Evaluation of Impact of Serial Hepatitis B Virus DNA Levels on Development of Hepatocellular Carcinoma

JOURNAL OF CLINICAL MICROBIOLOGY, June 2009, p. 1830 1836 Vol. 47, No. 6 0095-1137/09/$08.00 0 doi:10.1128/jcm.00029-09 Copyright 2009, American Society for Microbiology. All Rights Reserved. Evaluation of Impact of Serial Hepatitis B Virus DNA Levels on Development of Hepatocellular Carcinoma Henry Lik-Yuen Chan,* Vincent Wai-Sun Wong, Grace Lai-Hung Wong, Angel Mei-Ling Chim, Larry Hin Lai, and Joseph Jao-Yiu Sung Department of Medicine and Therapeutics and Institute of Digestive Disease, The Chinese University of Hong Kong, Shatin, Hong Kong Received 6 January 2009/Returned for modification 30 March 2009/Accepted 10 April 2009 We aimed to investigate the impact of hepatitis B virus (HBV) DNA on the development of hepatocellular carcinoma (HCC). We conducted a case/control study based on 506 chronic HBV patients followed up since 1997. Forty-one patients developed HCC, and each of them was age and gender matched with two simultaneously recruited controls without HCC. HBV DNA was measured at the initial visit, at yearly intervals, and at the last visit. Patient age at the time of HCC development was 55 9 years. Forty-nine (40%) patients experienced antiviral treatment. The median time from diagnosis to the development of HCC was 17 months, and the control patients were followed for 92 months. At the trough level (defined as lowest level among all studied visits), more (27 patients; 66%) HCC patients had HBV DNA levels of >10,000 copies/ml than the controls (17 patients; 21%). The area under the receiver operating characteristic curve of the trough log HBV DNA level for HCC was 0.79 (95% confidence interval [CI], 0.69 to 0.89). Trough log HBV DNA (odds ratio, 11.4; 95% CI, 3.6 to 37.6; P < 0.0001) and liver cirrhosis (odds ratio, 11.4; 95% CI, 3.6 to 36.2; P < 0.0001) levels were independently associated with HCC after an adjustment for age, gender, antiviral treatment, and HBV genotype. The difference in the trough HBV DNA level was more obvious among untreated patients (5.7 1.4 log copies/ml in HCC patients versus 3.2 1.3 log copies/ml in control patients; P < 0.0001) than among those who had received antiviral treatment (3.0 1.4 log copies/ml in HCC patients versus 2.5 0.9 log copies/ml in control patients; P 0.38). A high trough HBV DNA level was associated with a higher risk of HCC. Whether antiviral treatment could prevent HCC was uncertain. * Corresponding author. Mailing address: Department of Medicine and Therapeutics, 9/F Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, Hong Kong. Phone: 852-26323593. Fax: 852-26373852. E-mail: hlychan@cuhk.edu.hk. Published ahead of print on 22 April 2009. Chronic hepatitis B virus (HBV) infection is the commonest cause of hepatocellular carcinoma (HCC) in Southeast Asia (8). In addition to the host factors, including an older age and male gender, viral factors such as being positive for HBV e antigen (HBeAg) and having high HBV DNA levels, genotype C HBV (particularly subgenotype Ce), and basal core promoter mutations have been suggested to associate with a higher risk of HCC (7, 9, 10, 11, 14, 28). In several cohort studies in Taiwan, China, and Hong Kong, a single high HBV DNA level higher than 4 log to 4.5 log copies/ml was associated with an increased HCC risk in the subsequent 10 years (7, 9, 10, 11). In addition, patients who had persistently high HBV DNA levels at the last follow-up visit also had an increased risk of developing HCC (10). In all previous studies, there was no information on the HBV DNA levels during the years between the first and last follow-up visits. As HBV DNA levels tend to fluctuate, particularly in HBeAg-negative patients, an assessment at a single time point cannot accurately reflect the severity of liver disease (6, 12, 13). In recently published management guidelines, antiviral treatment is recommended in patients with liver cirrhosis when the HBV DNA level is higher than 4 log copies/ml (15, 21, 23). This recommendation was based primarily on a multicentered, placebo-controlled trial among Asian chronic HBV patients who suffered from severe liver fibrosis (20). In this study, lamivudine treatment was demonstrated to reduce the risk of liverrelated complications in 3 years. However, the benefit of prevention of HCC development among lamivudine-treated patients became dubious after excluding patients who developed HCC within the first year. In an Italian series, HBV DNA suppression by lamivudine and adefovir could not prevent the development of HCC among patients with decompensated liver cirrhosis (18). In this study, we aimed to investigate the impact of changes in HBV DNA on the development of HCC, either in the natural course of disease or as a result of antiviral treatment. To address this question, we studied the HBV DNA levels at different time intervals of follow-up among a cohort of chronic HBV patients before the development of HCC compared to those of simultaneously recruited controls. MATERIALS AND METHODS Patients. This case/control patient study was based on two cohorts of patients followed up at the hepatitis clinic at Prince of Wales Hospital. One cohort consisted of 426 consecutive chronic HBV patients who were recruited between December 1997 and July 2000 and were followed up thereafter (1, 7). Another cohort consisted of 80 chronic HBV patients who have participated in a placebocontrolled, randomized trial of lamivudine since 1999 (20). All patients were ethnically Chinese and were positive for HBV surface antigen for over 6 months. All patients had no history of alcoholism over 20 g per week or hepatitis C virus coinfection. Patients who had other possible causes of hepatitis or liver cirrhosis, including autoimmune liver disease, primary biliary cirrhosis, Wilson s disease, and hemochromatosis, were also excluded. At initial presentation, an ultrasound of the abdomen was performed to exclude any preexisting HCC. Patients were prospectively followed up every 3 to 6 months, or more frequently if clinically indicated, with monitoring of liver biochemistry and HBeAg and anti-hbe status as well as alpha-fetoprotein levels. An ultrasound of the abdomen, computerized tomography, hepatic angiogram, and/or liver biopsy would be performed when- 1830

VOL. 47, 2009 SERIAL HBV DNA AND HCC 1831 ever the alpha-fetoprotein level was on a rising trend over 20 g/liter to confirm the diagnosis of HCC. For patients with normal alpha-fetoprotein levels, an ultrasound of the abdomen was performed every 1 to 2 years. Clinical liver cirrhosis was defined as ultrasonic features of liver cirrhosis plus evidence of hypersplenism (splenomegaly with a platelet count of less than 100 10 9 /liter or a white cell count of less than 4 10 9 /liter), clinical ascites, varices, and/or hepatic encephalopathy (1). Antiviral treatment was started according to the physician s decision or the clinical trial protocol. The database was frozen on 30 December 2007. Forty-one patients developed HCC during the follow-up period. For each case patient, we randomly selected two control subjects from the same cohort of chronic HBV patients who were alive and had not been diagnosed with HCC throughout the follow-up period. As the age of the patient at the time of HCC development is a major confounding factor, the control subjects were individually matched to the case patients by the gender and age of the patient at the last follow-up (or when HCC was diagnosed) within 5 years. In other words, these patients were recruited at the same period of time, with the time of HCC diagnosis matched to that of the last follow-up of the controls. Other clinical factors, including liver cirrhosis and antiviral treatment, were not matched due to an insufficient number of matched patients to serve as controls in our database. HBV DNA was measured at the initial visit, at yearly intervals, and at the last follow-up visit (or when HCC developed if serum samples were available). Laboratory tests. Residual serum samples were stored in a 80 C freezer. HBV DNA was measured in the initial visit, then every year until the last follow-up visit. HBV genotyping was performed with the initial serum sample at the time of patient enrollment. HBV DNA was extracted by using a Qiagen QIAamp DNA minikit (Qiagen, Inc., Chatsworth, CA) according to the instructions of the manufacturer. Serology assays. HBV surface antigen (HBsAg) and anti-hepatitis C virus antibodies (third-generation assay) were tested using commercially available enzyme-linked immunosorbent assay kits (Abbott GmBH Diagnostika, Wiesbaden-Delkenheim, Germany). HBeAg and anti-hbe were measured by using an enzyme-linked immunosorbent assay (Sanofi Diagnostics, Pasteur, France). HBV DNA. HBV DNA was quantified by using a TaqMan real-time PCR assay as described previously (3, 22). This assay was standardized by serial dilution of EUROHEP genotype D HBV standard which contained 2.7 10 9 viral copies/ ml. The range of HBV DNA detection was from 10 2 to 10 9 copies/ml with a correlation coefficient of the standard curve routinely greater than 0.990. HBV genotyping. HBV genotyping was determined by PCR amplification and restriction fragment length polymorphism as described previously (4). All samples with inconclusive findings were confirmed by bidirectional sequencing of the HBV surface gene and compared to the published sequences in GenBank (9). Statistical analysis. Statistical analysis was performed by using SPSS version 13.0 (Chicago, IL). Continuous variables were expressed as the mean with the standard deviation or the median with the interquartile range as appropriate. HBV DNA and alanine aminotransferase (ALT) levels were analyzed at the first visit, the peak, the trough, and the last visit (or the visit when HCC was first diagnosed). The peak level was defined as the highest level of HBV DNA and ALT among all the studied visits, and the trough level was defined as the lowest level of HBV DNA and ALT among all the studied visits. Continuous variables were compared by using Student s t test or the Mann-Whitney U test as appropriate, and categorical variables were compared by using the chi-square test. Logistic regression analysis was used to determine the independent effects of HBV DNA and liver cirrhosis on the risk of HCC. The area under the receiver operating characteristic (ROC) curve (with a 95% confidence interval [95% CI]) was used to evaluate the predictive levels of HBV DNA and ALT to diagnose HCC. All statistical tests were two-sided, and s of less than 0.05 were considered statistically significant. RESULTS Clinical characteristics. The age of patients at the time of HCC development in the case cohort was 55 9 years. One hundred two (83%) of the patients were male. Thirteen (32%) case patients and 36 (44%) control patients had experienced antiviral treatment. Twelve HCC patients had received lamivudine treatment, which was changed to/supplemented with adefovir for 4 patients and changed to entecavir for 1 patient, and one HCC patient had received telbivudine treatment. All 36 control patients had received lamivudine treatment, which TABLE 1. Clinical characteristics of patients Variable a HCC (n 41) Non-HCC (n 82) Age of first visit (yr) 52 9 48 9 0.072 Age of last visit (yr) 55 9 55 9 0.52 No. (%) of males 34 (83) 68 (83) 1.00 Cirrhosis present at recruitment 27 (66) 20 (24) 0.001 Initial hemoglobin (g/dl) 14.4 1.8 14.8 1.5 0.24 Initial platelet (10 9 /mm 3 ) 125 74 161 53 0.001 Initial albumin (g/liter) 36 4 40 4 0.001 Initial bilirubin (mmol/liter) 15 12 12 11 0.001 Initial international normalized ratio 1.2 0.4 1.1 0.1 0.001 Creatinine ( mol/liter) 89 21 86 14 0.81 ALT (IU/liter) level at: First visit 63 (55 104) 61 (40 96) 0.126 Last visit 57 (37 121) 38 (25 59) 0.002 Peak 210 (111 379) 152 (84 268) 0.063 Trough 35 (23 44) 26 (20 32) 0.005 HBV DNA (log copies/ml) level at: First visit 6.4 1.4 5.8 1.7 0.059 Last visit 5.5 1.8 3.9 2.0 0.001 Peak 6.9 1.3 6.7 1.7 0.88 Trough 4.8 1.8 2.9 1.2 0.001 Positive for HBeAg b First visit 15 (37) 20 (24) 0.13 Last visit 14 (35) 12 (15) 0.01 HBV genotype C (vs B) c 33 (81) 53 (65) 0.10 Antiviral treatment 13 (32) 36 (44) 0.19 Follow-up duration (mo) 17 (6.5 64.5) 92 (78 99) 0.001 No. of visits 3 (2 7) 9 (8 9) 0.001 a Continuous variables are expressed as means standard deviations or medians (interquartile ranges) as appropriate, except where indicated otherwise. b One patient with HCC was missing HBeAg data at all visits. c Two patients in the control group had negative HBV DNA in all visits, and no genotyping results were available. was changed to/supplemented with adefovir for 12 patients, changed to entecavir for 1 patient, and changed to telbivudine for 1 patient. At the time of recruitment, case patients had a higher proportion of clinical liver cirrhosis and tended to have more advanced liver disease (lower serum albumin, higher serum bilirubin, worse clotting profile, lower platelet count) (Table 1). There were more patients infected by genotype C HBV among patients with HCC than controls but the difference did not reach statistical significance. The median time from diagnosis to HCC was 17 months, and 14 (34%) patients had HCC diagnosed during the first year of follow-up. The median number of serum samples available for the case patients was three, while nine (22%) case patients had only one

1832 CHAN ET AL. J. CLIN. MICROBIOL. FIG. 1. ROC curves of log HBV DNA at different time points for HCC. The area under the ROC curve for the first log HBV DNA (LogDNA_first) is 0.65 (95% CI, 0.50 to 0.71; P 0.059). The area under the ROC curve for the last log HBV DNA (LogDNA_last) is 0.72 (95% CI, 0.62 to 0.81; P 0.0001). The area under the ROC curve for the peak log HBV DNA (LogDNA_peak) is 0.51 (95% CI, 0.41 to 0.61; P 0.88). The area under the ROC curve for the trough log HBV DNA (LogDNA_trough) is 0.79 (95% CI, 0.69 to 0.89; P 0.0001). serum sample for analysis. The control patients had a significantly longer follow-up duration (median, 92 months) and more serum samples (median, nine) for analysis than the case patients. All control patients were followed up for more than 12 months. Relationship of HBV DNA and ALT with HCC. Patients who developed HCC had higher HBV DNA and ALT levels at the trough and the last visit than the control patients (Table 1). The higher HBV DNA at the last visit was accompanied by a higher proportion of HBeAg among patients with HCC. The peak and first HBV DNA and ALT levels had no association with HCC. Comparing the areas under the ROC curves, the trough log HBV DNA level offered the best prediction for HCC (0.79; 95% CI, 0.69 to 0.89; P 0.0001), followed by the last log HBV DNA level (0.72; 95% CI, 0.62 to 0.81; P 0.0001) (Fig. 1). The areas under the ROC curves for the trough ALT level (0.66; 95% CI, 0.55 to 0.76; P 0.005) and the last ALT level (0.68; 95% CI, 0.57 to 0.78; P 0.002) were generally lower than those of the corresponding log HBV DNA levels (Fig. 2). At the trough, more (27 patients; 66%) HCC patients had HBV DNA levels of 4 log copies/ml than control patients (17 patients; 21%) (Table 2). Although the median trough ALT level was within the laboratory limit for both case and control patients, significantly more HCC patients (29 patients; 71%) had serum ALT levels of 0.5 times the upper limit of the laboratory normal than the controls (31 patients; 38%). A similar but less contrasting difference between the two groups was observed for HBV DNA and ALT levels at the last visit. To avoid the potential bias of occult HCC, we performed a sensitivity analysis on HBV DNA by excluding the 14 patients who developed HCC within the initial 12 months of follow-up. In this analysis, the trough log HBV DNA level (4.5 1.9 log FIG. 2. ROC curves of ALT at different time points for HCC. The area under the ROC curve for the first ALT (ALT_first) is 0.59 (95% CI, 0.48 to 0.69; P 0.13). The area under the ROC curve for the last ALT (ALT_last) is 0.67 (95% CI, 0.57 to 0.77; P 0.002). The area under the ROC curve for the peak ALT (ALT_peak) is 0.60 (95% CI, 0.50 to 0.71; P 0.063). The area under the ROC curve for the trough ALT (ALT_trough) is 0.66 (95% CI, 0.55 to 0.76; P 0.005). copies/ml) of HCC patients was still significantly higher than that of the control subjects (2.9 1.2 log copies/ml; P 0.0001). Fifteen of 27 (56%) HCC patients had trough HBV DNA levels of 4 log copies/ml (versus 21% in control patients; P 0.001). The log HBV DNA level at the first visit became statistically significantly higher among the HCC patients (6.7 1.4 log copies/ml) than the control patients (5.8 1.7 log copies/ml; P 0.024), but the peak log HBV DNA levels were still comparable between the two groups (7.3 0.8 log copies/ml in HCC patients versus 6.7 1.7 log copies/ml in control patients; P 0.23). Relationship of HBV DNA and liver cirrhosis. As patients who had HCC tended to have more liver cirrhosis and more advanced liver disease than control patients, we further analyzed the independent effect of the trough HBV DNA level and presence of clinical liver cirrhosis on HCC. On multivariate analysis adjusted for age, gender, and antiviral treatment, the trough log HBV DNA level (odds ratio, 11.4; 95% CI, 3.6 to 37.6; P 0.0001) and presence of liver cirrhosis (odds ratio, 11.4; 95% CI, 3.6 to 36.2; P 0.0001) were independent factors associated with HCC. Patients who had either liver cirrhosis or trough HBV DNA levels of 4 log copies/ml had a moderately increased risk (odds ratio, 14.8 to 20.2), while patients who had both liver cirrhosis and high HBV DNA levels had a markedly increased risk (odds ratio, 201.5) of HCC (Table 3). Effect of antiviral treatment on HCC. The effect of antiviral treatment on the trough HBV DNA levels and HCC is shown in Table 4. Among patients who did not receive any antiviral treatment, HCC patients had significantly higher trough HBV DNA and ALT levels than the control patients. Thirteen HCC patients and 36 control patients had received antiviral treatment for 40 32 months and 58 31 months, respectively (P 0.092). Among patients who had received antiviral treat-

VOL. 47, 2009 SERIAL HBV DNA AND HCC 1833 Visit HBV DNA (log copies/ml) TABLE 2. Categories of HBV DNA and ALT levels with respect to status of HCC a ALT level HCC (n 41) Non-HCC (n 82) (ULN) HCC (n 41) Non-HCC (n 82) First 4 2 (5) 16 (20) 0.47 0.5 0 (0) 9 (11) 0.27 4 to 6 11 (27) 26 (32) 0.5 1 15 (37) 30 (37) 6 28 (68) 40 (49) 1 2 18 (44) 28 (34) 2 5 7 (17) 13 (16) 5 1 (2) 2 (2) Last UD 4 (10) 29 (35) 0.001 0.5 6 (15) 25 (31) 0.014 UD to 4 5 (12) 19 (23) 0.5 1 15 (37) 34 (42) 4 to 6 12 (29) 48 (22) 1 2 8 (20) 17 (21) 6 20 (49) 16 (20) 2 5 11 (27) 6 (7) 5 1 (2) 0 (0) Peak 4 1 (2) 6 (7) 0.25 0.5 0 (0) 2 (2) 0.30 4 to 6 6 (15) 19 (23) 0.5 1 2 (5) 10 (12) 6 34 (83) 57 (70) 1 2 9 (22) 18 (22) 2 5 15 (37) 34 (42) 5 15 (37) 18 (22) Trough UD 8 (20) 46 (56) 0.0001 0.5 12 (29) 52 (63) 0.001 UD to 4 6 (15) 19 (23) 0.5 1 27 (66) 25 (31) 4 to 6 16 (39) 16 (20) 1 2 2 (5) 5 (6) 6 11 (27) 1 (1) 2 5 0 (0) 0 (0) 5 0 (0) 0 (0) a UD, undetectable; ULN, upper limit of laboratory normal. ment, the differences in trough log HBV DNA and ALT levels between the HCC and control patients were less obvious. Numerically, the log HBV DNA was higher among the HCC patients, but the proportions of patients with undetectable HBV DNA were comparable between the two groups. Eight of 13 (62%) HCC patients and 20 of 36 (56%) patients without HCC had undetectable amounts of HBV DNA at the trough level (P 0.71). Among the 41 patients who developed HCC, 13 of them received antiviral treatment and 28 patients did not. Treated patients have significantly lower trough log HBV DNA levels (3.0 1.4 copies/ml) than the untreated patients (5.7 1.4 copies/ml) (P 0.001). However, all (100%) of the treated patients but only 14 (50%) of the untreated patients had liver cirrhosis (P 0.001). Although the duration of follow-up was longer for the treated patients (54 33 months) than the untreated patients (24 31 months; P 0.004), there was no difference in their ages at the time of HCC development (53 7 versus 55 4 years; P 0.40). The male gender ratio (10 patients [77%] versus 24 patients [86%]; P 0.66) and levels of HBV genotype C (12 patients [92%] versus 21 patients [75%]; P 0.40) and first log HBV DNA (6.4 1.5 versus 6.5 1.4 copies/ml; P 0.87) were comparable between the treated and untreated patients. DISCUSSION In this case/control patient study among patients who were prospectively followed up before the development of HCC, we have provided supportive evidence that high HBV DNA and ALT levels were associated with a higher cancer risk. As we have studied the serial HBV DNA levels during the follow-up period and our control patients had a median follow-up of up to 8 years, we were able to demonstrate that the trough HBV DNA level was more important than the peak level or a random time point (such as the first or last visit) measurement to predict HCC. The impact of a high HBV DNA level on HCC was more prominent among patients who have not received any antiviral treatment, as reported by most of the previous studies (10, 11). However, our results have cast a slight doubt on the effectiveness of antiviral treatment to prevent HCC as the difference of the trough HBV DNA levels, which reflected treatment efficacy, between the case and control patients became less obvious. To study the effect of HBV DNA on HCC development, longitudinal studies with HBV DNA measurements well before the development of HCC are preferred. This study design was ideal but difficult due to the low incidence of HCC. The annual incidence of HCC was approximately 500 to 1,000 per Amt of trough HBV DNA TABLE 3. Relationship between trough HBV DNA level and presence of liver cirrhosis and the risk of HCC a No. of cases/ no. of controls With cirrhosis Adjusted odds ratio (95% CI) No. of cases/no. of controls Without cirrhosis Adjusted odds ratio (95% CI) 4 log copies/ml 2/50 Referent 12/29 14.8 (2.8 79.0) 4 log copies/ml 12/26 20.2 (3.8 106.2) 15/18 201.5 (19.4 2,090.0) a Adjusted for age of last visit (continuous variable), gender (male or female), and whether or not the patient was receiving antiviral therapy.

1834 CHAN ET AL. J. CLIN. MICROBIOL. Treatment TABLE 4. Trough HBV DNA and ALT levels with respect to antiviral treatment among patients with and without HCC a HBV DNA (log copies/ml) ALT level (ULN) HCC (n 28) Non-HCC (n 46) HCC (n 28) Non-HCC (n 46) None b UD 0 (0) 19 (41) 0.0001 0.5 5 (18) 30 (65) 0.0001 UD to 4 5 (18) 14 (30) 0.5 1 21 (75) 15 (33) 4 to 6 12 (43) 12 (26) 1 2 2 (7) 1 (2) 6 11 (39) 1 (2) Mean SD 5.7 1.4 3.2 1.3 0.0001 39 14 28 13 0.001 Antiviral c UD 8 (62) 27 (75) 0.25 0.5 7 (54) 22 (61) 0.28 UD to 4 1 (8) 5 (14) 0.5 1 6 (46) 10 (28) 4 to 6 4 (31) 4 (11) 1 2 0 (0) 4 (11) 6 0 (0) 0 (0) Mean SD 3.0 1.4 2.5 0.9 0.38 28 10 30 14 0.81 a UD, undetectable; ULN, upper limit of laboratory normal. b The numbers of patients in the HCC and non-hcc groups were 28 and 46, respectively. c The numbers of patients in the HCC and non-hcc groups were 13 and 36, respectively. 100,000 patients in tertiary referral centers where the prevalence of active hepatitis and liver cirrhosis was much higher than that in the general population (7, 19, 25). In most longitudinal series, a very large number of patients were studied with a long follow-up and yet only a small minority developed HCC (9, 10, 11). As the risk of HCC could be influenced by various host (age, gender), disease (ALT level, cirrhosis, antiviral treatment), and virus (HBeAg, HBV DNA, HBV genotype) factors, it was almost impossible to study the independent effect of HBV DNA at different time intervals in detail. With the low event rate, it would be undesirable to study the entire cohort of patients as the statistical adjustment might overpower the model. To address these challenges, we have decided to study the newly diagnosed HCC patients in prospective databases against simultaneously recruited age- and gender-matched controls. Unfortunately, we were unable to match the liver cirrhosis status, treatment status, and HBV genotype due to the availability of patients in the database. Although the proportion of patients receiving antiviral treatment could not be matched, nearly all treated patients received lamivudine as the initial antiviral treatment, and the importance of the trough HBV DNA level remained significant after adjustment for the treatment factor. We were not able to match the follow-up as all patients were recruited at the same time frame while data for HCC patients were censored at the time of tumor development. In other words, patients with HCC inevitably had a shorter duration of follow-up and fewer clinic visits than the controls. As most Asian patients acquire HBV infection at birth or at a very early age, the matching of the age at the last visit has matched the duration of infection until HCC development. As liver biopsy was not a routine procedure, most patients lacked liver fibrosis scores for adjustment. Our small sample size might be the reason why we could not demonstrate the difference in HCC development between genotype B and C HBV infection (9, 10). In line with previous reports, HBV DNA levels higher than 10,000 copies/ml were found to be associated with an increased risk of HCC in this study (9, 10, 11). Furthermore, a high trough HBV DNA level was most significantly associated with an increased HCC risk. In other words, patients who had intermittent viremia and disease reactivation probably had a lower risk of HCC than others who had persistently active viral replication. The observation that HBV DNA and liver cirrhosis were independent risk factors of HCC with comparable odds ratios supported direct viral mechanisms for HCC in addition to liver necroinflammation and fibrosis (8). On the other hand, the presence of liver cirrhosis is probably secondary to previous hepatic necroinflammation, which is an indirect effect of a high level of HBV replication. One particular point of note was that most HCC patients were HBeAg negative and in their 50s, as in other series. Although patients in these studies have been followed up for up to 10 years, we could at most draw the conclusion that a high HBV DNA level was associated with a higher HCC risk among middle-aged chronic HBV patients. Nonetheless, we believe that it would be inappropriate to extrapolate our findings to young immunotolerant chronic HBV patients who usually are positive for HBeAg and have very high HBV DNA levels (5). ALT is a laboratory parameter commonly used to screen for disease activity of liver disease (17). In HBeAg-negative, chronic HBV patients, elevated ALT levels are usually associated with more active disease progression (2, 26). However, patients with ALT at a high range of reference levels were found to have increased liver-related mortality in a study of 142,055 healthy Korean individuals 35 to 59 years old in an insurance program (16). In an Italian study, lowering the upper limit of the ALT normal standard to 30 IU/liter for men and 19 IU/liter for women could differentiate normal healthy subjects from those suffering from chronic hepatitis C and nonalcoholic fatty liver disease (24). Among a cohort of 1,197 Chinese HBeAg-negative chronic HBV patients, ALT levels higher than 0.5 times the upper limit of the laboratory reference range were associated with an increased risk of liver cirrhosis (27). In a large-scale longitudinal study in Hong Kong, a single ALT level between 0.5 and two times the upper limit of the laboratory reference range was associated with an increased risk of

VOL. 47, 2009 SERIAL HBV DNA AND HCC 1835 liver-related complications among chronic HBV patients in the subsequent follow-up (29). In this study, we have shown that higher ALT levels, especially the trough ALT level, were also associated with increased HCC risk. Also, the risk started to increase when the ALT was greater than 0.5 times the upper limit of the normal laboratory reference range. Therefore, our results supported the need to reevaluate the normal range of ALT levels among patients suffering from chronic HBV. In most treatment guidelines, antiviral treatment was recommended when HBV DNA levels were elevated, particularly when there was clinical evidence of active hepatic necroinflammation and/or liver cirrhosis (15, 21, 23). There was little controversy on the benefit of successful viral suppression for the prevention of hepatic decompensation and progression of liver cirrhosis (18, 20). However, the lowering of HBV DNA levels by antiviral treatment did not seem to effectively prevent the development of HCC. In this study, patients who developed HCC also had a dramatic reduction of HBV DNA, though to a slightly lesser extent than the controls. One possible explanation was the delayed commencement of treatment after hepatocarcinogenesis had been triggered by the virus. In our patients, the mean duration of antiviral treatment prior to HCC development was only 40 months, and it might be too short to prevent HCC development. Another possible reason would be the carcinogenic effect of the integrated HBV in the host genome, which might not respond to viral suppression by antiviral treatment. In summary, we have demonstrated that high levels of HBV DNA at the trough, which reflected a persistently active viremia, were associated with a higher risk of HCC, independent of the presence of liver cirrhosis among middle-aged chronic HBV patients. Whether antiviral treatment commenced at this time of life could effectively prevent the development of HCC was uncertain. Our findings raised a query about the optimal timing of commencement of antiviral treatment. Future studies should address whether antiviral treatment should be started at an earlier age among patients who failed to clear the virus spontaneously before histologic evidence of severe fibrosis or cirrhosis has set in. And we recommend close HCC surveillance for patients at high risk even if they have good response to antiviral treatment. ACKNOWLEDGMENTS This study is supported by a research fund for the Control of Infectious Diseases, Food, and Health Bureau, Hong Kong, Special Administrative Region (application number 06060122) to H.L.-Y.C. H.L.-Y.C. is an advisory board member of Schering-Plough, Bristol- Myers Squibb, Pharmasset, and Novartis Pharmaceutical. REFERENCES 1. Chan, H. L. Y. 2002. Changing scene in hepatitis B serology interpretation. Hosp. 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