Impact of Wisteria floribunda Agglutinin-Positive Mac-2-Binding Protein in Patients with Hepatitis C Virus-Related Compensated Liver Cirrhosis

We aimed to examine the effect of Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+-M2BP) level on survival comparing with other laboratory liver fibrosis markers in hepatitis C virus (HCV)-related compensated liver cirrhosis (LC) (n = 165). For assessing prognostic performance of continuous fibrosis markers, we adapted time-dependent receiver operating characteristics (ROC) curves for clinical outcome. In time-dependent ROC analysis, annual area under the ROCs (AUROCs) were plotted. We also calculated the total sum of AUROCs in all time-points (TAAT score) in each fibrosis marker. WFA+-M2BP value ranged from 0.66 cutoff index (COI) to 19.95 COI (median value, 5.29 COI). Using ROC analysis for survival, the optimal cutoff point for WFA+-M2BP was 6.15 COI (AUROC = 0.79348, sensitivity = 80.0%, specificity = 74.78%). The cumulative five-year survival rate in patients with WFA+-M2BP ≥ 6.15 COI (n = 69) was 43.99%, while that in patients with WFA+-M2BP < 6.15 COI (n = 96) was 88.40% (p < 0.0001). In the multivariate analysis, absence of hepatocellular carcinoma (p = 0.0008), WFA+-M2BP < 6.15 COI (p = 0.0132), achievement of sustained virological response (p < 0.0001) and des-γ-carboxy prothrombin < 41 mAU/mL (p = 0.0018) were significant favorable predictors linked to survival. In time-dependent ROC analysis in all cases, WFA+-M2BP had the highest TAAT score among liver fibrosis markers. In conclusion, WFA+-M2BP can be a useful predictor in HCV-related compensated LC.


Baseline Characteristics
The baseline characteristics in the current analysis are presented in Table 1 (n = 165). There are 93 males and 72 females with the median age of 67 years. For the entire cohort, the WFA + -M2BP value ranged from 0.66 cutoff index (COI) to 19.95 COI (median value, 5.29 COI). Sixty-eight out of 165 patients (41.2%) had HCC on radiological findings, which included stage I in 22, stage II in 26, stage III in 10 and stage IV in 10. The median observation period in the present analysis was 3.852 years (range: 0.219-9.241 years). As for HCV genotype and HCV viral load, genotype 1 (84.8% (140/165)) and high viral load as defined by HCV-RNA ≥ 5 log copies/mL (87.3% (144/165)) were in the majority at study entry. During follow-up period, SVR was achieved in 68 patients (41.2%) by antiviral therapies. Of these, 34 patients were treated with interferon-based antiviral therapies and the remaining 34 patients were treated with interferon-free antiviral therapies.

Comparison of WFA + -M2BP Level in Patients with Stage I or II HCC and Those with Stage III or IV HCC
The difference of WFA + -M2BP levels in patients with stage I or II HCC (median: 7.57 COI, range: 0.8-17.95 COI) and those with stage III or IV HCC (median: 6.92 COI, range: 0.66-16.93 COI) did not reach significance (p = 0.2544) ( Figure 3B).

Comparison of WFA + -M2BP Level in Patients with Stage I or II HCC and Those with Stage III or IV HCC
The difference of WFA + -M2BP levels in patients with stage I or II HCC (median: 7.57 COI, range: 0.8-17.95 COI) and those with stage III or IV HCC (median: 6.92 COI, range: 0.66-16.93 COI) did not reach significance (p = 0.2544) ( Figure 3B).

Causes of Death
During the observation period, 50 patients (30.3%) died. The causes of death were HCC progression in 24 patients, liver failure in 19 patients and miscellaneous causes in seven patients.

Time-Dependent ROC Analyses for OS in all Cases
Results for time-dependent ROC analyses at one-, two-, three-, four-, five-, six-and seven-year of WFA + -M2BP, APRI, FIB-4 index, platelet count and hyaluronic acid in all cases are presented in Table 3. The plots of annual AUROCs of five liver fibrosis markers as shown in Figure 5. The total sum of AUROCs in all time-points (TAAT score) in WFA + -M2BP was the highest among five liver fibrosis markers (TAAT score = 4.93226), followed by hyaluronic acid (TAAT score = 4.74996).

Causes of Death
During the observation period, 50 patients (30.3%) died. The causes of death were HCC progression in 24 patients, liver failure in 19 patients and miscellaneous causes in seven patients.

Time-Dependent ROC Analyses for OS in all Cases
Results for time-dependent ROC analyses at one-, two-, three-, four-, five-, six-and seven-year of WFA + -M2BP, APRI, FIB-4 index, platelet count and hyaluronic acid in all cases are presented in Table 3. The plots of annual AUROCs of five liver fibrosis markers as shown in Figure 5. The total sum of AUROCs in all time-points (TAAT score) in WFA + -M2BP was the highest among five liver fibrosis markers (TAAT score = 4.93226), followed by hyaluronic acid (TAAT score = 4.74996).

Time-Dependent ROC Analyses for OS in HCC Patients
Results for time-dependent ROC analyses at one-, two-, three-, four-, five-, six-and seven-year of WFA + -M2BP, APRI, FIB-4 index, platelet count and hyaluronic acid in HCC patients are shown in Table 3. The plots of annual AUROCs of five liver fibrosis markers are shown in Figure 6. The TAAT score in hyaluronic acid was the highest among five liver fibrosis markers (TAAT score = 4.47631), followed by FIB-4 index (TAAT score = 4.19352).

Time-Dependent ROC Analyses for OS in Non-HCC Patients
Results for time-dependent ROC analyses at three-, four-, five-, six-and seven-year of WFA + -M2BP, aspartate aminotransferase to platelet ration index (APRI), FIB-4 index, platelet count and hyaluronic acid in non-HCC patients are shown in Table 3. The plots of annual AUROCs of five liver fibrosis markers are shown in Figure 7. The one-and two-year cumulative survival rate were both 100% in non-HCC patients and thus AUROCs in one-and two-year were not available. The TAAT score in WFA + -M2BP was the highest among five liver fibrosis markers (TAAT score = 3.97663), followed by FIB-4 index (TAAT score = 3.81393).

Discussion
HCV-related LC involves a highly heterogeneous condition with a broad spectrum of clinical characteristics, ranging from compensated stage to decompensated stage, each of which is characterized by a different prognosis [7][8][9]. It is obvious that decompensated LC patients had worse prognosis. We therefore conducted this study in limited patients with compensated LC. To our knowledge, this is the first report regarding the effect of WFA + -M2BP on clinical outcomes in HCV-related compensated LC patients. Furthermore, outcomes for CLDs are usually time-dependent. However, few studies have analyzed clinical data using time-dependent ROC analysis [26,27]. Thus, for detailed examination of the effect of WFA + -M2BP on clinical outcomes, we used both multivariate analysis and time-dependent ROC analysis, which took time-dependence into account [26,27].
In our results, WFA + -M2BP level ≥ 6.15 COI as determined by ROC analysis for survival was an independent adverse predictor linked to OS and for the entire cohort and non-HCC patients, it had the highest TAAT scores among liver fibrosis markers. In addition, cumulative survival rates were well stratified by WFA + -M2BP level irrespective of presence or absence of HCC. These results demonstrated that WFA + -M2BP can be helpful for predicting clinical outcome in HCV-related compensated LC. Our baseline data of WFA + -M2BP (range: 0.66-19.95 COI) may reflect the fact that HCV-related compensated LC patients had heterogeneous patients population. The insignificant correlation between WFA + -M2BP level and AFP or DCP values indicate that WFA + -M2BP could not be an alternative tumor marker to AFP or DCP.
In our previous study, we reported that WFA + -M2BP correlated with not only the degree of liver fibrosis but also the degree of liver inflammation activity [20,21,25]. Indeed, the proportion of A3 in patients with WFA + -M2BP ≥ 6.15 COI was significantly higher than that in patients with WFA + -M2BP < 6.15 COI in this study (p = 0.0293), and this may be associated with worse clinical outcome in patients with higher WFA + -M2BP level [7,28]. The statistical significance between non-HCC and HCC patients in terms of WFA + -M2BP level can be also explained by liver inflammation, although the difference of the proportion of A3 in HCC and non-HCC patients with available liver biopsy data did not reach significance (p = 0.2299, data not shown).
In HCC patients, hyaluronic acid had the highest TAAT score among five liver fibrosis markers. In this population, the presence of HCC itself can affect prognosis. On the other hand, in our previous study, we reported that serum hyaluronic acid could predict protein-energy malnutrition (PEM) in HCV-related liver disease [29]. Especially in this patient population, sufficient management for PEM may be essential for ameliorating outcome since HCC therapy can accelerate progression of PEM.
Notably, an achievement of SVR during follow-up period was the strongest favorable predictor linked to OS with the HR of 23.833 (p < 0.0001) in our multivariate analysis. A recent meta-analysis demonstrated that CHC subjects with SVR have a substantially reduced risk for HCC incidence (HR: 0.1-0.25), liver-related mortality (HR: 0.03-0.2) and all-cause mortality (HR: 0.1-0.3) as compared with no antiviral treatment or treatment failure, which are in agreement with our current results [30]. In compensated LC patients, antiviral therapy can be strongly recommended for improving clinical outcomes.
We acknowledge several limitations in this study. First, since this longitudinal study had a retrospective nature, our results should be cautiously interpreted; Second, in 52 patients (31.5%), LC was diagnosed not by liver biopsy but radiological findings alone, leading to bias; Third, liver biopsy involves restriction being prone to errors in a statistical analysis arising from the unrepresentativeness of the sample taken for assessing the degree of liver fibrosis, also leading to bias; Fourth, there were several missing values in our analysis; Fifth, calculating TAAT score is not a well established assessment method for predicting survival and this is only a proposal. However, our current results demonstrated that WFA + -M2BP performed well as a prognostic marker in HCV-related compensated LC patients.
Finally, we concluded that WFA + -M2BP can be a useful predictor in patients with HCV-related compensated LC.

Patients
Between March 2007 and June 2015, a total of 165 individuals with HCV-related compensated LC with available stored serum samples were admitted to the Division of Hepatobiliary and Pancreatic disease, Department of Internal Medicine, Hyogo College of Medicine, Hyogo, Japan and they were subjected to this analysis. Compensated LC was defined as Child-Pugh A LC. Subjects with HCV-related liver disease are defined as those with HCV antibody positive and hepatitis B surface antigen negative. In patients who did not receive liver biopsy (n = 52), LC was diagnosed through radiological findings: clinical features suggestive of portal hypertension such as splenomegaly, varices, ascites and a shrunken or deformed and nodular liver as identified on computed tomography (CT) or ultrasonography (US) findings [7]. The primary endpoint in our study is overall survival (OS). Firstly, we examined the relationship between WFA + -M2BP level and baseline characteristics or clinical outcomes. Subsequently, we investigated variables linked to OS using univariate and multivariate analyses. Finally, we compared the effect of WFA + -M2BP level on survival with those of other liver fibrosis markers including APRI, FIB-4 index, platelet count and hyaluronic acid using time-dependent ROC analysis. In principle, in patients with serum albumin level ≤3.5 g/dL, branched chain amino acids were administered [7]. In patients without HCC or those with curative treatment for HCC, antiviral therapies including interferon-based therapies or interferon-free therapies were also considered [7].
The ethics committee of Hyogo college of medicine approved the current study protocol (approval number: 1831; date: 1 March 2016) and this study protocol adhered to all provisions of the Declaration of Helsinki.

HCC Surveillance and HCC Therapy
Follow-up consisted of periodical evaluation by imaging studies including US, CT and/or magnetic resonance imaging for HCC incidence or HCC recurrence and periodical blood tests such as tumor markers. For cases with HCC, most adequate therapy for each case was selected according to Japanese guidelines through discussion with radiologists, surgeons and hepatologists [35][36][37].

Statistical Analysis
In continuous parameters, groups were compared by using Student's t-test or Mann-Whitney U test, or Spearman's rank correlation coefficient r s , as applicable. In categorical parameters, groups were compared by using Fisher's exact tests or Pearson χ 2 test, as applicable. In continuous parameters, we performed ROC curve analysis of survival for selection of the optimal cutoff value that is associated with maximal total value of specificity and sensitivity and we classified them into two groups using these cutoff values and treated them as nominal covariates. Kaplan-Meier curves were generated and compared by using the log-rank test. Parameters with p value less than 0.05 in the univariate analysis were entered into the multivariate analyses (Cox proportional hazard model). Furthermore, we analyzed time-dependent ROC curves of WFA + -M2BP, APRI, FIB-4 index, platelet count and hyaluronic acid for survival and compared between area under the ROCs (AUROCs) for these liver fibrosis markers in each time point (one-, two-, three-, four-, five-, six-, and seven-year) [26,27]. We also calculated the total sum of AUROCs in all time-points (TAAT score) in each liver fibrosis marker.
OS was calculated as the time interval from the date at which we obtained stored serum sample during hospitalization with the purpose of liver biopsy, HCC therapy or others until death (due to any cause) or the last follow-up visit. Data are presented as median (range) unless otherwise stated. We regarded variables with p value less than 0.05 as statistically significant variables. Statistical analysis was performed using JMP 11 (SAS Institute Inc., Cary, NC, USA).