Older Age and High Α-fetoprotein Predict Higher Risk of Hepatocellular Carcinoma in Chronic Hepatitis B-Related Cirrhotic Patients Receiving Nucleos(T)ide Analogue Therapy: A Retrospective Cohort Study

Background: Nucleos(t)ide analogues (NUCs) were proved to reduce hepatocellular carcinoma (HCC) development in patients with chronic hepatitis B (CHB) infection, but data was limited on the ecacy in the CHB patients with cirrhosis. Methods: We retrospectively analyzed data from 447 patients with CHB-related cirrhosis, who initiated tenofovir/entecavir therapy during April 2007 and August 2013. They were divided into HCC (n=48) and non-HCC (n=399) groups. The mean follow-up period was 63.2 ± 34.2 months. Results: Forty-eight patients (10.7%) developed HCC during surveillance. The annual incidence rate of HCC was 2.04 (95% CI: 1.52–2.68) per 100 person-year. The cumulative incidence of HCC was 0.9%, 9.8% and 22.1% at the 1, 5 and 10 years, respectively. Signicant predictors for HCC identied using multiple Cox regression analysis were age ≥ 50 years (hazard ratio [HR]: 2.34, 95% condence interval [CI] = 1.08– 5.1) and α-fetoprotein (AFP) ≥ 8 ng/ml (HR: 2.05, 95% CI = 1.1–3.84). The incidence rate of HCC was further analyzed in subgroups according to the risk factors identied by multivariate cox regression. The incidence rate of HCC was 8.67-fold higher in patients with age ≥ 50 years and AFP ≥ 8 ng/ml (3.14 per 100 person-year, 95% CI = 1.99–4.72) than those with age <50 years and AFP <8 ng/ml (0.36 per 100 person-year, 95% CI = 0.06–1.19). Conclusion: The cirrhotic CHB patients with age <50 years and AFP <8 ng/ml have the lowest annual incidence of HCC. However, the cirrhotic patients with age ≥ 50 years or/and AFP ≥ 8 ng/ml have signicantly higher risk for HCC and


Introduction
Hepatocellular carcinoma (HCC), accounts for most of liver cancer, is the sixth most common cancer in the world. It is also the third leading cause of cancer-related mortality, causing more than 800,000 death per year (1,2). Chronic hepatitis B virus (HBV) infection causes global health problem and more than 240 million people have the disease (3). Without treatment, 40% of the chronically infected patients will progress to cirrhosis, which increases the risk of HCC. Chronic HBV infection is also the most common cause of HCC and is associated with more than 50% of HCC cases (4,5). Long-term therapy with nucleos(t)ide analogs (NUCs) has been well demonstrated to result in improvement of liver necroin ammation and brosis, and regression of cirrhosis (6-9). The risk of HCC development was also reported to be reduced signi cantly in the NUCs-treated patients with advanced brosis or cirrhosis. The risk reduction was more prominent in patients with maintained viral suppression than in those with a virological breakthrough (10)(11)(12). Because of low drug resistance rate and high potency of viral suppression, entecavir (ETV) and tenofovir disoproxil fumarate (TDF) have become the rst-line NUC regimen for CHB treatment. Previous studies have shown that antiviral therapy with ETV reduce the risk of HCC in cirrhotic patients, particularly among those with maintained viral suppression. (12). The rate of reduction of HCC incidence was also more in the ETV-treated than those with LAM-treated cirrhotic patients (13). However, the risk of HCC is not completely eliminated by NUCs therapy.
The long-term use of NUCs therapy for cirrhotic CHB patients was reimbursed by the Taiwan's national health insurance system since 2010. There are few studies focusing on assessing the predictors of ontreatment HCC development in CHB-related cirrhotic patients with NUCs therapy. Therefore, this retrospective study was conducted to elucidate the risks and predictors of HCC development during NUCs therapy, and to identify high-risk patients that warrant intensive surveillance during therapy.

Study population
The patients diagnosed with CHB-related cirrhosis and initiated long-term ETV monotherapy (0.5 mg daily) or TDF monotherapy (300 mg) in our liver research unit during April 2007 and August 2013 were enrolled in this study. Chronic HBV infection was de ned as being seropositive for HBsAg for more than 6 months. Baseline clinical and biochemical data were recorded on the initiation of ETV or TDF therapy.
Diagnosis of liver cirrhosis was made by liver biopsy specimen with an Ishak modi ed histology activity index score ≥5 or Metavir score =4, or ultrasonography using the previously described cirrhosis scoring system (14) with splenomegaly or esophageal/gastric varices. All patients had serum HBV DNA ≥2000 IU/ml at baseline. Patients with HCC diagnosed before and within six months after the beginning of therapy or with a follow up duration less than 6 months were excluded from the study. We also excluded CHB patients coinfected with chronic hepatitis C or human immunode ciency virus, toxic hepatitis, autoimmune hepatitis, primary biliary cirrhosis, or Wilson's disease. This study was approved by the Medical Ethics Committee of Chang Gung Memorial Hospital (Institutional Review Board approval number: 104_9790B), and was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Follow-up for HCC surveillance
All patients were observed from the beginning of the NUCs therapy to the date of HCC diagnosis, last-visit or death. Liver ultrasonography and laboratory examination were routinely checked every three months. The diagnosis of HCC was con rmed by the histological evaluation of a needle biopsy sample or surgically resected specimens, two typical image studies such as dynamic liver computed tomography or magnetic resonance imaging, or one image study plus an increased serum AFP level of more than 400 ng/ml (15).

Statistical analysis
The continuous variables were reported as mean ± standard deviation. The categorial variables were summarized as a number (percentage). The difference between continuous and categorical variables was compared using the Student t test and the chi-square test. Cox proportional hazards regression model was used to assess the clinical, biochemical and virological variables associated with the risk of HCC development. The cumulative incidence of HCC was evaluated using the Kaplan−Meier method and compared with log-rank test. All statistical tests were two-tailed, with P values <0.05 considered statistically signi cant. Data were analyzed using SPSS 23 software for Windows (SPSS, Inc, Chicago, IL).

Patient characteristics
There was a total of 457 CHB patients with cirrhosis enrolled in our study. Six patients receiving ETV therapy and 1 patient receiving TDF therapy with a follow up duration less than 6 months, and 3 patients with HCC development within the rst 6 months enrollment were excluded. Finally, 447 patients were included in our nal analysis. They were divided into HCC (n = 48) and non-HCC (n = 399) groups. The baseline clinical and biochemical data of the patients on initiation of the NUC therapy was presented in Table 1. The mean age of the cirrhotic patients at initiation of NUC therapy was 55.3 ± 11.6 years, and 339 (75.8%) patients were men. Patients with HCC development during NUC therapy had lower pretreatment serum aspartate aminotransferase and higher HBV DNA levels. There was also a high percentage of patients exhibited a baseline AFP level ≥ 8 ng/ml.  Fig. 1, the cumulative incidence of HCC was 0.9%, 9.8% and 22.1% at the 1, 5 and 10 years, respectively.
However, different NUCs did not affect the risk of HCC development.
Baseline clinical and biochemical factors identi ed through univariate Cox regression analysis were then entered into stepwise multiple regression analysis.

Incidence of HCC according to risk factors
The analysis of incidence rate of HCC development was then strati ed into subgroup according to the risk factors identi ed by multivariate analysis ( Table 3). As compared to the patients with treatment age < 50 years and AFP < 8 ng/ml (0.36, 95% CI = 0.06-1. 19), the incidence rates of HCC per 100 person-years were signi cantly higher in patients with age ≥ 50 years and AFP ≥ 8 ng/ml (3.14, 95% CI = 1.99-4.72), P = 0.004. The patients with age ≥ 50 years and AFP ≥ 8 ng/ml had 8.67-fold higher rate of HCC than those with age < 50 years and AFP < 8 ng/ml. Among patients with either age ≥ 50 years or AFP ≥ 8 ng/ml, the incidence rates of HCC was also higher than those with age < 50 years and AFP < 8 ng/ml.

Discussion
In the present study, we demonstrated that long-term NUC therapy could not fully eliminate the risk of HCC development in CHB-related cirrhotic patients. Age and AFP were identi ed to be predictors associated with HCC development. From previous studies, the cumulative incidence of HCC at year 5 was 7-18% in NUC-treated cirrhotic patients (12,13,(16)(17)(18). Our data showed similar results that the 5-year cumulative incidence of HCC was 9.8% with an annual incidence of 2.04 per 100 person-year.
The risk factors associated with HCC development in CHB-related cirrhotic patients under NUC therapy, including older age, male gender, HBeAg positivity, statin use, platelet count, AFP and hemoglobin levels, variceal bleeding history, and 1-year virological response, had been reported by previous studies (19)(20)(21).
Our study demonstrated that an older age (≥ 50 years) and AFP ≥ 8 ng/ml were predictors for risk of HCC in those patients with long-term NUCs treatment. Previous reports have indicated that the risk of HCC in cirrhotic patients under NUC therapy was age-dependent (19)(20)(21). Tsai et al. proved that a higher risk for HCC development manifested at age 60 or higher. In our study, we showed that age ≥ 50 years was a predictor for HCC development. Serum AFP was determined as a serological biomarker for detection of HCC; therefore, it has commonly been used for HCC surveillance (22,23). A randomized controlled trial of surveillance among CHB patients has indicated the sensitivity and speci city of AFP level to detect HCC were 64% and 91%, respectively, using a serum cut-off point of 20 ng/ml (24). In our study, the incidence rates of new HCC case per 100 person-year were signi cantly higher in patients with AFP ≥ 8 ng/ml (3.03, 95% CI = 2.07-4.3) than to those with AFP < 8 ng/ml (1.36, 95% CI = 0.84-2.09), P = 0.007. The patients with AFP ≥ 8 ng/ml had a 2.19-fold rate of HCC than those with AFP < 8 ng/ml. For further analysis of the risk for HCC affected by the predictors, we strati ed the patients into four groups according to the predictors identi ed by multivariate analysis. The incidence rate of HCC in patients with age < 50 years and AFP < 8 ng/ml was 0.36 (95% CI = 0.06-1.19) per 100 person-years. It was lower than the pooled rate of HCC incidence rate CHB patients with Child-Turcotte-Pugh A cirrhosis demonstrated by the previous meta-analysis study (25). Although the risk of HCC development in CHB-related cirrhotic patients could not be completely eliminated by long-term NUC therapy, we found this subgroup of patients obtained more bene ts from therapy with signi cant lower rate of HCC than other subgroup of patients. On the contrary, patients with age ≥ 50 years or/and AFP ≥ 8 ng/ml had signi cantly higher risk for HCC, compared with those with age < 50 years and AFP < 8 ng/ml; therefore, these patients should be closely monitored for HCC occurrence during NUC therapy.
Although male patients were determined to possessed a greater risk for HCC in previous study (19), we detected contrasting result that no signi cant differences in HCC risk between sexes in CHB cirrhotic patients. According to a meta-analysis performed by Zhang et al in 2019, antiviral therapy for CHB patients with TDF signi cantly reduced the incidence of HCC compared to those with ETV (26). However, yet other studies have reported contrasting results (27)(28)(29). As shown by Fig. 2E, our data showed similar results that the incidence of HCC in cirrhotic CHB patients didn't affect by different oral anti-viral agent (20,27).
There was limitation of our study. The mean duration of follow-up was 47.4 ± 26.5 months in TDF-treated patients and 67.0 ± 34.8 months in ETV-treated patients, respectively. The duration was relatively shorter in the TDF group and warrant a longer follow-up period in further study to clarify the long-term effect against HCC development.

Conclusions
Long-term NUC therapy could not completely eliminate the risk for HCC development in CHB-related cirrhotic patients. Cirrhotic CHB patients with age < 50 years and AFP < 8 ng/ml have the lowest annual incidence of HCC, which is lower than the pooled incidence of HCC in CHB patients. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.

Funding: Not applicable
Authors' contributions: Ching-Chih Hu, Chih-Lang Lin and Rong-Nan Chien conceived the study. Ching-Chih Hu, Pei-Hung Chang and Chih-Lang Lin contributed to data collection. Ching-Chih Hu, Man-Chin Hua and Cheng-Hao Weng analyzed the data. Ching-Chih Hu, Cheng-Hao Weng and Rong-Nan Chien contributed to writing, reviewing, and revising the paper. All authors interpreted the data and approved the nal manuscript