total protein, albumin and pre-albumin were individually determined between the patients with HCC and those with CH, there were no significant differences among those with B2, C2 and genotype mixture, except that alanine aminotransferase was different between HCC patients infected with HBV B2 and HBV C2 (P = 0.040) (Table III).


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  		Table III. Clinical features of the patients with HCC and controls infected with HBV B2, C2 and genotype mixture

 
Age-related changes of serum viral load and HBeAg seroconversion
Average viral load was significantly lower in HCC patients with HBV C2 than those with HBV genotype mixture (P = 0.012) (Table III). Serum viral load went up in HCC patients younger than 35 years and older than 65 years, and young patients (<30 years) with HBV B2 infection had higher viral load than those with C2 (P = 0.001) (Figure 1A). Average viral load was significantly lower in CH patients with HBV C2 than those infected with HBV B2 and HBV genotype mixture (P = 0.042, P = 0.000) (Table III). Serum viral load in CH patients decreased with age. CH patients with HBV genotype mixture infection had higher viral load than those with HBV C2 (P = 0.013) and HBV B2 (P = 0.044) at 30–39 years age group (Figure 1B). Average viral load was significantly lower in ASCs with HBV C2 than those with HBV B2 (P = 0.004) (Table III). Serum viral load was significantly higher in ASCs with HBV B2 than those with HBV C2 at the age groups <30 and 40–49 years (Figure 1C).


Figure 1
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  		Fig. 1. Age-related changes of serum viral load in HCC patients (A), CH patients (B) and ASCs (C) infected with HBV B2, C2 and genotype mixture (MIX).

 
HBeAg expression was more prevalent in HCC patients with HBV genotype mixture than in those with B2 (P = 0.016) (Table III). HBeAg expression decreased with age in HCC patients with either HBV B2 (<40 versus 40–60 years, P = 0.007) or genotype mixture (<40 versus 40–60 years, P = 0.019; 40–60 versus >60 years, P = 0.045), but not in those with HBV C2. In HCC patients younger than 40 years, HBeAg was more prevalent in those infected with genotype mixture than with HBV B2 (P = 0.040) and C2 (P = 0.008). AntiHBe was more prevalent in the patients with HCC than those with CH (HBV B2, P = 0.003; HBV C2, P = 0.001; genotype mixture, P = 0.006). AntiHBe seropositivity increased with age in HCC patients infected with HBV B2 (<40 versus 40–60 years, P = 0.018; 40–60 versus >60 years, P = 0.003). HBeAg expression decreased with age in CH patients with genotype mixture (<40 versus 40–60 years, P = 0.011), whereas AntiHBe seropositivity increased with age in those with C2 (<40 versus 40–60 years, P = 0.035) (Figure 2). Age-related changes of HBeAg seroconversion were not consistent with serum viral load in HCC patients with HBV B2 and genotype mixture, quite in contrast to CH patients.


Figure 2
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  		Fig. 2. Age-related changes of HBeAg expression and AntiHBe seropositivity in HCC patients (A and B), CH patients (C and D) and ASCs (E and F) infected with HBV B2, C2 and genotype mixture. Data of genotype mixture in ASCs, HBV B2 and genotype mixture in CH patients and ASCs at >60 years age groups are not shown because of small sample size.

 
Factors independently associated with the development and the recurrence of HCC
Independent factors contributing to HCC were evaluated in multivariate regression analysis. Age 40 years or older and serum viral load (≥10 000 copies/ml) were independently associated with hepatocarcinogenesis if the patients with CH were used as controls. Age 40 years or older, viral load (≥10 000 copies/ml), HBeAg expression and AntiHBe seropositivity were independent risk factors for HCC if ASCs were used as controls (Table IV). HBV C2 was not an independent risk factor for HCC. Female gender was a protective factor.


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  		Table IV. Multivariate regression analysis for factors independently associated with HCC

 
Of the 462 patients with HCC, 298 had intact records with or without histologically confirmed liver cirrhosis. HBV subgenotype B2 was more frequent in HCC patients without liver cirrhosis than those with liver cirrhosis (19.05 versus 9.36%, P = 0.032). No differences in age, gender ratio, serum viral load, HBeAg seropositivity and frequency of HBV C2 and genotype mixture were determined between HCC patients with liver cirrhosis and those without liver cirrhosis, as shown in the supplementary Table II, available at Carcinogenesis Online. A total of 168 patients with HCC were treated with surgical resection. HCC patients infected with HBV C2 had a higher chance to receive surgical treatment than those with B2 (137/337 versus 14/62, {chi}2 = 7.27, P = 0.007). HCC patients infected with HBV B2 (14 cases) and with HBV C2 (119 cases) were followed up for a year after surgical resection. The recurrence of HCC in the patients with HBV B2 was more frequent than in those with HBV C2 infection (9/14 versus 40/119, {chi}2 = 5.06, P = 0.024). In HCC patients infected with HBV B2, serum viral load was higher in the patients with recurrent tumor than those without the recurrence (5.33 ± 1.36 versus 3.80 ± 0.90 log10 copies/ml, P = 0.012). Independent factors associated with the recurrence of HCC were evaluated in multivariate regression analysis. Age 50 years or younger [odds ratio 2.307 (95% confidence interval 1.109–4.796), P = 0.025] and HBV B2 infection [3.779 (1.112–12.839), P = 0.030] were independently associated with the recurrence of HCC.


   Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
In this study, all participants were of the Han nationality and from Shanghai and surrounding provinces with similar distribution of HBV subgenotypes in ASCs. This study shows that HBV genotypes C2 and B2 are highly prevalent in this area. In determining the role of HBV C2, B2 and genotype mixture on clinical outcome of chronic HBV infection, major confounding factors were age and gender because average age and gender ratios were significantly different between the cases and the controls. After the adjustments for age and gender, HBV B2 was less frequent in the HCC patients than in ASCs at several age groups. Nevertheless, HBV C2 was more frequent in HCC patients than in ASCs at 40–49 years age group. Multivariate logistic regression analysis indicated that HBV C2 infection was not independently associated with hepatocarcinogenesis. The association of HBV C2 and HCC might not be genuine. High prevalence of HBV C2 in the patients with HCC is probably due to the high prevalence of this subgenotype in this area or the decreases in the proportion of HBV B2 in HCC patients at older age groups. This conclusion is different from other studies (19, 20) and consistent with a study in Hong Kong (25). The reasons for the differences are unknown but it cannot be ruled out that HBV C2 from Eastern China and Hong Kong may differ in some important molecular characteristics of HBV C2 from other areas.

For the first time, this study revealed that genotype C-based genotype mixtures were associated with higher viral load and more severe course in the patients from Eastern China where HBV C2 and B2 were highly prevalent. Genotype mixtures were more frequently encountered in CH patients and HCC patients in comparison with ASCs, indicating that coinfections with different genotypes might interfere with the host immune system and lead to a more severe liver damage. A recent study showed that coinfection with other HBV genotypes in Vietnam was associated with altered pathogenesis (26).

Young HCC patients with HBV B2 infection had higher viral loads than those with HBV C2 infection (Figure 1). Our data partially agree with a recent report (27). Viral and clinical characteristics of HBV B2 are different from those of HBV B1 (28). Viral load of HCC patients with HBV B2 and genotype mixture went up in older age groups, quite in contrast to the patients with CH, indicating that the ability of the host’s immune system to clear HBV was different between the patients with HCC and those with CH.

HBeAg expression decreased with age in HCC patients with either HBV B2 or genotype mixture (Figure 2A), whereas serum viral load went up in those older than 65 years (Figure 1A). The age-associated HBeAg seroconversion was significantly different between HBV B2 and C2 and not consistent with serum viral load. We assume that mutations in the precore/core promoter region may partially be responsible for decreased HBeAg positivity and elevated viral load in the old patients with HCC. The mutations were frequently reported in HCC patients with HBV infection (25, 29), especially in those with HBV genotype C (13, 30–32). Our results imply that HBV subgenotype B2 and HBV genotype mixture also generate mutations in the precore region in hepatocarcinogenesis.

Multivariate regression analysis indicated that the viral load was an independent risk factor for the development of HCC. This result is in accordance with many other studies (15–17, 20). In this assay, serum viral load at the concentration of 10 000 copies/ml was selected as a reference based on previous studies (15, 20). Serum viral load was suggested to be an independent risk factor for hepatocarcinogenesis, either compared with CH patients or compared with ASCs. HBV B2 infection and age at 50 years or younger were independent risk factors for the recurrence of HCC after surgical resection, probably because viral load was high in young HCC patients with HBV B2, and/or HCC patients with HBV B2 had a higher rate of multinodal tumors not accessible for curative resection than those with HBV C2. The level of serum viral load at diagnosis was reported as an independent prognostic factor for HBV-related HCC in Japan (33). Our data further revealed that an elevated viral load in HCC patients with HBV B2, not in those with C2, was independently associated with the recurrence of HCC after surgical resection. This result is not in agreement with a recent study from Taiwan (34). This disagreement can be explained by a different follow-up period and unknown differences between HBV subgenotypes from Taiwan and Eastern China. HBV genotype B had already been related to early onset of HCC (10, 18). HBV B2 was associated with HCC without liver cirrhosis, probably because HBV genotype B2 was associated with early onset of HCC, as demonstrated by a linear trend in the proportion assay (Table II). Young patients with HCC are associated with poor prognosis.

In conclusion, HBV subgenotypes C2 and B2 were highly prevalent in Eastern China. HCC patients with HBV coinfections with two or three genotypes had a higher viral load than those with HBV C2 infection. HBV genotype coinfections were associated with more severe course of the disease. HBV C2 predominance in HCC patients was probably due to high prevalence of this subgenotype in this area. HBV B2 was related to an elevated viral load in the young patients and associated with the recurrence of HCC after surgical resection. This study provides important evidences on the role of HBV B2, C2 and genotype mixture, as well as viral load and HBeAg seroconversion, on the development and the recurrence of HCC. Together with other studies, these results show that the virological characteristics of HBV genotype mixture and subgenotypes B2 and C2 lead to a drastically different clinical courses.


   Supplementary material
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables I and II are found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Chinese National 10th 5-Year Plan (2004BA718B01 to G.C.); DFG grant Scha (778-2-2 to S.S.)


   Acknowledgments
 
The authors thank Ms Yun Zhou (International Cooperation Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China) for her technical assistance and Ms Jie Xu (Department of Foreign Languages, Second Military Medical University, Shanghai, China) for proofreading.


   References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received October 3, 2007; revised November 23, 2007; accepted December 22, 2007.


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