To investigate the level of hepatitis B virus (HBV) DNA in Chinese chronic hepatitis B (CHB) patients below which hepatocellular carcinoma (HCC) is unlikely to occur.
Methods A total of 92 CHB patients diagnosed with HCC were recruited; 184 CHB patients without HCC, matched for age, sex and HBeAg status were included as controls. HBV DNA levels were performed at the time of HCC development and at the same age time points for control group.
Results The median HBV DNA level in HCC patients was 1.7 × 106 copies/mL compared with 2.2 × 105 copies/mL in controls (P = 0.006). In HCC patients, 21 (22.8%) were HBeAg(+), with no significant difference in HBV DNA levels compared with controls. Seventy-one (77%) HCC patients were HBeAg(−) with median HBV DNA level of 3.2 × 105 copies/mL, compared with 6.0 × 104 copies/mL in controls (P = 0.006). In HBeAg(−) patients, the control group had significantly greater proportion of patients having HBV DNA levels <105 and <104 copies/mL compared with HCC patients. Fifteen per cent of all HCC patients had HBV DNA levels <103 copies/mL.
Conclusions In HBeAg(+) patients, HBV DNA levels were high in both HCC and control patients. In HBeAg(−) patients, HCC was more likely to develop in patients with HBV DNA level >104 copies/mL. However, 15% of the patients with HCC had HBV DNA levels <103 copies/mL.
An estimated 400 million people worldwide are infected with chronic hepatitis B (CHB), 40% of whom may develop complications of cirrhosis including hepatocellular carcinoma (HCC).1, 2 Several viral factors have been shown to be associated with increased risk of HCC in patients with CHB, including genotype C and core promoter mutations.3–6 One study also showed that the HBeAg(+) CHB was associated with higher risk of HCC. However, in this study, the HBeAg status was only obtained at the time of study entry and not at the time of HCC development.7 Furthermore, the majority of patients who develop HCC are in fact negative for HBeAg with previous seroconversion to antibody against HBeAg (anti-HBe).8
The exact mechanism for the pathogenesis of hepatitis B virus (HBV) in HCC is unknown. It may be partly related to the oncogenic property of HBV, including activation of proto-oncogenes and inhibition of tumour suppressor genes by the HBV X protein.9 It may also be partly related to the development of cirrhosis. Both mechanisms would be dependent on the level of HBV DNA in the patients. Recent studies have indeed shown that higher levels of serum HBV DNA are associated with increased risk of HCC development.10–14 In a study of 3653 CHB patients evaluating the relationship between viral load and HCC (the Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer [REVEAL]-HBV study), the incidence of HCC increased with HBV DNA levels in a dose–response relationship, rising from 108/100 000 person-years in patients with HBV DNA <300 copies/mL to 1152/100 000 person-years in patients with HBV DNA ≥1 000 000 copies/mL.10
However, the majority of these studies assess the HBV DNA levels at the time of enrolment into the studies and not at the time of HCC development. In addition, although the current treatment guidelines include HBV DNA levels as one of the criteria for treatment, the optimal level of HBV DNA suppression to minimize the development of cirrhotic complications and HCC is unknown.15–17 In this study, we aimed to evaluate the serum HBV DNA levels at the time of HCC development, and to determine whether there is a safe level below which the chance of HCC development becomes minimal.
This was a retrospective analysis. All 92 treatment-naïve CHB patients diagnosed with HCC in the Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong, between 1997 and 2006 were recruited into this study. All had HBV DNA levels available at the time of the diagnosis of HCC. A total of 184 treatment-naïve CHB patients, matched for age, sex and HBeAg status, and without the complication of HCC were recruited as controls at a 2:1 ratio. Patients with hepatitis C co-infection, primary biliary cirrhosis, autoimmune hepatitis, significant alcohol intake (>20 g/day) and Wilson’s disease were excluded.
Diagnosis of HCC
The diagnosis of HCC was made using a combination of elevated α-fetoprotein (>400 ng/mL) and typical appearance on at least two radiological imaging techniques, including ultrasonography, computerized tomography, magnetic resonance imaging and hepatic angiography with lipiodol.
HBV DNA measurements
HBV DNA levels were determined at the time of diagnosis of HCC for HCC patients and at the same age time points for the control patients. HBV DNA was measured by the COBAS Taqman HBV DNA assay [lower limit of detection 60 copies/mL (12 IU/mL); Roche Diagnostics, Branchburg, NJ, USA]. In addition, 20 patients had serum samples available for HBV DNA assay at 1 year prior to development of HCC to determine whether there were any significant changes in HBV DNA levels with the development of HCC.
All statistical analyses were performed using the Statistical Program for Social Sciences (spss 14.0; SPSS Inc., Chicago, IL, USA). Categorical variables were analysed using chi-squared test and Fisher’s exact test when appropriate. Continuous variables with skewed distribution were analysed using the Mann–Whitney U-test. The receiver operating characteristic (ROC) curve and the area under ROC (AUROC) curve were used to determine the predictive value of serum HBV DNA in the development of HCC, and also to determine an optimal cut-off level of HBV DNA associated with minimal risk of HCC, with the best possible prediction having an area of 1, while the worst would be 0.5, running along the diagonal reference line. A P-value of ≤0.05 was considered statistically significant.
The baseline demographics of HCC and control patients are shown in Table 1. The median HBV DNA level at the time of HCC development was 1.7 × 106 copies/mL compared with 2.2 × 105 copies/mL in the control group (P = 0.006), as shown in Table 2. Overall, patients with and without HCC who were positive for HBeAg had a significantly higher medium HBV DNA level than those negative for HBeAg (6.5 × 106 vs. 1.3 × 105 copies/mL, respectively, P < 0.001).
|Parameters||HCC patients||Control patients|
|Total HCC patients||92||184|
|Male||69 (75%)||138 (75%)|
|Female||23 (25%)||46 (25%)|
|Age (median) (years)||61 (range, 32–84)||61 (range, 32–84)|
|Positive||21 (23%)||42 (23%)|
|Negative||71 (77%)||142 (77%)|
|Median HBV DNA (copies/mL)||8.6 × 106||5.2 × 106|
|HCC (copies/mL)||Control (copies/mL)||P-value|
|Total||1.7 × 106 (60–6 × 108)||2.2 × 105 (35–8 × 109)||0.006|
|HBeAg(+)||8.6 × 106 (263–5 × 108)||5.2 × 106 (2642–8 × 109)||0.73|
|HBeAg(−)||03.2 × 105 (60–6 × 108)||6.0 × 104 (60–1 × 109)||0.006|
Twenty-one patients (27%) were positive for HBeAg. There was no significant difference in the median HBV DNA level in patients with HCC and in the control group (8.6 × 106 vs. 5.2 × 106 copies/mL, respectively, P = 0.73). There was also no significant difference observed between HCC patients and control patients in the different strata of HBV DNA levels, as shown in Table 3. The AUROC curve for HBV DNA in predicting HCC was not significant, as shown in Figure 1.
|HBV DNA (copies/mL)||Patients with HCC (%)||Controls (%)||P-value|
In the 71 HCC patients negative for HBeAg (77%), the median HBV DNA level of 3.2 × 105 (range, 60–6 × 108) copies/mL was significantly higher than that of the control HBeAg(−) patients [6.0 × 104 (range, 35–1 × 109) copies/mL, P = 0.006]. The comparison between HCC and control patients stratified according to the HBV DNA level is shown in Table 3. The control group had a significantly greater proportion of patients with HBV DNA levels <105 and <104 copies/mL compared with the patients with HCC (36% vs. 23% and 53% vs. 38%, respectively, both P < 0.05). Thirty-eight per cent and 15% of HCC patients had HBV DNA levels <105 and <103 copies/mL respectively. There was no significant difference in the median HBV DNA level in the 20 patients with serum HBV DNA determined 1 year prior to the diagnosis of HCC compared with HBV DNA performed at the time of diagnosis of HCC (9.1 × 105 vs. 1.5 × 106 copies/mL, respectively, P = 0.82). Of these 20 patients, eight (40%) had ≤1 log change in the HBV DNA levels, whereas five (25%) and seven (35%) had a decrease and increase in HBV DNA level of >1 log, respectively. The AUROC curve for HBV DNA in predicting HCC in HBeAg(−) patients was 0.62 (0.53–0.70, 95% CI, P = 0.006) (Figure 1), indicating that HBV DNA levels had poor predictive value for the development of HCC.
Previous studies have shown viral load to be an important and independent risk factor for the development of HCC in patients with CHB.10–14 Long-term follow-up studies have shown that the risk of HCC increases significantly with HBV DNA level >104 copies/mL at the time of recruitment. The question remains whether there is a threshold level of HBV DNA which is not associated with hepatocarcinogenesis and the development of HCC. The REVEAL-HBV study does provide some clues.10 The hazard ratio for the development of HCC in subjects with HBV DNA levels >105 copies/mL both at enrolment and at follow-up was 10.1, compared with subjects whose HBV DNA levels was <104 copies/mL at enrolment. The hazard ratio for subjects whose HBV DNA levels had decreased from >105 copies/mL at enrolment to <104 copies/mL at follow-up was 3.8, a still substantial risk factor. The incidence rates of HCC per 100 000 person-years for subjects with HBV DNA levels at enrolment of 300–9999 copies/mL and <300 copies/mL were 111 and 108, respectively. Thus, even though the emphasis of the REVEAL-HBV study is on the greater relative risk of HCC for subjects with high HBV DNA levels, subjects with HBV DNA levels <104 copies/mL either at enrolment or at follow-up were still at considerable risk for HCC.10 Current guidelines include viral load as one of the criteria for treatment. Identifying a threshold HBV DNA level would be of importance both in selecting patients for treatment and for cessation of treatment.15–17
Our current study assessed the HBV DNA level at the time of HCC development to determine whether there is any safe threshold below which HCC is unlikely to develop. We stratified patients according to their HBeAg status for our analysis. In HBeAg(+) patients, the HBV DNA levels were predictably high in both patients with and without HCC. There were no significant differences between these two groups across different HBV DNA gradients. This may be partly due to the small number of HBeAg(+) patients with low HBV DNA levels. In this present study, 86% of HCC patients and 62% of control patients had HBV DNA levels >106 copies/mL. It remains a possibility that the risk of HCC may not be much different with higher levels of HBV DNA. The overall results in HBeAg(+) patients suggest that HBV DNA levels are likely to be high, and no safe threshold can be defined whereby the HCC risk is minimal.
The majority (77%) of HCC patients were HBeAg(−). These HCC patients had a significantly higher HBV DNA level at the time of HCC development compared with HBeAg(−) patients without HCC. There was also a significant difference across a gradient of HBV DNA level identified, with greater proportion of patients with HBV DNA >104 copies/mL developing HCC. This is consistent with results from the recent large-scale prospective studies.10, 11
We assessed HBV DNA in a subgroup of patients with HBV DNA available 1 year prior to the diagnosis of HCC. There was no significant difference in the level of HBV DNA performed 1 year prior to the diagnosis of HCC and at the time of diagnosis, suggesting that the HBV DNA levels do not change significantly with the development of HCC. However, the number of patients with HBV DNA levels at 1 year prior is small (n = 20) and a single year interval may not be sufficient to exclude changes in HBV DNA levels after the development of HCC. To determine changes in the HBV DNA levels from HBeAg seroconversion to the development of HCC, quantification of HBV DNA over a longer period of time is required.
We used the ROC curve to demonstrate the inherent trade-off between sensitivity and specificity as different HBV DNA levels were taken as cut-off points to predict HCC occurrence. The AUROC curve represents the overall accuracy of the test and ranges between 0 and 1.0, with an area of 0.5 representing test accuracy not better than chance alone. The AUROC for HBV DNA in predicting HCC in HBeAg(+) patients was not significant. In contrast to HBeAg(+) patients, the AUROC was significant in HBeAg(−) patients. However, the AUROC curve at 0.62 was of little predictive value. This means that the level of HBV DNA at any single time point is a poor predictor of HCC development. The non-significant AUROC curve in HBeAg(+) patients and the poor predictive value of the AUROC curve in HBeAg(−) patients would suggest that there is no useful cut-off level for HBV DNA below which HCC risk is minimal.
Fifteen per cent of our patients with HCC had HBV DNA level of <103 copies/mL. This finding is consistent with one of our previous studies showing that 29% of patients with cirrhosis complications, including HCC, had undetectable HBV DNA levels as defined by <200 copies/mL at the time of complications.8 Both studies confirm that there is probably no safe threshold below which HCC is unlikely to occur. The association between occult hepatitis B infection and HCC again suggests that there is unlikely to be a ‘safe’ threshold for HBV DNA levels with respect to HCC development.18, 19 Therefore, we speculate that long-term maximal viral suppression with antiviral therapy is desirable not only in decreasing the chance of drug-resistant mutants from emerging, but also in minimizing the risk of HCC development.
In conclusion, in HBeAg(+) patients, HBV DNA levels were high in both patients with and without HCC. In HBeAg(−) patients, HCC was more likely to develop in patients with HBV DNA >104 copies/mL. However, even low levels of HBV DNA (<103 copies/mL) were associated with HCC development. There was no ‘safe’ HBV DNA level below which HCC is unlikely to occur.
Declaration of personal interests: None. Declaration of funding interests: This study was funded in part by Novartis.