Analysis of high-risk factors for hepatocellular carcinoma after sustained virological suppression of chronic hepatitis B

Abstract

Chronic hepatitis B (CHB) patients who have sustained virological suppression (SVS) still develop hepatocellular carcinoma(HCC), and the risk factors for developing HCC in these patients are not fully understood. This study included a total of 5234 patients who achieved SVS. After SVS, the incidence rate of HCC was 1.9% in 1–8 years and 1.33% in 9–16 years. There was a significant difference between the two periods (P < 0.001). After 1–8 years and 9–16 years of SVS, after multivariate analysis and IPTW adjustment, the factors related to the occurrence of HCC were men, hypertensive patients, diabetes patients and high FIB-4 scores. In summary, patients with CHB who have achieved SVS may still develop HCC. Among them, men, hypertensive patients, diabetes patients and high FIB-4 scores should be listed as the key monitoring objects of HCC.

Clinical trial number: Not applicable.

Introduction

Hepatocellular carcinoma(HCC) is a cancer with a high mortality rate [1]. It is estimated that 72% of HCC occurs in Asia, while China accounts for over 50% [2]. At present, many patients with CHB have sustained virological suppression (SVS) after receiving nucleoside analogues(NA) drugs.Sustained virological suppression (SVS) means that after 24 weeks of taking NA drugs, the serum HBV DNA was continuously lower than the lower limit of the detection value for two times and continuously lower than the lower limit of the detection value (HBV DNA < 500IU/ml), without no virological rebound.The use of NA can reduce the risk of HCC, but the hepatitis B virus can persist in the DNA of liver cells, promoting the occurrence and development of HCC [3]. Therefore, monitoring these patients is necessary, and screening biomarkers or scoring mechanisms that can predict HCC is urgently needed. Our database records 16 years of data on SVR in CHB patients. We hope to find out the risk factors that can predict HCC by analyzing these data.

Materials and methods

Ethical approval

The whole scheme of this retrospective study was approved by the institutional review board of the First Affiliated Hospital of Wenzhou Medical University, and was carried out in accordance with the Declaration of Helsinki.

Study population

From October 2008 to October 2024, a total of 11,150 patients with CHB visited our hospital for treatment. The screening process is shown in Fig. 1.

Fig. 1

Flowchart of the patient selection process. Abbreviations: HBV, hepatitis B virus; CHB, chronic hepatitis B; NA, nucleoside (t) analog; HCC, hepatocellular carcinoma; SVS, sustained virological suspression

The selection criteria include: age ≥ 18 years old; Hepatitis B surface antigen (HBsAg) positive for more than 6 months or defined as CHB based on clinical history; Take NA virus drugs(including lamivudine, tibivudine, entecavir, adefovir dipivoxil, tenofovir) and obtain SVS.

The exclusion criteria include: follow-up time < 1 year; Not achieving sustained virological suppression; Metabolic associated steatohepatitis - related liver disease (MASLD); Merge human immunodeficiency virus, hepatitis C virus, hepatitis D virus or hepatitis E virus infections; Alcoholic liver disease; Autoimmune hepatitis; Decompensated cirrhosis (such as ascites, esophageal or gastric variceal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, hepatorenal syndrome); Occurrence of hepatocellular carcinoma at baseline or within one year of enrollment; History of hepatocellular carcinoma in the past; Liver transplantation; Use interferon needles; Patients without regular 3-month follow-up.

In the end, 5234 patients were enrolled. The information about baseline patient characteristics and clinical outcomes is obtained from complete inpatient and outpatient medical records [4]. These enrolled patients are all patients who have regular follow-up at our hospital. On average, they come to our hospital every 3 months for routine blood tests, HBV-DNA and liver ultrasound contrast/liver enhanced MRI/liver enhanced CT examinations. Regularly monitor for the occurrence of hepatocellular carcinoma.

The diagnostic criteria for MASLD include the patient’s liver ultrasound or CT indicating MASLD, or liver steatosis defined as controlled attenuation parameter (CAP) ≥ 248 dB/m [5].

Cohort 1 included 5234 patients 1–8 years after SVS. Queue 2 included 1196 patients who had SVR for 9–16 years, all of whom were from case queue 1 (Fig. 1).

The first recording time of the patient taking NA and obtaining SVS was the first observation time point. The date of first discovery of HCC or the date of last visit was the last observation time point. The last observation date of this study was Oct 31, 2024. The observation indexes included liver function, blood routine test, coagulation function, renal function, FIB-4 score, APRI score, ALBI score, alpha fetoprotein (AFP), fasting glucose (GLU), hepatitis B virus e antigen (HBeAg). FIB-4 score: Age(year)×AST(U/L)/Platelet count (10^9/L)×√ALT(U/L)]. The APRI (aspartate aminotransfer to platelet ratio index) score can be used for the evaluation of liver cirrhosis, and the calculation formula is: [AST/upper limit of normal, ULN]×100]/platelet count(×10^9/L). Albumin bilirubin (ALBI) calculation formula: (log10 bilirubin µMol/L×0.66)+(Albumin/L×− 0.085). The diagnosis of diabetes (DM) is based on the criteria of the China diabetes Association: (i) random blood glucose ≥ 11.1 umol/L, (ii) Glucose test#Fasting blood sugar ≥ 7.0 umol/L or (iii) use of any hypoglycemic drugs [6]. According to international guidelines, confirm the diagnosis of hepatocellular carcinoma (HCC) using radiology (dynamic computed tomography and/or magnetic resonance imaging) [7]: mainly based on the “fast in and fast out” enhancement method of dynamic enhanced scanning [8].

Serum HBV DNA levels were quantified using the COBAS^® TaqMan HBV test (Roche, Branchburg, NJ, USA), which has a lower detection limit of 500 IU/mL.

Statistical analysis

Continuous variables are represented by the median (Quartile range). The Mann Whitney U test was used for continuous variables. The categorical variable is tested by Chi-squared test or Fisher exact test. We analyzed 13 covariates previously reported as risk factors for HCC development: sex (female or male), age (< 40 or ≥ 40), hypertension (presence or absence), diabetes (presence or absence), body mass index (BMI) (≥ 25 or < 25 kg/m2),γ- GT (≥ 56U/L or < 56U/L), FIB-4 score (≤ 3.25 or > 3.25), APRI score (< 2 or ≥ 2), ALBI score ( < − 2.60 or ≥ − 2.60) eGFR (≥ 60 ml/min/1.73m2 or < 60 ml /min/1.73m2), AFP (≥ 5.0ng/ml or < 5.0ng/ml), HBeAg (present or not present). Actuarial analysis was performed on the cumulative incidence of HCC development using the 1 minus Kaplan Meier method, and potential imbalances between groups were adjusted using treatment weighted inverse probability (IPTW). The probability (propensity) was calculated by multivariate logistic regression analysis of the 13 covariates mentioned above. The statistical significance is defined as p < 0.05. All statistical analyses were conducted using Stata (version 17.0) and R language (R version 4.2.2).

Results

Patient characteristics

5,234 cases met the screening criteria for inclusion in this study, and a total of 573 cases (10.95%) were diagnosed with HCC during the follow-up period. After SVS, the incidence of HCC in 1–8 years was 1.90%, and that in 9–16 years was 1.33%. There was a significant difference in the incidence of HCC between 1 and 8 years and 9–16 years after SVS (p < 0.001) (Fig. 2).

Fig. 2

Cumulative incidence of HCC development after SVS. The annual incidence rate of HCC in 1–8 years after SVS was 1.90%, and 1.33% in 9–16 years. There is a significant difference in the incidence of HCC between 1–8 years and 9–16 years (p < 0.001). Abbreviation: HCC, hepatocellular carcinoma; SVS, sustained virology suppression

In cohort 1, 5,234 patients were divided into HCC group or no HCC group after 1–8 years of SVS. The baseline data of patients not taking anti hepatitis B drugs are shown in Supplementary Table 1. The baseline characteristics of patients are shown in Table 1. Table 1 showed a total of 5,234 patients, of which 573 developed HCC. Patients with diabetes and hypertension were more likely to develop HCC. Patients with higher γ-GT, FIB-4, APRI, and ALBI scores were more likely to develop HCC. In contrast, women and patients with higher platelet counts and creatinine clearance rates were less likely to develop HCC.

Table 1 Baseline characteristics of patients after 1–8 years of SVS (cohort 1)

Table 2 Baseline characteristics of patients after 9–16 years of SVS (cohort 2)

Cohort 2 included 1,196 patients with or without HCC 9 to 16 years after SVS, all of whom were from patients in cohort 1. See Table 2 for the evaluation of characteristics of patients with or without HCC 9 to16 years after SVS. Male, over 40 years old, with diabetes and hypertension, patients with higher γ-GT, FIB-4 scores, ALBI scores, and AFP scores, as well as those with lower platelet counts, were more likely to develop HCC.

Relevant factors between SVR 1–8 years and HCC

Table 3 showed the correlation factors between various indicators of patients and the occurrence of HCC 1–8 years after SVS. In univariate analysis, we analyzed 13 covariates previously reported as risk factors for the development of HCC, and the results showed that except for creatinine clearance rate, the remaining 12 covariates were significantly correlated with the development of HCC.

Table 3 Related factors of HCC occurring 1–8 years after SVS

Table 4 Related factors of HCC occurring 9–16 years after SVS

Figure 3A showed that the cumulative incidence of HCC development after IPTW adjustment for patient sex was 9.29% and 1.09% at 4 years, and 22.83% and 2.62% at 8 years (aHR8.000, 95% CI 5.800–11.000, P < 0.001). Figure 3B showed that the cumulative incidence of HCC development adjusted for IPTW in patients’ age stratification was 6.29% and 1.32% at 4 years, 15.59% and 3.45% at 8 years (aHR 4.500, 95% CI 3.200–6.200, P < 0.001). Figure 3C showed that whether the patient has hypertension, the cumulative incidence of HCC development after IPTW adjustment was 12.83% and 1.71% at 4 years, 34.01% and 4.15% at 8 years (aHR 7.700, 95% CI 6.100–9.700, P < 0.001). Figure 3D showed that if the patient had diabetes, the cumulative incidence of HCC development adjusted by IPTW was 12.59% and 1.76% respectively in four years, and 38.24% and 4.12% respectively in eight years (aHR 8.400,95% CI 6.600–11.000, P < 0.0001). Figure 3E showed that the cumulative incidence of HCC development adjusted for IPTW in patients with FIB-4 score was 13.98% and 1.47% at 4 years, and 35.43% and 3.58% at 8 years (aHR 9.500, 95% CI 7.600–12.000, P < 0.001). Figure 3F showed that the AFP score of patients was stratified, and the cumulative incidence of HCC development after IPTW adjustment was 12.41% and 1.01% at 4 years, 27.59% and 3.3% at 8 years (aHR 6.500, 95% CI 4.700-9.000, P < 0.001).

Fig. 3

Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment. (A) Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment (by sex). (B) Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment(whether the age is greater than 40 years). (C) Cumulative incidence of HCC (hypertension) in 1–8 years of SVS after IPTW adjustment. (D) Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment (diabetes). (E) Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment (stratified according to FIB-4 score). (F) Cumulative incidence of HCC in 1–8 years of SVS after IPTW adjustment (stratified according to AFP). Abbreviation: IPTW, inverse probability of treatment weighting; HCC, hepatocellular carcinoma; SVR, sustained virological suppression; FIB-4, Fibrin-4; AFP, alpha fetoprotein; DM, diabetes; HT, hypertension

Relevant factors between SVR for 9–16 years and HCC

Table 4 showed the relevant factors of HCC in patients with CHB during the 9–16 years of SVS. In univariate analysis, male, age > 40 years old, diabetes, FIB-4 score > 3.25, APRI score ≥ 2, ALBI score≥ -2.60, AFP ≥ 5 were significantly related to the development of HCC.

Figure 4A showed that the cumulative incidence of HCC development adjusted for IPTW in patients’ sex was 7.36% and 0.15% at 12 years, and 9.19% and 0.51% at 16 years (aHR 35.000, 95% CI 12.000-110.000, P < 0.001). Figure 4B showed that the cumulative incidence of HCC development adjusted for IPTW in patient age stratification was 2.09% and 1% at 12 years, 3.45% and 1.93% at 16 years (aHR1.500, 95% CI 0.540-4.000, P = 0.4400). Figure 4C showed that the cumulative incidence of HCC development adjusted for IPTW was 8.33% and 0.74% at 12 years and 17.06% and 0.78% at 16 years, respectively, with or without hypertension (aHR 6.000, 95% CI 2.300–15.000, P = 0.0002). Figure 4D showed that the cumulative incidence of HCC development adjusted by IPTW was 17.06% and 0.78% respectively in 12 years and 17.06% and 1.99% respectively in 16 years (aHR 15.000,95% CI 6.400–37.000, P < 0.0001). Figure 4E showed that the cumulative incidence of HCC development adjusted for IPTW in patients with FIB-4 scores was 13.51% and 0.6% at 12 years, 15.47% and 1.77% at 16 years (aHR 15.000, 95% CI 6.400-35000, P < 0.001). Figure 4F showed that the AFP score of patients was stratified, and the cumulative incidence of HCC development after IPTW adjustment was 10.06% and 0.74% at 12 years, 15.88% and 2.06% at 16 years (aHR 2.900, 95% CI 0.790–11000, P = 0.1100).

Fig. 4

Cumulative incidence of HCC in 9–16 years of SVS after IPTW adjustment

(A) Cumulative incidence of HCC in 9–16 years of SVS after IPTW adjustment (by sex). (B) Cumulative incidence of HCC in 9–16 years of SVS after IPTW adjustment(whether the age is greater than 40 years). (C) Cumulative incidence of HCC in 9–16 years of sustained virology response after IPTW adjustment(hypertension). (D) Cumulative incidence of HCC in 9–16 years of SVS after IPTW adjustment (diabetes).(E)The Cumulative incidence of HCC in 9–16 years of SVS after IPTW adjustment (stratified according to FIB-4 score). (F) The Cumulative incidence ofHCC in 9–16 years of SVS after IPTW adjustment (stratified according to AFP). Abbreviation: IPTW, inverse probability of treatment weighting; HCC, hepatocellular carcinoma; SVS, sustained virological suspression; FIB-4, Fibrin-4; AFP, alpha fetoprotein; DM, diabetes; HT, hypertension

Discussion

This study observed and statistically analyzed 5234 CHB patients who received long-term NA therapy and achieved SVS from October 2008 to October 2024. The results showed that the annual incidence rate of HCC in 9–16 years after SVR was 1.33%, significantly lower than 1.90% in 1–8 years (P < 0.001).

After SVS for 1–8 years, after multifactor analysis and IPTW adjustment, the factors related to the occurrence of HCC were men, older than 40 years, patients with hypertension, diabetes, patients with high FIB-4 score and high AFP score. After SVS for 9–16 years, after multivariate analysis and IPTW adjustment, the factors related to the occurrence of HCC were men, hypertensive patients, diabetes patients and patients with high FIB-4 scores. High AFP scores were significant in multivariate analysis, but after adjustment for IPTW, there was no correlation with the occurrence of HCC.

Men from different ethnic groups and over 40 years old, need to be monitored for HCC [9]. Yip, T. C. et al. analyzed the age, gender, and incidence of HCC in patients with CHB who received medical care at the Hong Kong Hospital Authority from January 2000 to August 2016. The results showed that males were an independent predictive risk factor for HCC [10].

Primary hypertension is associated with an increase in mortality from HCC [11]. The Renin-angiotensin system regulates blood pressure by retaining water and electrolytes, regulating blood volume and perfusion of paraglomerular organs, and also affects tumor behavior by regulating and changing its microenvironment. Angiotensin II can promote tumor progression and proliferation by activating adhesion molecules within the vascular endothelium, stimulating angiogenesis, stimulating tumor growth factors, and remodeling of the parenchyma. Hypertension is a potential risk factor for liver injury and fibrosis through glucose intolerance and the reduction of IL-10 mediated HO-1 induced anti-inflammatory mechanism [12]. Kasmari et al. showed that there is an independent risk association between hypertension and HCC in the absence of cirrhosis [13]. In non cirrhotic patients, there is still an independent risk association between hypertension and HCC after multivariate analysis and IPTW calculation.

Insulin resistance is believed to be the main mechanism triggered by various pathogenic factors such as pro-inflammatory cytokines, chemokines, and immune mediated effects, leading to damage to insulin receptors [14]. Hsu, Y. C. and other researchers developed and validated a risk score, and found that diabetes was a risk determinant for predicting HCC in Asian patients with CHB receiving antiviral treatment [15]. A South Korean study using the National Health Insurance Service Health Screening Queue (NHIS-HEALS) reported that diabetes was associated with a higher risk of HCC [16, 17]. A recent Korean study on 214,167 male patients with hepatitis B virus reported that the risk of HCC was significantly associated with diabetes [18]. In a systematic review and meta-analysis of 36 studies, diabetes was associated with an HR of 1.36 for HCC development in patients with hepatitis B virus [19]. To sum up, diabetes increases the risk of HCC [20]. The use of drugs to treat diabetes, such as Metformin, is associated with a reduced risk of HCC [11].

The FIB-4 score, first proposed by Sterling et al., is a simple and widely accepted non-invasive indicator for evaluating liver fibrosis [21]. Tseng, T. C. et al. followed up a total of 2075 patients with CHB who received treatment for an average of 16.02 years. The results showed that a low FIB-4 index as clinical supplementary data was a favorable predictor for determining patients with the lowest risk of HCC [22]. Goh, M. J., et al. proved that in multivariate analysis, FIB-4 index is an independent risk factor related to the development of HCC in patients with CHB younger than 40 years [23]. NA therapy can reduce the occurrence of HCC, but the occurrence of HCC can still be observed [24]. Cox proportional risk model shows that high FIB-4 index and men are independently related to the incidence rate of HCC [24]. In the above studies, high FIB-4 index has a predictive effect on HCC. These conclusions are consistent with our research results, that is, patients continue to take NA to obtain SVR, but the increase of FIB-4 index still has a certain predictive effect on HCC.

Although this study studied clinical data from 5234 patients for 16 years, as a single center retrospective study, it still had some limitations. Firstly, it was essentially a retrospective study. Secondly, since the information digitization of our institution started around 2007, considering the integrity of the data, we used Cox to analyze the proportional risk model of factors related to the development of HCC within 16 years after the SVR, with an interval of 8 years. Although after 16 years of follow-up, the incidence of HCC in patients who used oral anti hepatitis B drugs and continued to respond was very low, only 40 cases, but the data for 16 years were from the same database, and the positive outcome data from other institutions were not introduced like other studies, thus maintaining the consistency and uniformity of the data. Third, our follow-up period is only 16 years, and we need a longer follow-up period to observe the correlation between the continuous response of virus to hepatitis B patients and HCC. Fourthly, although there are many cases in this study, they all come from the same institution, and the conclusion still needs further confirmation from data from other institutions. Fifthly, there is no data on HBV genotyping in this study. The data for this paper has been recorded since 16 years ago, however, at that time, there was no data on HBV genotyping in our center. Sixth, fibrosis is a non-invasive assessment. However, the paper did not use methods such as elastography or liver biopsy to evaluate liver fibrosis.

In conclusion, in patients with CHB, excluding MASLD, the incidence of HCC in 9–16 years after SVS was 1.33%, significantly lower than that in 1–8 years. However, the risk of HCC cannot be completely eliminated after 9–16 years of SVS. Male, patients with hypertension and diabetes, and patients with high FIB-4 score should be monitored.