Role of hepatitis D virus infection in development of hepatocellular carcinoma among chronic hepatitis B patients treated with nucleotide/nucleoside analogues

Hepatitis D virus (HDV) infection increases the risk of hepatocellular carcinoma (HCC) in the natural course of chronic hepatitis B (CHB) patients. Its role in patients treated with nucleotide/nucleoside analogues (NAs) is unclear. We aimed to study the role of hepatitis D in the development of HCC in CHB patients treated with NAs. Altogether, 1349 CHB patients treated with NAs were tested for anti-HDV antibody and RNA. The incidence and risk factors of HCC development were analyzed. Rates of anti-HDV and HDV RNA positivity were 2.3% and 1.0%, respectively. The annual incidence of HCC was 1.4 per 100 person-years after a follow-up period of over 5409.5 person-years. The strongest factor association with HCC development was liver cirrhosis (hazard ratio [HR]/95% confidence interval [CI] 9.98/5.11–19.46, P < 0.001), followed by HDV RNA positivity (HR/ CI 5.73/1.35–24.29, P = 0.02), age > 50 years old (HR/CI 3.64/2.03–6.54, P < 0.001), male gender (HR/CI 2.69/1.29–5.60, P: 0.01), and body mass index (BMI, HR/CI 1.11/1.03–1.18, P = 0.004). The 5-year cumulative incidence of HCC was 7.3% for patients with HDV RNA negativity compared to that of 22.2% for patients with HDV RNA positivity (P = 0.01). In the subgroup of cirrhotic patients, the factors associated with HCC development were HDV RNA positivity (HR/CI 4.45/1.04–19.09, P = 0.04) and BMI (HR/CI 1.11/1.03–1.19, P = 0.01). HDV viremia played a crucial role in HCC development in CHB patients who underwent NA therapy.

www.nature.com/scientificreports/ been the most crucial determinant of HCC. The incidence of HCC increases significantly after the development of liver cirrhosis. The incidence of HCC has been reported to be 0.2 per 100 person-years in inactive carriers, 0.6 per 100 person-years in non-cirrhotic patients, and 3.7 per 100 person-years in patients with compensated cirrhosis 4 . However, antiviral therapy including nucleotide/nucleoside analogues (NAs) is reported to result in an improvement in hepatic inflammation and fibrosis, which in turn reduces the risk of HCC development and mortality 5,6 . Hepatitis D virus (HDV) is a single-stranded RNA virus and its propagation depends on HBV 7 . The worldwide prevalence of HDV ranges from < 1% to > 50% 8 . Among chronic hepatitis B (CHB) patients, the hepatitis D antibody (anti-HDV) positivity rate is reported to be 2-5% in the general population 9,10 and up to 68.9% in persons who inject drugs in Taiwan 11 . Chronic hepatitis D (CHD) infection may result in a deterioration of liver function and may also lead to a threefold increase in the risk of HCC in the typical course of the disease 12,13 . Notably, the impact of HDV on HCC occurrence in CHB patients treated with NAs remains unclear. Hence, we aimed to address this issue by enrolling a well-characterized CHB cohort treated with NAs. HDV serology and virology in this cohort were also studied and considered for the evaluation of its impact on HCC development.

Methods
Patients. CHB patients allocated to NAs were consecutively enrolled in a medical center in Taiwan from June 2000 to July 2018 in the retrospective study. Patients were followed up from the beginning of NA treatment. The treatment indications for NAs were based on the national health insurance reimbursement regulations of the Ministry of Health and Welfare in Taiwan 2,14 , Briefly, the criteria included (1) HBeAg-negative patients with HBV DNA greater than 2000 IU/mL with persistent alanine aminotransferase abnormality greater than two times the upper limit of normal (ULN) on two occasions 3 months apart, and (2) HBeAg-positive patients with HBV DNA greater than 20,000 IU/mL with ALT abnormality greater than two times the ULN for two occasions 3 months apart, or with ALT > 5 times the ULN, (3) cirrhotic patients with HBV DNA greater than 2000 IU/ mL, and (4) liver decompensation, denoted as prothrombin time prolongs > 3 s or a total bilirubin > 2 mg/ dL 2 . Patients were excluded if they had any of the following conditions: alcoholism (≥ 20 g daily), coinfection with human immunodeficiency virus or hepatitis C virus (HCV), ongoing interferon-based therapy, pre-existing HCC before the use of NAs, and the use of NAs for chemotherapy prophylaxis. The study was conducted in accordance with the principles of the Declaration of Helsinki of 1975, as revised in 2008. The ethics committee of Kaohsiung Medical University Hospital approved the study. All patients provided informed consent before enrollment.
Laboratory analyses. Biochemical analyses were performed using a multichannel autoanalyzer (Hitachi Inc., Tokyo, Japan). Hepatitis B surface antigen (HBsAg) was detected using standard quantitative chemiluminescent microparticle immunoassay (ARCHITECT HBsAg, Abbott Diagnostics, Chicago, IL, USA). Serum HBV DNA was examined using a standardized, automated quantitative polymerase chain reaction assay (COBAS TaqMan HBV test, Roche Diagnostics, Branchburg, NJ; detection limit 12 IU/mL) 15 . Anti-HDV antibody was verified at the initiation of treatment with anti-HBV agents. Screening for anti-HDV immunoglobulin G was performed using the anti-HDV enzyme-linked immunosorbent assay kit (General Biologicals Corporation, Hsinchu, Taiwan) 7,10 . HDV RNA was tested in patients with anti-HDV positivity using the LightMix Kit HDV (Roche Life Science, Berlin, Germany) on the Roche LightCycler (Roche Life Science, Berlin, Germany; detection limit: 10 copies/mL) 16 . The fibrosis-4 index was calculated by using the following formula: age (years) × aspartate aminotransferase [U/L]/platelets [10 9 /L] × alanine transaminase [U/L]) 1/2 . Liver cirrhosis was diagnosed using transient elastography (FibroScan; Echosens, Paris, France; >12 kPa) 17 , histology, or the presence of laboratory, radiological, endoscopic, or clinical evidence of portal hypertension and/or cirrhosis. HCC was confirmed by clinical or histological diagnosis according to the guidelines of the American Association for the Study of Liver Diseases 18 and the Asian Pacific Association for the Study of the Liver 19 . Patients visited the clinics every 1 to 3 months depending on the physician's discretion. Laboratory tests, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alpha-fetoprotein (AFP), and hepatitis B virus serology/virology were assessed every 3 to 6 months if the patient's condition was stable. HCC surveillance by abdominal sonography was performed every 3 to 6 months based on the severity of the liver disease.

Statistical analyses.
Frequencies were analyzed between the groups using the chi-square (χ 2 ) test with Yates correction or Fisher's exact test. Data are presented as mean ± standard deviation. Data were compared using analysis of variance, Student's t-test, or the nonparametric Mann-Whitney U test. A stepwise logistic regression analysis was applied to analyze the characteristics associated with anti-HDV and HDV RNA positivity by analyzing the covariates with P values < 0.1 in the univariate analysis. Kaplan-Meier analysis and a log-rank test was performed for the comparison of the cumulative incidence of HCC with respect to various determinants. Cox regression analysis was applied to analyze the factors independently associated with HCC development by analyzing the covariates with P-values < 0.2 in the univariate analysis or the factors considered to have potential and clinical relevance. Statistical analyses were performed using IBM SPSS Statistics, version 20 (IBM Corp., Armonk, NY, USA). All statistical analyses were based on two-sided hypothesis tests with statistical significance set at P < 0.05.

Results
Patient characteristics. Altogether, 2500 CHB patients who received NAs were initially recruited. After excluding patients with pre-existing HCC (n = 343), patients using NAs for chemotherapy prophylaxis (n = 771), patients with anti-HCV positivity (n = 27), and patients receiving interferon-based therapy (n = 10), 1349 www.nature.com/scientificreports/ patients were enrolled for analysis ( Fig. 1). The mean age was 48.0 years and 72.4% of the patients were male. The mean HBV DNA level was 5.9 log 10 IU/mL. Patients with liver cirrhosis accounted for 29.1% (n = 392) of the study population. The rates of anti-HDV and HDV RNA positivity were 2.3% and 1.0%, respectively. The most commonly used NAs were entecavir (45.2%) and tenofovir disoproxil fumarate (12.2%) ( Table 1).

Discussion
In the present study, we observed that the annual incidence of HCC was 1.4% in CHB patients treated with NAs. As expected, liver cirrhosis was the most important risk factor associated with HCC occurrence. We demonstrated that coinfection with HDV also played an essential role in HCC development, although the prevalence was low in Taiwanese patients. Compared to patients without concurrent HDV infection, those with HDV viremia had a nearly sixfold risk of HCC development.
HBV infection induces hepatic inflammation, which leads to liver cirrhosis and HCC. The HBV DNA level has a dose-dependent association with HCC development in the typical course of the disease 20 . The other risk factors of HBV-related HCC include liver cirrhosis, age, gender, and metabolic factors 21,22 . Several models have assessed the risk of HCC in CHB patients without antiviral therapy 23,24 . Moreover, emerging evidence has shown that HBV DNA suppression with NAs 25,26 or interferon 27,28 significantly reduced the HCC risk when compared with untreated controls. Currently, NAs are the mainstream agents for CHB therapy. They have reduced the HCC risk by 34-78% according to various studies 29 . However, HCC continues to develop under viral suppression. Some HCC prediction models have also been created in patients undergoing treatment with NAs 30,31 and the pretreatment viral loads were not found to be as crucial as those in NA-naïve patients. Underlying liver cirrhosis was reported to be the most critical determinant of HCC 32 , which is consistent with the results of the present study. The reported annual incidence of HCC was 0.3-1.2% in non-cirrhotic CHB patients and 1.8-6.0% in cirrhotic CHB patients undergoing NA therapy 33 . We observed a similar incidence of HCC in our patient cohort, with an annual incidence of 0.4% in non-cirrhotic patients and 4.6% in cirrhotic patients. Continuous surveillance of HCC remains mandatory in CHB patients receiving antiviral therapy, and it is crucial in cirrhotic patients. www.nature.com/scientificreports/ The prevalence of HDV in the general population is low. It has been decreasing in Taiwan in recent decades 9,10 . The seroprevalence of anti-HDV and HDV RNA was 2.3% and 1%, respectively, in the present study, which is similar to that observed in a recent report in Taiwan 9 . HDV would increase the risk of HCC in the typical course of the disease 13,22 . However, debates continue regarding this issue 9,34 . There is no potent treatment against HDV infection. To date, interferon has been the standard of care 35 and NAs are not effective in treating CHD 36 . However, NAs remain the mainstay for managing CHB. The impact of HDV on HCC has rarely been elucidated in NA-treated cohorts. Brancaccio et al. conducted a case-control study that disclosed that despite long-term NAs use, an increased risk of HCC remained in HDV-infected patients compared to those with HBV mono-infection 37 . In the current long-term observational study with a larger Asian cohort, we consistently demonstrated that HDV viremia increases the risk of HCC. This finding was also in line with the results from another recent study 38 . The aforementioned study by Kamal et al. suggested that HDV viremia might increase the incidence of liver-related events such as HCC, hepatic decompensation, and death. Notably, the majority of the patients were treated with interferon, while only a few patients received NAs 38 . In the present NA-based cohort study, we observed that one-fifth of the patients with HDV viremia had developed HCC at the 5-year follow-up. Moreover, 50% of the HDV-RNA positive cirrhotic patients eventually developed HCC. Currently, there is no potent antiviral agent to treat HDV infection. Given the high risk of HCC development, we suggest that each HDV-infected subject should be monitored regularly and closely until effective or curative anti-HDV agents can be prescribed in the clinical setting.
The present study has some limitations. Due to the low prevalence of HDV infection in the region where the study was conducted, the retrospective cohort was limited by relatively few index patients with HDV infection, particularly in non-cirrhotic subjects. This may have restricted the cumulative clinical impact of HDV on HCC.
As with the majority of other studies, the current study aimed to identify baseline factors including HDV status rather than the sequential parameters predictive of HCC. We failed to provide information regarding cirrhotic development or resolution at the time of event occurrence or patient censoring. In addition, the follow-up duration was more condensed in HDV-infected patients. Although the follow-up duration was condensed in the HDV group, the HCC incidence was significantly higher than that in HBV mono-infected patients during the relatively brief observation period. The difference in HCC incidence between the groups was postulated to be more pronounced if the follow-up duration was prolonged in the HDV group.
In conclusion, the results of the present study suggest that HDV viremia plays an essential role in HCC development in CHB patients treated with NAs. HDV testing should be proposed for each HBV subject even when HBV replication is under control by NAs. We hope that these results will promote further validated studies, particularly in regions where HDV infection is widespread.