Hepatitis B Virus DNA Levels and Outcomes in Chronic Hepatitis B

Transcription

1 Hepatitis B Virus DNA Levels and Outcomes in Chronic Hepatitis B Chien-Jen Chen, 1,2 Hwai-I Yang, 1 and Uchenna H. Iloeje 3 for The REVEAL-HBV Study Group 4 Serum hepatitis B virus (HBV) DNA levels can fluctuate markedly during the course of chronic HBV infection. Both case-control and cohort studies have shown a significant, dose-response association between serum HBV DNA levels measured at the time of initial evaluation and the subsequent risk of cirrhosis. A similar direct relationship has been shown for the risk of hepatocellular carcinoma (HCC) in cross-sectional, case-control, and cohort studies. Interventional studies have shown a strong correlation between the indices of disease activity seen on liver biopsy and levels of serum HBV DNA. These studies have also shown that reduction in HBV DNA levels correlate strongly with improvements in liver histology. For patients with HCC, prognosis (including risk of death, metastasis, and recurrence following surgery) is worse with higher serum HBV DNA levels. The preponderance of the evidence in the published literature demonstrates that serum HBV DNA level is an important and independent risk factor for disease progression in chronic hepatitis B. The relative importance of serial HBV DNA measurements, the loss of hepatitis B e and surface antigens, as well as the emergence of HBV mutants in the progression of chronic hepatitis B, especially in young patients, is an important need for future research. (HEPATOLOGY 2009;49:S72-S84.) Introduction Chronic hepatitis B virus (HBV) infection is a global public health concern. There are an estimated 350 million carriers of hepatitis B surface antigen (HBsAg) in the Abbreviations: ALT, alanine aminotransferase; HAI, histology activity index; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; REVEAL-HBV, Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-HBV study. From the 1 Genomics Research Center, Academia Sinica and 2 Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan; 3 Global Epidemiology and Outcomes Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., Wallingford, CT; 4 Other members of the RE- VEAL-HBV (Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer HBV) Study Group are listed in the Acknowledgment. Received November 28, 2008; accepted January 26, This study was supported by grants from Academia Sinica, Taiwan; Department of Health, Executive Yuan, Taiwan; and Bristol-Myers Squibb Co., USA. Address reprint requests to: Chien-Jen Chen, Sc.D., Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan. chencj@gate.sinica.edu.tw; cjchen@ntu.edu.tw; fax: Copyright 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Dr. Iloeje is employed by Bristol-Myers Squibb Co. A grant was provided by Bristol-Myers Squibb to Dr. Chen for conducting the laboratory tests for this study. The other authors declare that they have no potential conflicts of interest. Role of the Sponsors: Dr. Iloeje (employed by Bristol-Myers Squibb) was involved in the study design, development of the analysis plan, and interpretation of the data and writing of the report. All data handling was done by the staff at the Academia Sinica and National Taiwan University. At no time did the funding sources have access to the data. The corresponding author had final responsibility for the decision to submit for publication. world among whom there are more than 500,000 deaths annually attributable to cirrhosis and liver cancer (World Health Organization and Centers for Disease Control and Prevention fact sheets are available at and Despite the success of HBV vaccination in reducing the prevalence of chronic HBV infection as well as the incidence of acute hepatitis B, fulminant hepatitis, and hepatocellular carcinoma (HCC) in vaccine recipients, 1,2 there remains a large group of chronically infected persons at continued risk of developing cirrhosis or HCC. A central pathway in the pathogenesis of liver diseases from viral and nonviral etiologies is the transformational change seen in the function of the hepatic stellate cells from vitamin A storage cells to activated, profibrogenic cells. 3 The process of hepatic fibrogenesis is a dynamic one, and removal of the insult (viral and nonviral) may lead to reversal of fibrosis. 3-5 In chronic hepatitis B, presence of circulating virus is a marker of active infection and signifies the potential for persistent insult to the liver. The importance of serum HBV DNA levels as a predictor of the development of cirrhosis and HCC has been extensively reviewed recently. 6 Several hospital-based and community-based case-control and cohort studies have consistently found significant associations between elevated HBV DNA levels and risk of liver cirrhosis and HCC. However, many of these studies were limited by small number of and controls, inadequate matching S72

2 HEPATOLOGY, Vol. 49, No. 5, Suppl., 2009 CHEN, YANG, AND ILOEJE S73 or adjustment of confounding factors, lack of causal temporality, and analysis of risk predictors at study entry only. The progression of chronic hepatitis B to HCC is a multistage process and nonviral risk factors may include aflatoxin exposure, alcohol drinking, and host susceptibility factors. 7,8 A population-based, long-term follow-up study with repeated measurements of key risk factors would be the best approach to address the complex questions around the dynamics of HBV infection markers and their impact on disease progression risk. 9 The variation in HBV DNA level by various risk predictors of chronic hepatitis B progression and the importance of HBV DNA level in the determination of clinical outcomes will be reviewed in this article. Variation in Serum HBV DNA Levels Among Patients with Chronic Hepatitis B The natural course of chronic HBV infection is highly variable from patient to patient as well as over time and with increasing age. 6 Early life or perinatal infection is characterized by a period of immune tolerance where the host coexists with the virus without apparent injury to the host. During this period, serum HBV DNA levels are persistently high. Following the immune tolerance phase, infected patients can progress through an immune active phase or immune clearance phase, where hepatitis becomes active and it appears that the host immune system tries to clear infected hepatocytes. This phase is marked by hepatic inflammation, serum alanine aminotransferase (ALT) elevations, and a modest to moderate reduction of the circulating HBV DNA levels. The immune clearance phase is highly variable in duration and frequency, but a prolonged phase or recurrent episodes of acute liver inflammation may result in repeated cycles of injury and regeneration resulting in necroinflammation/fibrosis and an increased risk of progression to cirrhosis and HCC. The progression to a state of detectable liver injury is characterized by the presence of HBsAg and hepatitis B e antigen (HBeAg) in serum, moderate to high levels of circulating HBV DNA, elevation of serum ALT levels, and absence of antibody against HBeAg (anti-hbe). In some patients, seroconversion to anti-hbe seropositive status occurs with ongoing viral replication (the precore or basal core promoter mutant virus infections). These HBeAg-negative and anti-hbe positive patients have serum HBV DNA levels usually lower than those in patients who are HBeAg-positive with chronic hepatitis B. Another group of infected persons is able to inactivate the infection and go into the nonreplicative phase or inactive carrier state. Patients in this phase are characterized by the continued presence of HBsAg in serum, absence of HBeAg and presence of anti-hbe, low levels of serum HBV DNA and normal serum ALT values. These patients usually do not progress to worsening liver disease, cirrhosis, or end-stage liver diseases, but these endpoints may have occurred during the immune clearance phase and the patient with the inactive carrier state may have residual liver fibrosis. In a recent nested case-control study of 58 HBeAg-seroconverted children with chronic HBV infection enrolled in community-based and clinic-based cohorts, 10 the mean age ( standard deviation) at HBeAg seroconversion was years; mean peak HBV DNA levels were before seroconversion and after seroconversion. Among patients infected with HBV as adults, the majority resolve the infection spontaneously, clearing HBV DNA and HBsAg and developing anti-hbs. In a small proportion ( 5%), HBV infection becomes chronic. These adult-acquired infections usually progress directly into an immune active or immune clearance phase without experiencing a period of immune tolerance. The REVEAL-HBV Study In the Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer-HBV study (REVEAL-HBV study) on 4155 HBsAg-seropositive adults aged years at enrollment in and followed through 2004, serum samples collected and frozen at study entry and during follow-up were retrieved for analyses of HBV DNA level, genotype, and HBV mutant types. In the analyses of HBV viral levels and chronic hepatitis B outcomes, only subjects with adequate serum samples for HBV DNA quantification at study entry and who were negative for antibody to hepatitis C virus (anti-hcv) by immunoassay technique were included (n 3653). As shown in Fig. 1, elevated baseline HBV DNA levels were associated with HBeAg-seropositivity (P 0.001), liver cirrhosis at study entry (P 0.001), male sex (P 0.001), younger ages (P 0.001), increasing serum ALT levels (P 0.001), and habitual cigarette smoking (P 0.002). There was no significant association between habitual alcohol drinking and serum HBV DNA level at study entry (P 0.15). Participants infected with HBV genotype C had higher average HBV DNA levels at study entry than those infected with genotype B(P 0.001). The distributions of serum HBV DNA levels at study entry for participants who were HBeAg-seronegative and with normal ALT levels ( 45 U/L) without cirrhosis are shown in Fig. 2. As in the analysis of patients with HBeAg, serum HBV DNA levels at study entry were significantly associated with male sex, younger age, higher (normal) ALT levels, and cigarette smokers (compared to nonsmokers). Again, HBV DNA levels were not associ-

3 S74 CHEN, YANG, AND ILOEJE HEPATOLOGY, May 2009 Fig. 1. Distributions of serum HBV DNA levels at study entry by various characteristics of all REVEAL-HBV Study participants (N 3653). ated with a history of alcohol use. Interestingly, participants infected with HBV genotype C had lower serum HBV DNA levels than those infected with genotype B (P 0.001). Serum HBV DNA Level and Liver Cirrhosis The findings of case-control and cohort studies on the association between serum HBV DNA levels and risk of cirrhosis are summarized in Table 1. In a case-control study of 79 patients with cirrhosis and 158 controls matched on age, sex, and HBeAg serostatus, the presence of cirrhosis was associated with higher levels of serum HBV DNA. 15 In a follow-up study of 2763 HBsAg-seropositive adults in China, elevated serum HBV DNA levels at study entry were associated with an increased mortality from chronic liver diseases and an increased morbidity of

4 HEPATOLOGY, Vol. 49, No. 5, Suppl., 2009 CHEN, YANG, AND ILOEJE S75 Fig. 2. Distributions of serum HBV DNA levels at study entry by various characteristics of REVEAL-HBV Study participants with HBeAg-negative serostatus, normal ALT level, and no liver cirrhosis (N 2925). severe liver diseases among survivors. 16 In the REVEAL- HBV study, the incidence of cirrhosis (per 100,000 person-years) increased with increasing serum HBV DNA levels () at study entry: ranging from 339 ( 300), 430 ( ), 774 ( ), 1879 ( ) to 2498 ( ). The biological gradient remained significant in stratified analyses across a variety of baseline characteristics such as sex, age, and habits of cigarette smoking and alcohol drinking. In multivariate Cox regression analyses of risk factors predicting progression to liver cirrhosis, increasing HBV DNA level was the strongest independent predictor. 13 Serum HBV DNA Levels and HCC The findings from case-control and case-cohort studies on the association between serum HBV DNA levels and risk for HCC are summarized in Table 2. A significant association between elevated serum HBV DNA levels and increased risk of HCC was observed in all studies despite differences in study design (cross-sectional versus longitudinal and community/hospital based), health status of controls, method and detection limit for the determination of HBV DNA levels, HBV DNA levels selected as the referent group, and variables chosen for adjustment in the analyses In a community-based, nested case-control study of 44 HBeAg-negative patients with newly developed HCC and 86 matched controls who were selected from a cohort of 1991 male HBeAg-negative HBV carriers in Taiwan, a significant dose-response relationship between serum HBV DNA levels at study entry and HCC risk was observed. 17 Compared with serum HBV DNA levels 2.5 pg/ml as the referent group, the multivariateadjusted odds ratio was 2.3 and 6.0, respectively, for se-

5 S76 CHEN, YANG, AND ILOEJE HEPATOLOGY, May 2009 Table 1. Case-Control and Cohort Studies of Serum HBV DNA Level and Liver Cirrhosis Study (Year) Study Population Endpoint Testing Method HBV DNA Levels, () Main Findings Adjustment Variables Case-control study Yuan et al. 79 with (2005) 15 chronic hepatitis B and cirrhotic complications (including HCC); 158 controls from hepatitis clinic without cirrhotic complications Cohort studies Chen et al HBsAg-positive (2006) 16 adults Iloeje et al. (2006) HBsAgpositive, anti-hcv negative adults Cirrhotic complications 85 deaths from chronic liver disease 367 current of severe liver disease (incl. HCC) of 1683 survivors Diagnosis of cirrhosis by ultrasound Digene Hybrid Capture II assay*; Cobas Amplicor Monitor test Cirrhosis, n Controls, n (OR) (referent) 32 Matched for (2.15) 17 age, sex and 10,000 99,999 8 (1.46) 25 HBeAg status 100, , (3.05) 21 1,000, (3.05) 63 Cumulative mortality/ 100,000 Adjusted RR (95% CI) Age and sex , ( ) 100, ( ) Cases/survivors (prevalence) Adjusted OR (95% CI) /151 (13.2%) Age and sex , /772 (17.6%) 1.3 ( ) 100, /716 (29.5%) 2.7 ( ) Cobas Amplicor HBV Monitor test Incidence of cirrhosis per 100,000 person-years Adjusted HR (95% CI) Age, sex, habits ( ) of cigarette 10,000 99, ( ) smoking and 100, , ( ) alcohol 1,000, ( ) drinking, HBeAg status, and ALT level *Detection limit: 140,000 (Digene Diagnostics, Gaithersburg, MD). Detection limit: 200 (Roche Diagnostics, Branchburg, NJ). Detection limit: Detection limit: 300 (Roche Diagnostics, Indianapolis, IN). ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; OR, odds ratio; PCR, polymerase chain reaction. rum HBV DNA levels of and 13.0 pg/ml. In a hospital-based nested case-control study on 48 patients with HCC and 48 age-matched and sex-matched controls with cirrhosis in Japan, patients with HCC had higher serum HBV DNA levels at study entry than patients with cirrhosis who remained HCC-free during follow-up. 18 In three case-control studies in which the serum HBV DNA levels at study entry were dichotomized into two groups, the elevated HBV DNA level group in each study had an increased risk of developing HCC: the multivariate-adjusted odds ratio comparing high versus low HBV DNA levels being 15.6 ( 83 versus 83 ) in a study from Senegal; ( 83 versus 83 ) in a study from China, 19 and 2.5 ( 10 5 versus 10 5 copies/ ml) in a study from Taiwan. 21 Nested in a large-scale hospital-based long-term follow-up study of 4841 male HBV carriers in Taiwan, a case-control study of 154 newly developed HCC and 316 matched HBV carrier controls found a significantly increasing risk of HCC across the biological gradient of serum HBV DNA levels at study entry. 20 In the further analysis of HBV DNA levels in serial serum samples collected during follow-up, a significant dose-response relationship was observed between the risk of

6 HEPATOLOGY, Vol. 49, No. 5, Suppl., 2009 CHEN, YANG, AND ILOEJE S77 Table 2. Case-Control and Case-Cohort Studies of Serum HBV DNA Level and Hepatocellular Carcinoma Study (Year) Study Design Study Population Endpoint Testing Method HBV DNA Levels HCC Cases, n Controls, n OR (95% CI) Adjustment Variables Yang et al. (2002) 17 Community-based, nested Ikeda et al. (2003) 18 Hospital-based, nested Tang et al. Community-based, (2004) 19 nested Yu et al. (2005) 20 Hospital-based, nested Liu et al. (2006) 21 Hospital-based, cross-sectional Tsai et al. (2007) 22 Hospital-based, cross-sectional Liu et al. Community-based, (2008) 23 nested Wu et al. Hospital-based, (2008) 24 case-cohort 44 with HCC and 86 matched controls nested in a cohort of 1991 men (HBsAg positive and HBeAg negative) 48 with HCC and 48 age-sex-matched controls nested in 160 consecutive pts with cirrhosis (HBsAg positive, anti-hcv negative) who received no antiviral therapy 14 with HCC and 28 matched controls in Senegal 55 with HCC and 55 matched controls in China (undetectable HBV DNA by dot blot hybridization) 154 with HCC and 316 controls nested in 4841 Taiwanese men from clinical settings, followed for 14 years 200 with HCC and 160 chronic HBV carriers 26 with HCC and 155 chronic HBV carriers( 40 years of age) 157 with HCC and 47 chronic HBV carriers( 40 years of age) 170 with HCC and 276 with chronic hepatitis B with normal ALT level at study entry 112 HCC and 1031 HBV carriers HCC incident HCC incident HCC death HCC incident Noncirrhotic HCC HCC incident HCC incident HCC incident Branched-chain 2.5 pg/ml DNA assay (Quantiplex)* pg/ml 13.0 pg/ml TMA and hybridization protection assay Real-time TaqMan PCR assay assay assay Fluorescein quantitative- PCR (LOQ: 500 copies/ ml) assay (LOQ: 215 copies/ ml) Continuously low Decreasing in 3 years Intermittently high Persistently high ( ) 6.0 ( ) ( ) 3.1 ( ) Age, anti-hcv status, and habits of cigarette smoking and alcohol drinking None Age, sex, HBsAg status, year of cohort entry, and military rank Age, sex, HBsAg status, dot blot result, year of study entry, and township of residence 3.62 log Age, date of blood collection, log ethnicity, history ( ) of cigarette smoking and log ( ) alcohol drinking log ( ) log 10 copies/ ml ( ) Age, sex, HBV genotype, precore 10 5 copies/ A1896 mutant, ml 2.5 ( ) and basal core promoter T1762/ A1764 mutant log 10 titre ( ) Age, sex, ALT level, and HBV genotype log 10 titre ( ) Undetectable Age, cigarette log smoking, alcohol ( ) consumption, and family history of log ( ) chronic liver diseases log ( ) log ( ) 7.00 log ( ) No follow-up Age, total number of sample 4.39 visits, HBV log 10 copies/ genotype, frequency ml One follow-up sample 4.39 log 10 Two or more follow-up samples but 50% of follow-up samples 4.39 log 10 50% of followup samples 4.39 log ( ) 3.2 ( ) 5.0 ( ) of ALT elevation, and HBeAg status *Detection limit: 2.5 pg/ml (Chiron Diagnostics, Emeryville, CA ). Detection limit: 3.7 log 10. Detection limit: 83 (Applied Biosystems, Foster City, CA). Detection limit: 100. ALT, alanine aminotransferase; anti-hcv, antibodies to hepatitis C virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LOQ, limit of quantification; OR, odds ratio; PCR, polymerase chain reaction; TMA, transcription-mediated amplification.

7 S78 CHEN, YANG, AND ILOEJE HEPATOLOGY, May 2009 Table 3. Cohort Studies of Serum HBV DNA Level and Hepatocellular Carcinoma Study (Year) Study Design Study Population Endpoint Ohata et al. (2004) 25 Hospital-based 73 patients with chronic hepatitis B, anti-hcv negative, followed 6mo Mahmood et al. Hospital-based 91 patients with (2005) 26 cirrhosis Ikeda et al. (2005) 18 Hospital-based 57 patients with chronic hepatitis B receiving IFN therapy Chen et al. Community-based 2763 HBsAgpositive (2006) 16 adults Chen et al. (2006) 11 Community-based 3653 HBsAgseropsitive, anti- HCV-negative adults Chan et al. Hospital-based 1006 patients with (2008) 27 chronic hepatitis B Yang et al. (2008) 28 Community-based 2762 HBsAgpositive, anti- HCV-negative adults with detectable HBV DNA levels 21 HCC incident 23 HCC incident 13 HCC incident 231 deaths from HCC 164 HCC incident 86 HCC incident 153 HCC incident Testing Method HBV DNA Levels Main Findings Adjustment Variables Cumulative Adjusted RR (95% CI) incidence TMA* /35 (17%) Age, sex, habitual heavy /28 (54%) 3.08 ( ) drinking, ALT level, IFN therapy, fibrosis stage, and inflammation grade Amplicor 2.6 log 10 1/20 (5%) ALT level Monitor kit log 10 5/21 (23.8%) 5.0 log 10 17/47 (36.2%) 2.33 ( ) TMA and HBV DNA loss None hybridization ( 5000 protection ) assay* Without HBV DNA 2/25 (8%) loss 11/32 (34.4%) 4.3, unadjusted % Age and sex , % 1.7 ( ) 100, % 11.2 ( ) Incidence rate Adjusted HR (95% CI) per 100,000 person-years Cobas Amplicor HBV Monitor test (LOQ: 100 ) (LOQ: 100 ) Sex, age, cigarette and ( ) alcohol use, HBeAg, ALT level, and 10,000 99, ( ) cirrhosis at entry 100, , ( ) 1,000, ( ) Cumulative incidence Crude HR (95% CI) log 10 19/500 (3.8%) log 10 36/313 (11.5%) 1.62 ( ) 6.5 log 10 31/193 (16.1%) 2.73 ( ) Adjusted HR (95% CI) Three strata 1.79 ( ) Sex, age, cirrhosis, albumin, and HBV genotype Incidence rate Adjusted HR (95% CI) per 100,000 person-years Sex, age, habits of 10,000 99, ( ) cigarette smoking and alcohol drinking, 100, , ( ) ALT level, cirrhosis at entry, and HBV 1,000, ( ) genotype *Detection limit: 3.7 log 10. Roche Diagnostics, Tokyo, Japan. Detection limit: Detection limit: 300 (Roche Diagnostics, Indianapolis, IN). ALT, alanine aminotransferase; anti-hcv, antibodies to hepatitis C virus; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; IFN, interferon; LOQ, limit of quantification; mo, months; PCR, polymerase chain reaction; TMA, transcription-mediated amplification. HCC and the proportion of follow-up serum samples with elevated HBV DNA levels. 24 The significant doseresponse relationship was also observed in another community-based nested case-control study of 170 newly diagnosed of HCC and 276 HBV carrier controls with normal ALT levels at study entry. 23 In a hospitalbased cross-sectional case-control study of 183 of HCC and 202 HBV carrier controls, serum HBV DNA levels were sigificantly associated with HCC risk in the older ( 40 years) but not in the younger ( 40 years) age group. 22 The findings of cohort studies on the association between serum HBV DNA levels and HCC risk are summarized in Table 3. There was also consistency among these studies despite differences in sudy design and methodology. 11,16,18,25-28 The number of cohort participants was 100 in three hospital-based cohort studies, 18,25,26 and the adjustment for confounding factors was considered inadequate in one community-based study 16 and two hospital-based studies. 18,26 In a hospital-based cohort study of 1006 patients with chronic hepatitis B, serum HBV DNA levels at study entry were significantly associ-

8 HEPATOLOGY, Vol. 49, No. 5, Suppl., 2009 CHEN, YANG, AND ILOEJE S79 ated with subsequent development of HCC in a doseresponse relationship after adjstment for risk predictors including HBV genotype. 27 In the REVEAL-HBV study, the incidence of new onset HCC (per 100,000 person-years) increased with serum HBV DNA levels (in ) at study entry ranging from 108 ( 300), 111 ( ), 297 ( ), 962 ( ), to 1152 ( ). 11 In multivariate Cox regression analyses of risk factors predicting progression to HCC, increasing HBV DNA category was the strongest independent predictor of HCC risk after cirrhosis. In the subset analysis of last follow-up serum HBV DNA levels for individuals who had serum HBV DNA levels at study entry, HCC risk was highest among those who had persistently high HBV DNA levels of In a recent analysis, the independent effect of HBV viral levels on HCC was assessed after adjustment for HBV genotype and mutants. 28 HBV genotype was identifiable only for participants with detectable serum HBV DNA levels at study entry (n 2762), and HBV mutants were tested only for participants with serum HBV DNA levels 10 4 (n 1526). HBV genotype C infection was associated with a higher risk of HCC than HBV genotype B. The G1896A mutation in the precore region was associated with a lower risk of HCC compared to the wild-type virus, while the double mutation (A1762T/G1764A) in the basal core promoter region was associated with a higher risk than the wild-type virus. Elevated serum HBV DNA levels at study entry were associated with an increased risk of HCC after multivariate adjustment for risk predictors including HBV genotype and mutants (odds ratio 1.6 for HBV DNA levels 10 5 versus 10 4 to 10 5, P 0.04). Serum HBV DNA Level and Liver Histology The evidence for an association between serum HBV DNA levels and histopathological findings on liver biopsies are summarized in Table In a cross-sectional study of 55 patients who were HBeAg-negative with chronic hepatitis B, patients with detectable HBV DNA levels ( 0.5 meq/ml) had significantly higher necroinflammation scores in histology activity index (HAI) than those with undetectable HBV DNA levels. 29 In a crosssectional study of 94 chronic hepatitis B patients, 31 serum HBV DNA levels were positively correlated with necroinflammation scores of HAI (r 0.31, P 0.014), fibrosis scores (r 0.33, P 0.017), and total HAI scores (r 0.37, P 0.008) in patients who were positive for anti- HBe but not for those who were HBeAg-positive. In a cross-sectional study of 47 HBsAg-positive blood donors with detectable serum HBV DNA levels, serum HBV DNA levels significantly correlated with HAI-necroinflammation scores (r 0.59; P 0.001) and Ishak fibrosis stages (r 0.50; P 0.001). 32 In another crosssectional study of 213 patients with chronic hepatitis B with serum HBV DNA level 10 5, serum HBV DNA levels were not correlated with histological grade and stage of liver disease in either HBeAg-positive and HBeAg-negative patients. 33 In a large meta-analysis of 26 intervention studies including 3428 treated patients, histological grades were significantly associated with serum HBV viral level at study entry (r 0.78; P ) and at the end of treatment (r 0.71; P 0.003). 30 More importantly, improvement in histological grade was strongly correlated with a decrease in serum HBV DNA levels (r 0.96; P 0.001). Serum HBV DNA Level and Prognosis of HCC The findings of case-series and cohort studies of serum HBV DNA levels and metastasis, recurrence, and death from HCC are summarized in Table 5. 14,34-41 In four eries studies on the recurrence of HCC after surgical resection 34,38,39 or transarterial chemolipidolization, 37 patients with high serum HBV DNA levels at study entry had a significantly higher risk of HCC recurrence than those with low levels. In another three case-control studies on death from HCC, patients with high serum HBV DNA levels at study entry had a significantly higher risk of HCC death than those with low serum HBV DNA levels. 35,36,41 A doseresponse relationship between serum HBV DNA levels and the metastasis or recurrence of HCC was reported in another case-series study. 40 In the REVEAL-HBV study, 14 the mortality (per 100,000 person-years) increased with baseline HBV DNA level (in ) ranging from 9 ( 300), 48 ( ), 75 ( ), 143 ( ), to 267 ( ) for chronic liver disease and cirrhosis; and 73, 48, 174, 692, and 816, respectively, for liver cancer. In multivariate Cox regression analyses of risk factors predicting progression to mortality, increasing HBV DNA level was the strongest independent predictor of death from chronic liver disease and cirrhosis, and was second to cirrhosis in predicting death from HCC. There was no association between serum HBV DNA levels and non liver related mortality. Treatment-Induced Viral Suppression and Liver Disease Progression In what has become recognized as a landmark study, the impact of antiviral therapy on disease progression

9 S80 CHEN, YANG, AND ILOEJE HEPATOLOGY, May 2009 Table 4. Cross-Sectional and Intervention Studies of Serum HBV DNA Level and Liver Histology Study (Year) Study Design Study Population Endpoint Testing Method Main Findings Chan et al. (2002) 29 Cross-sectional 55 HBeAg-negative chronic hepatitis B patients Mommeja-Marin et al. (2003) 30 Meta-analysis of intervention studies 26 prospective studies involved 3428 patients Yuen et al. (2004) 31 Cross-sectional 94 patients with chronic hepatitis B Nabuco et al. (2007) 32 Cross-sectional 47 HBsAg-positive blood donors with detectable serum HBV DNA Shao et al. (2007) 33 Cross-sectional 213 patients with chronic hepatitis B and serum HBV DNA level 10 5 Histological activity index core for necroinflammation (HAI-NI) and fibrosis (HAI-F) DNA cross-linking assay (LOQ: 0.5 meq/ml) and PCR assay Patients with detectable HBV DNA levels had significantly higher HAI-NI score Histological activity index Various Histological grades significantly associated with HBV viral load at study entry (r 0.78; P ) and at the end of treatment (r 0.71; P 0.003) Change in median histological grades correlated significantly with change in median HBV DNA levels (r 0.96; P 10-6 ) Total histological activity index (HAI); necroinflammation (HAI-NI); and fibrosis (HAI-F) scores Histological diagnosis and Ishak classification Histological grade and stage Cobas Amplicor HBV Monitor test* Amplicor (LOQ: 1000 ) Cobas Amplicor HBV Monitor test* In HBeAg-positive patients, no correlation between HBV DNA levels and HAI-NI, HAI- F, and total HAI scores In anti-hbe-positive patients, HBV DNA levels positively correlated with HAI-NI (r 0.31; P 0.014), HAI-F (r 0.33; P 0.017), and total HAI score (r 0.37; P 0.008). Patients with histologically diagnosed chronic hepatitis and cirrhosis had higher serum HBV DNA than those without the histological liver diseases (median HBV DNA: 25,260,000 vs 9480 ) Serum HBV DNA levels significantly correlated with necroinflammatory score (r 0.59; P 0.001) and fibrosis stage (r 0.50; P 0.001) Serum HBV DNA levels were not correlated with histological grade and stage of liver disease in both HBeAg-positive and -negative patients *Detection limit: 200 (Roche Diagnostics, Branchburg, NJ). HAI-F, histology activity index fibrosis score; HAI-NI, histology activity index necroinflammation score; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; LOQ, limit of quantification; meq, milliequivalent; PCR, polymerase chain reaction. was tested in a randomized, placebo-controlled study in patients with advanced fibrosis or cirrhosis due to hepatitis B. 42 As shown in Table 6, 436 patients received lamivudine and 215 patients received placebo. The trial was terminated after 36 months on the recommendation of an independent data safety and monitoring board, based on a significant differences in clinical outcomes between lamivudine-treated and placebo-treated patients. Antiviral therapy significantly lowered the risk of liver disease progression (17.7% versus 7.8%), and led to a lower rate of progression in Child-Turcotte-Pugh score (8.8% versus 3.4%). Even after the relatively brief treatment period of 36 months, lamivudine treatment was associated with fewer of HCC than was placebo treatment (7.4% versus 3.9%), although this difference was just short of statis-

10 HEPATOLOGY, Vol. 49, No. 5, Suppl., 2009 CHEN, YANG, AND ILOEJE S81 Table 5. Case-Series and Cohort Studies of Serum HBV DNA Level and Metastasis/Recurrence/Death of Hepatocellular Carcinoma Study (Year) Study Population Endpoint Testing Method HBV DNA Levels Main Findings Adjustment Variables Kubo et al. 40 HCC patients 19 patients with (2000) 34 underwent HCC recurrence surgical resection Branched-chain DNA assay (Quantiplex)* 0.7 meq/ml 0.7 meq/ml Ohkubo et al. (2002) HCC patients HCC death TMA 3.7 LGE/mL 3.7 LGE/mL Yeo et al. 125 HCC patients HCC death 5.65 log 10 (2007) 36 underwent (LOQ: 10 systemic ) for 5.65 log 10 copies/ chemotherapy pretreatment ml HBV DNA levels Jang et al. (2007) HCC patients underwent transarterial Iloeje et al. (2007) HBsAgpositive, Anti- HCV-negative adults from community 32 patients with HCC recurrence chemolipiodoliza-tion Local recurrence Kim et al. 157 patients with Recurrence of HCC (2008) 38 HCC underwent surgical resection (excluded patients with fluctuating DNA) Hung et al. 72 HCC patients 30 patients with (2008) 39 underwent HCC recurrence surgical resection Huang et al. (2008) HCC patients 69 patients with HCC metastasis or recurrence in two years of follow-up Branched DNA assay (Versant 3.0) Cumulative recurrence 64% 100% Median survival time (years) Adjusted HR (95% CI) 5.1 ( ) Adjusted OR (95% CI) 3.9 ( ) 1.7 ( ) Median recurrence-free Adjusted HR (95% CI) time (months) ( ) 5.2 ( ) Positive surgical margin Tumor size Total bilirubin and HCV infection HBeAg, Child-Pugh classification, number of tumors, and Lamivudine use Age, AFP, Child-Pugh classification, tumor size, number of tumors, and Lamivudine therapy Distant recurrence ( ) Liver cancer mortality Adjusted HR (95% CI) rate per 100,000 person-years 119 HCC deaths Cobas Amplicor Sex, age, cigarette smoking, HBV Monitor test Digene hybrid capture assay Cobas Amplicor HBV Monitor test (LOQ: 1000 ) ( ) 10,000 99, ( ) 100, , ( ) 1,000, ( ) HBV DNA consistently 10 5 HBV DNA consistently log 10 4 log 10 alcohol consumption, HBeAg, ALT level, and cirrhosis status 5-yr cumulative Adjusted OR (95% CI) recurrence rate 54.7% Tumor size, tumor number, vascular invasion, and 72.9% 1.6 ( ) grades Cumulative recurrence 2/19 (10.5%) 28/53 (52.8%) Adjusted HR (95% CI) 22.3 ( ) Age, AFP, tumor size, tumor differentiation, lymphovascular permeation, and microsatellite lesions Cumulative incidence Adjusted OR (95% CI) 3 log 10 22% GGT, tumor capsule, portal 3 5 log vein thrombosis, tumor 10 60% 5.2 ( ) dimension, cirrhosis, BCLC 5 log 10 80% 14.0 ( ) stages, and BCP mutations Fattovich et al. (2008) patients with HBeAg-positive chronic hepatitis 11 patients with liver-related death (HCC and liver failure) and 1 with orthotopic liver transplantation Cobas Amplicor HBV Monitor test Sustained remission HBeAg-/HBV DNA or HBeAg reversion and HBeAg persistence Cumulative incidence 2/40 (5.0%) 10/30 (33.3%) Adjusted HR (95% CI) 27.0 ( ) Age, sex, duration of HBeAg positive phase, and cirrhosis at entry *Detection limit: 2.5 pg/ml (Chiron Diagnostics, Emeryville, CA ). Detection limit: 3.7 log 10. Detection limit: 100,000 (Digene Diagnostic Inc., Beltsville, MD). Detection limit: 300 (Roche Diagnostics, Indianapolis, IN). Detection limit: (Bayer HealthCare LLC, Tarrytown, NY). AFP, alpha-fetoprotein; ALT, alanine aminotransferase; HCV, hepatitis C virus; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; LGE, logarithm of genome equivalent; LOQ, limit of quantification; meq, milliequivalent; OR, odds ratio; PCR, polymerase chain reaction; TMA, transcription-mediated amplification. tical significance. The observed benefits of treatment was most likely driven by viral suppression, because disease progression among treated patients who developed lamivudine resistance (YMDD mutants) was 13%, which was higher than that in the group without drug resistance. In another intervention study of lamivudine therapy in 353 patients with chronic hepatitis B and 303 pa-

11 S82 CHEN, YANG, AND ILOEJE HEPATOLOGY, May 2009 Table 6. Intervention Studies of Antiviral Treatment on Long-Term Progression of Chronic Hepatitis B Study (Year) Study Population Endpoint Study Variable Main Findings Adjustment Variables Liaw et al. (2004) 42 Di Marco et al. (2004) patients with advanced fibrosis or cirrhosis receiving Lamivudine and 215 patients receiving placebo 353 patients with chronic hepatitis and 303 patients with cirrhosis receiving Lamivudine treatment Overall disease progression Increase in Child-Pugh score HCC HCC Liver-related death Treatment groups Lamivudine Placebo Lamivudine Placebo Lamivudine Placebo Response to lamivudine Maintained virological response Virological breakthrough Maintained virological response Virological breakthrough Percent with disease progression HR (95% CI) 0.5 ( ) 0.5 ( ) Country, sex, baseline ALT level, Child-Pugh score, and Ishak fibrosis score 0.5 ( ) HR (95% CI) Sex, age, initial diagnosis, baseline HBV DNA levels, baseline ALT 3.0 ( ) levels, hepatic flare after virological breakthrough, and previous IFN therapy Above excluding age 2.9 ( ) ALT, alanine aminotransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; IFN, interferon; HR, hazard ratio. tients with cirrhosis, the risk of developing HCC or liver-related death was three-fold higher among patients with cirrhosis who had virological breakthrough than in those who achieved persistent viral suppression on treatment. 43 Needs for Future Research There is ample and strong evidence linking elevations in serum HBV DNA levels and liver disease progression in chronic hepatitis B. Elevations in serum HBV DNA is not only a major risk factor for disease progression, but the risk factor most amenable to modification. These findings raise the question if there is enough evidence that persistent elevations in HBV DNA levels over time (rather than a single elevated value) is a stronger risk predictor for disease progression. Most studies have been based on determinations of serum HBV DNA levels at a single time at study entry rather than at multiple time points. The REVEAL-HBV study demonstrated that persistently elevated serum HBV DNA levels documented at two different time points (at study entry and last follow-up examination) is associated with the highest risk of HCC. Recent unpublished data from this study, using data on serum HBV DNA and ALT levels at multiple time points and time-dependent regression modeling, confirm that elevated serum HBV DNA at multiple time points is indeed a very strong independent predictor of HCC risk. 44 Because the REVEAL-HBV study population were infected in early life with HBV genotype B and C and recruited into this study at the age of at least 30, the results may not be directly applicable to other populations, especially younger adults and children and patients who were infected in adulthood. Other prospective noninterventional studies (especially in non-asian populations) that measure serum HBV DNA levels as well as other key risk factors at repeated time points should contribute to answering this question. The following studies are also needed to assess the importance of long-term change in serum HBV DNA levels in the development of liver cirrhosis and HCC: (1) The impact of long-term sequential changes in HBeAg serostatus, serum levels of HBV DNA and ALT, HBsAg serostatus, and HAI on the development of liver cirrhosis and HCC; (2) The importance of HBV genotypes and subtypes and long-term emergence of mutants in the development of liver cirrhosis and HCC; (3) The individual variation in the environmental and genetic susceptibility to disease progression of chronic hepatitis B; and (4) The assessment of development of end-stage liver diseases in patients with chronic hepatitis B who are treated with antivirals. Acknowledgment: The following institutions and investigators, in addition to the authors, participated in the REVEAL-HBV Study Group as a part of Taiwan Community-Based Cancer Screening Project: Chang-Gung Memorial Hospital and Chang-Gung University (Y. F. Liaw); Genomics Research Center (C. L. Jen, S. L. You); College of Public Health, National Taiwan University (T. H. H. Chen); Department of Gastroenterology,

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