Post-treatment Mac-2-Binding Protein is a Useful Predictor of Hepatocellular Carcinoma Development after Hepatitis C Virus Eradication


 Background and aims: Recent advances of direct-acting antiviral drugs for hepatitis C virus (HCV) have dramatically improved the sustained virologic response (SVR) rate, but hepatocellular carcinoma (HCC) development not rarely occurs even in patients who achieve an SVR. Wisteria ﬂoribunda agglutinin-positive Mac-2-binding protein (WFA+-M2BP) was recently developed as a noninvasive biomarker of liver fibrosis. However, the association between the WFA+-M2BP level and HCC development after the achievement of an SVR is unclear. Methods: We examined the association between WFA+-M2BP and HCC development in 552 HCV patients who achieved an SVR (Interferon [IFN]-based therapy, n=228; IFN-free therapy, n=294). Results: Multivariate analysis revealed that a high WFA+-M2BP level at SVR week 24 after treatment (SVR24) (hazard ratio [HR]=1.215, P=0.020), low platelet counts (HR=0.876, P=0.037) and old age (HR=1.073, P=0.012) were independent risk factors for HCC development regardless of the treatment regimen. Receiver operator characteristics curve analysis revealed that an WFA+-M2BP level at SVR24 of ≥1.62 cut off index (COI) was the cut-off value for the prediction of HCC development (adjusted HR = 12.565, 95% CI 3.501-45.092, P<0.001). The 3- and 5-year cumulative incidences of HCC were 0.7% and 0.7% in patients with low WFA+-M2BP at SVR24 (<1.62 COI), and 4.8% and 12.4% in patients with high WFA+-M2BP (≥1.62 COI) were, respectively (P<0.001).Conclusion: The assessment of liver fibrosis using the WFA+-M2BP level at SVR24 is a useful predictor of HCC development after HCV eradication even in the IFN-free therapy era.


Introduction
Hepatitis C virus (HCV) infections represent an important global health problem leading to liver cirrhosis and hepatocellular carcinoma (HCC). At present, the World Health Organization estimates that 71 million people are chronically infected with HCV and approximately 400,000 people die every year from the complications of cirrhosis and HCC [1]. In Japan, it is estimated that 30,000 people died of HCC and 65% of all HCC deaths were due to chronic HCV infection [2].
Interferon (IFN)-based therapy, which was the standard treatment for chronic HCV infection until 2011, provided a sustained virologic response (SVR) in only 50% of the patients infected with HCV genotype 1, which was dominant in Japan. In addition, it was poorly tolerated due to adverse events, particularly in elderly patients or those with advanced stage disease. However, recent advances of IFN-free therapy with oral direct-acting antivirals (DAAs) dramatically improved the SVR rates and tolerability, and a large number of HCV patients currently achieved an SVR with this treatment. Previous studies have shown that the eradication of HCV not only reduces the incidence of HCC, but also improves all-cause mortality [3,4]. However, HCC development is not rarely observed even in patients who achieved an SVR. Indeed, the annual incidence of HCC among patients who achieved an SVR with IFN-based therapy ranges from 0.4 to 2% [4][5][6][7][8].
Therefore, it is important to identify the risk factors for HCC development after HCV eradication. Previously, we reported that the pretreatment Wisteria oribunda agglutinin-positive Mac-2-binding protein (WFA + -M2BP) level was a useful predictor of HCC development in patients who achieved an SVR with IFN-based therapy [9]. WFA + -M2BP was originally identi ed as a glycobiomarker of liver brosis, and the serum WFA + -M2BP level is reportedly signi cantly associated with histologically con rmed liver brosis in patients with chronic liver disease [10]. At present, WFA + -M2BP is generally used in Japan as one of the noninvasive biomarkers for the assessment of liver brosis. However, the change in the WFA + -M2BP level after HCV eradication and its association with HCC development after the achievement of an SVR with IFN-free therapy remains uncertain. The aim of this study was thus to determine the impact of WFA + -M2BP on the prediction of HCC development after HCV eradication.

Patients
A total of 522 patients who achieved an SVR with anti-viral therapy between March 2004 and December 2019 were enrolled in this study. All participants met the following inclusion and exclusion criteria: (1) presence of persistent HCV infection; (2) negativity for hepatitis B surface antigen or human immunode ciency virus; (3) negative history of other chronic liver diseases (autoimmune hepatitis, primary biliary cirrhosis, hemochromatosis, and Wilson's disease); (4) absence of HCC or any suspicious lesions detected on ultrasonography, dynamic computed tomography, or magnetic resonance imaging at enrollment; (5) negative history of previous treatment for HCC or liver transplantation; (6) followed-up period for ≥ 6 months after the end of treatment (EOT); and (7) absence of HCC development within 6 months after the EOT. This study's protocol was approved by the Juntendo University Shizuoka Hospital's Ethics Committee, and the study was performed in accordance with the 2013 revision of the Declaration of Helsinki. WFA-M2BP Measurement All routine laboratory data were collected immediately before treatment. The FIB-4 index was calculated as previously described [11]. Serum WFA + -M2BP levels were measured using pre-treatment serum samples stored at − 20°C. WFA + -M2BP quanti cation was performed usung a WFA-antibody immunoassay using a commercially available kit (HISCL M2BPGi; Sysmex Co., Kobe, Japan) and a fully automatic immunoanalyzer (HISCL-5000; Sysmex Co.). A SVR was de ned as negativity for serum HCV RNA at SVR24.

Patient Follow-up
Serum tumor markers and ultrasonography were performed at least once every 6 months during the follow-up period. The negativity of serum HCV-RNA was con rmed annually. HCC diagnosis was con rmed predominantly via imaging studies, including dynamic computed tomography and magnetic resonance imaging. When typical imaging features were absent, a ne-needle aspiration biopsy was performed. The follow-up period was terminated on December 31 2019.

Statistical Analyses
Categorical data were compared using the corrected chi-squared method. Continuous variables were analyzed using the Mann-Whitney U test. Factors associated with HCC development were determined using Cox proportional hazard models, and the HR and 95% CI were calculated. The cumulative incidence of HCC development was determined by the Kaplan-Meier method, and differences were tested using the log-rank test. P < 0.05 was considered statistically signi cant. All statistical analyses were performed using PASW Statistics 18 (IBM SPSS, Chicago, IL, USA).

Patient Characteristics
A total of 522 HCV patients who achieved an SVR were enrolled in this study; the clinical characteristics are summarized in Table 1  There was a greater number of patients with HCV genotype 1 infection, females and elderly patients, and the serum aspartate aminotransferase (AST) level, alanine aminotransferase (ALT) level and platelet counts were lower in the IFN-free therapy group than in the IFN-based therapy group. The WFA + -M2BP levels did not differ between the two groups at baseline and signi cantly decreased at SVR week 24 (SVR24) in both groups compared to baseline (P < 0.001). The albumin and platelet counts were signi cantly increased at SVR24, while the AST, ALT, and alpha-fetoprotein (AFP) levels were signi cantly decreased at SVR24 compared to baseline (P < 0.001). HCC Development After Achievement Of An SVR Among the 522 patients, 14 (3.4%) developed HCC during a median follow-up period of 2.9 years (range, 0.5-13.4 years). The estimated cumulative incidences of HCC development were 1.7% and 3.3% at 3 and 5 years, respectively (Fig. 1). Further, the cumulative incidence of HCC development did not signi cantly differ according to the treatment regimen (Fig. 2). The albumin levels (P = 0.004), and platelet counts (P = 0.002) were signi cantly lower and the AFP levels (P = 0.034), FIB-4 index (P = 0.003), WFA + -M2BP level at baseline (P = 0.002), and WFA + -M2BP level at SVR24 (P < 0.001) were signi cantly higher in those who developed HCC than in those who did not (Table 2).

Discussion
The present study aimed to determine the utility of the post-treatment WFA+-M2BP level in the prediction of HCC development after HCV eradication. Our ndings revealed that age, platelet counts, and the WFA + -M2BP level at SVR24 were useful predictors of HCC development after HCV eradication, regardless of the treatment regimen. Among these factors, both the platelet count and WFA + -M2BP level were previously found to be signi cantly associated with the severity of histological liver brosis [10,12]. In addition, age is a well-known surrogate marker of disease duration and is associated with more advanced brosis [13]. Several previous studies also showed that old age and advanced liver brosis were signi cant risk factors for HCC development [4,6,14,15]. Based on these results, the European Association for the Study of the Liver (EASL) recommends that patients with advanced brosis and cirrhosis who achieve an SVR should undergo surveillance for HCC every 6 months [16]. Our ndings con rmed the clinical importance of assessing the severity of liver brosis in the development of HCC after HCV eradication. Although liver biopsy has been recognized as the gold standard for the assessment of brosis, it can exhibit sampling variability and risk of lethal complications such as liver bleeding. Therefore, noninvasive markers such as the WFA + -M2BP level are important and useful for assessing the severity of liver brosis. We previously reported that the pre-treatment WFA + -M2BP level was a useful predictor of HCC development in patients who achieved an SVR by IFN-based therapy [9]. However, the present study demonstrated that WFA + -M2BP level at SVR24 was more useful in predicting HCC development than pre-treatment WFA + -M2BP level. In our previous report, we showed that the WFA + -M2BP level is affected by necroin ammatory activity in the liver [9]. In this study, the WFA + -M2BP levels signi cantly decreased after HCV eradication.
These results suggest that the WFA + -M2BP level at SVR24 is more useful than the pre-treatment level for assessing liver brosis and predicting HCC development.
Another nding of the present study was that the incidence of HCC development after HCV eradication was comparable between IFN-based therapy and IFN-free therapy. Initially, some studies reported that the incidence of HCC development after HCV eradication was unexpectedly high despite the lack of long-term follow-up [17,18]. However, several recent studies have revealed that the incidence of HCC development did not signi cantly differ between IFN-based therapy and IFN-free therapy, and this phenomenon can be explained by patient characteristics such as age and liver function [19,20].
In our study, there were more elderly patients, females, patients with HCV genotype 1 infection, and patients with low platelet count, who were considered to be resistant to previous IFN-based therapy, in the IFN-free therapy group relative to the IFN-based therapy group. The present study has several limitations. First, the incidence of HCC development was low (9 of 228 patients treated with IFN-based therapy and 5 of 294 patients treated with IFN-free therapy) because our study was retrospectively performed in a single center. Second, the observation period was relatively short in patients treated with IFN-free therapy; the median observation period was only 3 years in the IFN-free therapy group, compared to 5.3 years in the IFN-based therapy group. Third, the patient background characteristics that might affect HCC development differed between patients treated with IFN-based therapy and IFN-free therapy. Therefore, a large-scale prospective study is required to validate our study ndings.
In summary, the incidence of HCC after HCV eradication is comparable between IFN-based therapy and IFN-free therapy. The WFA + -M2BP level at SVR24 is a useful predictor of HCC development after HCV eradication, regardless of the treatment regimen. Our results suggested that it was important to assess liver brosis using the WFA + -M2BP level at SVR24 for prediction of HCC development after achieving an SVR.