Post-treatment serum Wisteria floribunda agglutinin-positive mac-2-binding protein level is a useful predictor of hepatocellular carcinoma development after hepatitis C virus eradication.

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 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 AND
RESULTS: We examined the association between WFA+-M2BP and HCC development in 522 HCV patients who achieved an SVR (Interferon [IFN]-based therapy, n = 228; IFN-free therapy, n = 294). 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 a 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 1% and 1.6% in patients with low WFA+-M2BP at SVR24 (<1.62 COI), and 4.7% and 12.5% in patients with high WFA+-M2BP (≥1.62 COI) were, respectively (P < 0.001).
CONCLUSIONS: 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. In Japan, it is estimated that 30 000 people died of HCC and 65% of all HCC deaths were due to chronic HCV infection.

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., 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%., , , , Therefore, it is important to identify the risk factors for HCC development after HCV eradication. Previously, we reported that the pretreatment Wisteria floribunda 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. WFA+‐M2BP was originally identified as a glycobiomarker of liver fibrosis, and the serum WFA+‐M2BP level is reportedly significantly associated with histologically confirmed liver fibrosis in patients with chronic liver disease. At present, WFA+‐M2BP is generally used in Japan as one of the noninvasive biomarkers for the assessment of liver fibrosis. 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.

Methods

Patients

Between March 2004 and March 2018, a total of 1176 patients (IFN‐based therapy, n = 697; IFN‐free therapy, n = 479) received antiviral therapy at the Juntendo University Shizuoka Hospital. SVR was defined as undetectable serum HCV‐RNA at 24 weeks post‐treatment (SVR24). Among the 697 patients treated with IFN‐based therapy and 479 patients treated with IFN‐free therapy, 433 (62.1%) and 452 patients (94.4%), respectively, achieved SVR24. All participants met the following inclusion criteria: (i) a presence of persistent HCV infection and (ii) a follow‐up period of ≥6 months after achieving SVR24. The exclusion criteria are as follows: (i) testing positive for hepatitis B surface antigen or human immunodeficiency virus; (ii) presence of HCC or any suspicious lesions detected by ultrasonography, dynamic computed tomography, or magnetic resonance imaging with reference to tumor markers at enrollment; (iii) history of other chronic liver diseases, such as autoimmune hepatitis, primary biliary cirrhosis, hemochromatosis, and Wilson's disease); (iv) history of previous treatment for HCC or liver transplantation; and (v) HCC development within 1 year after the end‐of‐treatment (EOT). Thus, 45 patients who had a history of previous treatment for HCC, 13 patients who had developed HCC within 1 year after EOT, and 305 patients who were followed up for less than 6 months after achieving SVR24 were excluded. Finally, the data of 552 patients were retrospectively analyzed.

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 including the WFA+‐M2BP level were measured immediately before treatment and after achieving SVR24. The FIB‐4 index was calculated as described previously. The serum level of WFA+‐M2BP measured with a WFA‐antibody immunoassay using a commercially available kit (HISCL M2BPGi; Sysmex Co., Kobe, Japan) and a fully automatic immunoanalyzer (HISCL‐5000; Sysmex Co.).

Patient follow‐up

Serum tumor markers such as alpha‐fetoprotein (AFP) and des‐ɤ‐carboxy prothrombin, and ultrasonography were performed at least once every 6 months during the follow‐up period. The negativity of serum HCV‐RNA was confirmed annually. HCC diagnosis was confirmed predominantly via imaging studies, including dynamic computed tomography and magnetic resonance imaging. When typical imaging features were absent, a fine‐needle aspiration biopsy was performed. The follow‐up period was terminated on 31 December 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. The annual incidence of HCC development after achieving SVR24 was assessed using the person‐years method. P < 0.05 was considered statistically significant. All statistical analyses were performed using PASW Statistics 18 (IBM SPSS, Chicago, IL, USA).

Results

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. Among the 228 patients treated with IFN‐based therapy, 172 were treated with pegylated‐IFN plus ribavirin combination therapy for 24–72 weeks, while 56 were treated with pegylated‐IFN plus ribavirin combined with an NS3/4 protease inhibitor: 25 with telaprevir, 22 with simeprevir, 9 with faldaprevir. Among the 294 patients treated with IFN‐free DAA therapy, 53 were treated with daclatasvir/asunaprevir, 111 with ledipasvir/sofosbuvir, 99 with sofosbuvir/ribavirin, 10 with elbasvir/grazoprevir, 3 with ombitasvir/paritaprevir/ritonavir, and 18 with glecaprevir/pibrentasvir; a total of 87 (29.6%) patients had a history of previous IFN‐based therapy.

Table 1

Patient characteristics according to treatment regimen

Variables	All (n = 522)	IFN‐based therapy (n = 228)	IFN‐free therapy (n = 294)	P‐value
Age (years)†	62 (20–88)	58 (20–85)	67 (25–88)	<0.001‡
Sex (male/female)	278/244	137/91	141/153	0.004§
Genotype 1/2	292/230	111/117	181/113	0.002§
HCV‐RNA (logIU/L)†	6.3 (1.2–7.8)	6.2 (1.2–7.8)	6.4 (3.1–7.8)	0.025‡
At baseline
Albumin (g/dL)†	4.2 (2.9–5.2)	4.2 (3.3–4.8)	4.2 (2.9–5.2)	0.690‡
Total bilirubin (mg/dL)†	0.7 (0.3–2.9)	0.7 (0.3–2.1)	0.7 (0.3–2.9)	0.257‡
AST (IU/L)†	39 (10–499)	42 (13–499)	36 (10–281)	0.005‡
ALT (IU/L)†	42 (9–1071)	51 (11–1071)	35 (9–366)	<0.001‡
Platelet counts (×104/μL)†	16.9 (0.5–38.3)	17.6 (4.3–38.3)	16.1 (0.5–36.7)	0.019‡
AFP (ng/mL)†	4 (1–459)	5 (1–358)	4 (1–459)	0.004‡
FIB‐4 index†	2.46 (0.30–33.42)	2.24 (0.31–16.22)	2.68 (0.30–33.42)	<0.001‡
WFA+‐M2BP (COI)†	1.64 (0.20–19.81)	1.63 (0.24–18.11)	1.65 (0.20–19.81)	0.948‡
At SVR24
Albumin (g/dL)†	4.3 (3.2–6.6)	4.3 (3.2–5.5)	4.3 (3.2–6.6)	0.376‡
AST (IU/L)†	22 (4–111)	21 (11–111)	23 (4–110)	0.404‡
ALT (IU/L)†	16 (2–146)	17 (5–146)	15 (2–95)	0.027‡
Platelet counts (×104/μL)†	17.3 (0.2–42.5)	17.3 (5.5–39.0)	17.3 (0.2–42.5)	0.592‡
AFP (ng/mL)‡	3 (1–27)	3 (1–27)	3 (1–17)	0.095‡
FIB‐4 index†	2.10 (0.3–111.86)	1.81 (0.33–7.97)	2.26 (0.38–111.86)	<0.001‡
WFA+‐M2BP (COI)†	0.89 (0.13–16.65)	0.81 (0.13–7.08)	0.96 (0.17–16.65)	0.004‡

Data are expressed as the median (range),

Mann–Whitney U‐test.

Chi‐squared test.

AFP, alpha‐fetoprotein; ALT alanine aminotransferase; AST, aspartate aminotransferase, COI, cut‐off index; FIB‐4, fibrosis‐4; HCV, hepatitis C virus; IFN, interferon; SVR, sustained virologic response; WFA+‐M2BP, W. floribunda agglutinin‐positive Mac‐2‐binding protein.

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 significantly decreased at SVR week 24 (SVR24) in both groups compared to baseline (P < 0.001). The albumin and platelet counts were significantly increased at SVR24, while the AST, ALT, and AFP levels were significantly decreased at SVR24 compared to baseline (P < 0.001).

HCC development after achieving SVR

The median follow‐up period after achieving SVR24 was 2.9 years (range, 0.5–13.4 years), constituting a total of 1794.5 person‐years. During the follow‐up period, 14 patients (0.78% per 1 person‐year) developed HCC. The estimated cumulative incidences of HCC development were 1.7 and 3.3% at 3 and 5 years, respectively (Fig. 1a). Further, the cumulative incidence of HCC development did not significantly differ according to the treatment regimen (Fig. 1b). The albumin levels (P = 0.004), and platelet counts (P = 0.002) were significantly 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 significantly higher in those who developed HCC than in those who did not (Table 2).

Figure 1

Cumulative incidence of hepatocellular carcinomat after end‐of‐treatment in all patients (a) and according to treatment regimen (b). EOT, end‐of‐treatment; HCC, hepatocellular carcinoma.

Table 2

Patient characteristics according to hepatocellular carcinoma development

Variables	HCC (n = 14)	No‐HCC (n = 508)	P‐value
Age (years)†	72 (45–82)	62 (20–88)	0.056‡
Sex (male/female)	10/4	268/240	0.133§
Genotype 1/2	11/3	281/227	0.070§
HCV‐RNA (logIU/L)†	6.3 (1.6–7.8)	6.4 (1.2–7.1)	0.937‡
At baseline
Albumin (g/dL)†	3.9 (3.4–4.7)	4.2 (2.9–5.2)	0.004‡
Total bilirubin (mg/dL)†	0.8 (0.5–1.6)	0.7 (0.3–2.9)	0.060‡
AST (IU/L)†	46 (22–195)	39 (10–499)	0.238‡
ALT (IU/L)†	49 (15–209)	42 (9–1071)	0.427‡
Platelet counts (×104/μL)†	11.6 (6.1–20.2)	17.0 (0.5–38.3)	0.002‡
AFP (ng/mL)†	7 (1–142)	4 (1–459)	0.034‡
FIB‐4 index†	3.99 (1.69–16.22)	2.41 (0.30–33.42)	0.003‡
WFA+‐M2BP (COI)†	3.72 (0.88–15.87)	1.62 (0.20–19.81)	0.002‡
At SVR24
Albumin (g/dL)†	4.3 (3.2–4.7)	4.3 (3.4–6.6)	0.280‡
AST (IU/L)†	27 (14–53)	22 (4–111)	0.056‡
ALT (IU/L)†	20 (11–52)	16 (2–146)	0.029‡
Platelet counts (×104/μL)†	14.0 (3.4–19.0)	17.4 (0.2–42.5)	0.005‡
AFP (ng/mL)†	4 (1–15)	3 (1–27)	0.106‡
FIB‐4 index†	2.88 (1.46–9.66)	2.07 (0.33–111.86)	0.005‡
WFA+‐M2BP (COI)†	1.73 (0.52–5.90)	0.88 (0.13–16.65)	<0.001‡

Data are expressed as median (range).

Mann–Whitney U‐test.

Chi‐squared test.

AFP, alpha‐fetoprotein; ALT alanine aminotransferase; AST, aspartate aminotransferase; COI, cut‐off index; FIB‐4, fibrosis‐4; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; SVR, sustained virologic response; WFA + ‐M2BP, W. floribunda agglutinin‐positive Mac‐2‐binding protein.

Risk analysis

To identify noninvasive markers predicting HCC development in patients achieving an SVR, a Cox proportional hazard analysis was performed (Table 3). Univariate analysis revealed that male sex, serum albumin level, total bilirubin level, platelet count, FIB‐4 index, and the WFA+‐M2BP level at baseline and SVR24 were associated with HCC development. Multivariate analysis identified three independent risk factors: age (hazard ratio [HR] = 1.073, 95% confidence interval [CI] 1.016–1.133, P = 0.011), platelet counts (HR = 0.876, 95% CI 0.773–0.992, P = 0.036), and WFA+‐M2BP level at SVR24 (HR = 1.215, 95% CI 1.031–1.432, P = 0.020).

Table 3

Factors associated with hepatocellular carcinoma development after achievement of a sustained virologic response

Variables	Univariate analysis		Multivariate analysis
	HR (95% CI)	P value	HR (95% CI)	P value
Age every 1 year	1.069 (1.014–1.127)	0.211	1.073 (1.016–1.133)	0.012
Sex female vs. male	0.476 (0.149–1.521)	0.002
IFN‐based/IFN‐free therapy	1.682 (0.417–6.784)	0.465
HCV genotype 2 vs. 1	0.330 (0.092–1.185)	0.089
HCV‐RNA every 1.0 logIU/mL	0.961 (0.622–1.487)	0.860
At baseline
Albumin every 1 g/dL	0.159 (0.040–0.634)	0.009
Total bilirubin every 1 mg/dL	3.075 (1.030–9.178)	0.044
AST every 1 IU/L	1.004 (0.997–1.011)	0.246
ALT every 1 IU/L	1.000 (0.994–1.006)	0.970
Platelet counts every 1 × 104/μL	0.854 (0.771–0.946)	0.002	0.876 (0.774–0.992)	0.037
AFP every 1 ng/mL	1.005 (0.998–1.013)	0.164
FIB‐4 index every 1.00	1.140 (1.059–1.228)	0.001
WFA + ‐M2BP every 1 COI	1.200 (1.093–1.317)	<0.001
At SVR24
Albumin every 1 g/dL	0.143 (0.032–0.642)	0.011
AST every 1 IU/L	1.019 (0.992–1.047)	0.164
ALT every 1 IU/L	1.011 (0.988–1.034)	0.366
Platelet counts every 1 × 104/μL	0.855 (0.772–0.948)	0.003
AFP every 1 ng/mL	1.138 (0.984–1.316)	0.081
FIB‐4 index every 1.00	1.023 (0.982–1.065)	0.270
WFA + ‐M2BP every 1 COI	1.305 (1.140–1.494)	0.001	1.215 (1.031–1.432)	0.020

AFP, alpha‐fetoprotein; ALT alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; COI, cut‐off index; FIB‐4, fibrosis‐4; HCV, hepatitis C virus; HR, hazard ratio; IFN, interferon; SVR, sustained virologic response; WFA + ‐M2BP, W. floribunda agglutinin‐positive Mac‐2‐binding protein.

Analysis of the area under receiver operator characteristics curve (AUROC) revealed that the WFA+‐M2BP level at SVR24 was more predictive of HCC development than the WFA+‐M2BP level at baseline (AUROC = 0.805 and 0.744, respectively, Fig. 2). Furthermore, receiver operator characteristics curve analysis indicated that a 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 incidence rates of HCC development in patients with a WFA+‐M2BP level of <1.62 COI were 1 and 1.6%, respectively (0.40% per 1 person‐year), whereas those in patients with a WFA+‐M2BP level of ≥1.62 COI were 4.7 and 12.5%, respectively (2.68% per 1 person‐year, P < 0.001, Fig. 3).

Figure 2

Receiver operating characteristic curve for the prediction of hepatocellular carcinoma development. AUC, area under the curve; CI, confidence interval; SVR24, sustained virologic response at week 24; WFA + ‐M2BP, Wisteria floribunda agglutinin‐positive mac‐2‐binding protein.

Figure 3

Cumulative incidence of hepatocellular carcinoma according to the Wisteria floribunda agglutinin‐positive mac‐2‐binding protein at sustained virologic response week 24. EOT, end‐of‐treatment; HCC, hepatocellular carcinoma; SVR24, sustained virologic response at week 24; WFA + ‐M2BP, W. floribunda agglutinin‐positive mac‐2‐binding protein.

Among patients treated with IFN‐based therapy, the 3‐ and 5‐year cumulative incidence rates of HCC development in patients with a WFA+‐M2BP level of <1.62 COI were 0.6 and 1.3%, respectively (0.41% per 1 person‐year), whereas those in patients with a WFA+‐M2BP level of ≥1.62 COI were 3.1 and 12.6%, respectively (3.31% per 1 person‐year, P < 0.001, Fig. 4a). Among patients treated with IFN‐free therapy, the 3‐year cumulative incidence rate of HCC development was 1.4% in patients with a WFA+‐M2BP level of <1.62 COI (0.38% per 1 person‐year) and 5.8% in patients with a WFA+‐M2BP level of ≥1.62 COI (2.03% per 1 person‐year, P = 0.042, Fig. 4b).

Figure 4

Cumulative incidence of hepatocellular carcinoma according to the Wisteria floribunda agglutinin‐positive mac‐2‐binding protein at sustained virologic response week 24 in patients treated with interferon‐based therapy (a) and interferon‐free therapy (b). EOT, end‐of‐treatment; HCC, hepatocellular carcinoma; SVR24, sustained virologic response at week 24; WFA + ‐M2BP, W. floribunda agglutinin‐positive mac‐2‐binding protein.

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 findings 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 significantly associated with the severity of histological liver fibrosis., In addition, age is a well‐known surrogate marker of disease duration and is associated with more advanced fibrosis. Several previous studies also showed that old age and advanced liver fibrosis were significant risk factors for HCC development., , , Based on these results, the European Association for the Study of the Liver (EASL) recommends that patients with advanced fibrosis and cirrhosis who achieve an SVR should undergo surveillance for HCC every 6 months. Our findings confirmed the clinical importance of assessing the severity of liver fibrosis in the development of HCC after HCV eradication. Although liver biopsy has been recognized as the gold standard for the assessment of fibrosis, 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 fibrosis. We previously reported that the pretreatment WFA+‐M2BP level is a useful predictor of HCC development in patients who achieve an SVR by IFN‐based therapy. However, the present study demonstrates that WFA+‐M2BP level measured after achieving SVR24 is more useful than the pretreatment WFA+‐M2BP level for predicting HCC development. In our previous study, we showed that the WFA+‐M2BP level is affected by necroinflammatory activity in the liver. In this study, the WFA+‐M2BP levels significantly decreased after HCV eradication. These results suggest that the WFA+‐M2BP level measured after achieving SVR24 is more useful than the pretreatment level for assessing liver fibrosis and predicting HCC development.

Another finding 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., However, several recent studies have revealed that the incidence of HCC development did not significantly differ between IFN‐based therapy and IFN‐free therapy, and this phenomenon can be explained by patient characteristics such as age and liver function., 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 findings.

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 fibrosis using the WFA+‐M2BP level at SVR24 for prediction of HCC development after achieving an SVR.