In the current study, the risk of incident HCC in 2403 CHB patients was significantly increased with decreasing amount of hepatic steatosis and increasing burden of fibrosis. Every 10 dB/m decrease in CAP was associated with 6% increase in HCC risk. The 4-year cumulative HCC risk was 2.88%, 1.56% and 0.71% for patients with no steatosis, mild-to-moderate steatosis, and severe steatosis, respectively. The other independent risk factors for HCC in this study included older age, male gender, and higher ALBI score, which are well-reported and consistent with the literature. This prospective study involved a large cohort of CHB patients, and has been adjusted for the underlying metabolic risk factors including obesity, central obesity, hypertension and diabetes mellitus. The use of transient elastography to quantify hepatic steatosis and liver fibrosis aided the demonstration of the significant inverse relationship between steatosis and HCC development, which was synergistic with liver fibrosis.
Animal studies showed that viral antigen expression and HBV DNA levels were decreased in mice with concomitant NAFLD and HBV compared to HBV alone.25, 26 For clinical studies, our group previously showed that HBV viral load was inversely associated with hepatic steatosis,11 and suggests a possible inhibitory effect of hepatic steatosis on HBV viral replication. Similarly, in another paper, we found that hepatic steatosis was associated with lower quantitative HBsAg levels and higher chance of HBsAg seroclearance, although severe hepatic steatosis was associated with advanced fibrosis or fibrosis progression.14, 27 In the current study, advanced fibrosis/ cirrhosis is found to synergistically agonize the risk of HCC in patients without severe steatosis (i.e. group 4 - see text in the Results section), which suggests that there is complex interaction between hepatic steatosis and fibrosis in CHB patients. The correlation between CAP and HBV DNA was − 0.065 (p = 0.002). In patients with CAP ≥ 248 dB/m (i.e. any degree of hepatic steatosis), the proportion of serum HBV DNA detectability was 540/1156 (46.1%) vs. 564/1247 (45.2%) for patients without hepatic steatosis; p = 0.486. For the lower HBV DNA load in patients with HCC, it is likely to be due to the fact that 91.7% of them were on antiviral therapy compared to 56.4% for those without HCC (Supplementary table 2). For patients on NA, the proportion of HBV DNA detectability was 217/1372 (15.8%) vs 887/1031 (86%) for patients not on NA (p < 0.001). Taken together, the very weak negative association between CAP and HBV DNA could not fully explain the apparent protective effect of hepatic steatosis on HCC. However, serum HBV DNA is just one of the many viral biomarkers that could be evaluated, and it remains unknown whether the presence of hepatic steatosis inhibits other HBV-related activities including upstream transcriptional activity and DNA integration into the host genome. It is possible that in NA-treated patients with suppressed reverse transcriptase activity, the residual viral replication (including transcription, translation and eventually production of oncogenic proteins) is inhibited by the presence of hepatic steatosis. The exact step of viral replication affected by hepatic steatosis is unknown. In a study involving histological assessment of patients having concomitant CHB and NAFLD, viral antigens staining for HBsAg and HBV core antigen (HBcAg) in hepatocytes were lower compared to patients with CHB alone.28 Theoretically, translation for viral proteins or any upstream steps of viral lifecycle could be affected by hepatic steatosis, leading to reduced production of oncogenic products as well as DNA integration (Supplementary Fig. 4). This hypothesis would need further mechanistic studies to explore.
On the other hand, it is well known that patients with burnt-out non-alcoholic steatohepatitis (NASH) do not have excess hepatic fat any more.29, 30 Viewing from this perspective, reduction in CAP might signify building up of liver fibrosis, i.e. ‘burnt-out NASH’ which provides another explanation for the negative association between hepatic fat and HCC development observed in the current study (Supplementary Fig. 4).
Although liver biopsy was not performed for most patients, liver histology obtained at hepatic resection or liver transplantation around the time of HCC from 22 patients was studied, which showed that 5/22 (22.7%) and 10/22 (45.4%) patients had hepatic steatosis and advanced fibrosis/ cirrhosis, respectively, on liver histology. This highlighted that hepatic steatosis was retained in these patients even when HCC was formed. Moreover, the sensitivity of detection of hepatic steatosis and advanced fibrosis/ cirrhosis by transient elastography technique was 80% and 70%, respectively. It has been reported that liver stiffness could be influenced by many factors, including ALT,31 CAP,32 cholestasis,33 hepatic congestion,34, 35 and probe type.36 We minimized the confounding effect of ALT by adopting a different liver stiffness cut-off as per recommendation of the EASL-ALEH guidelines and using appropriate probes for patients with different BMI range (see Methods section). Moreover, the majority of patients in this study had BMI < 30 and therefore M probe was used (Table 1). While one report (n = 82) mentioned that CAP could be influenced by significant fibrosis in patients with NAFLD,37 a bigger study involving 450 patients with NAFLD showed that probe type and steatosis did not affect LSM.38 Although residual confounding between LSM and CAP could not be excluded, the independent effect of hepatic steatosis on HCC has been further elucidated by PSM analysis. After matching of age, gender, liver stiffness, platelet, HBV DNA, albumin, bilirubin, AST as continuous variables and antiviral treatment, hepatic steatosis was independent associated with reduced risk of HCC, with a hazard ratio of 0.41 (95% CI 0.21–0.83).
There are two limitations of our study. Firstly, liver biopsies were not done for most patients to assess the histological steatosis, NASH activity and actual fibrosis stage. However, liver biopsy is an invasive procedure, and is not feasible to be performed for a large cohort of patients, vast majority being stable and asymptomatic, due to the associated risks of the procedure. Transient elastography demonstrates excellent performance in diagnosing advanced fibrosis and hepatic steatosis with high accuracy with references to histological findings,39–41 which was similarly observed in the histology in 22 HCC patients around the time of HCC diagnosis. In addition, information on genotypes, known viral mutations (e.g. core promoter mutations)42 and family history of HCC were not available in the current study.
In conclusion, our study found that decreasing quantity of hepatic steatosis, as measured by CAP, and increasing burden of liver fibrosis, as measured by liver stiffness, were significantly and independently associated with a higher risk of incident HCC among CHB patients. Our present study findings highlight the importance of routine liver stiffness and CAP measurements in the risk stratification and monitoring of CHB patients.