Serum Wisteria Floribunda Agglutinin-Positive Mac-2 Binding Protein Values Predict the Development of Hepatocellular Carcinoma among Patients with Chronic Hepatitis C after Sustained Virological Response

Measurement of Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA+-M2BP) in serum was recently shown to be a noninvasive method to assess liver fibrosis. The aim of this study was to evaluate the utility of serum WFA+-M2BP values to predict the development of hepatocellular carcinoma (HCC) in patients who achieved a sustained virological response (SVR) by interferon treatment. For this purpose, we retrospectively analyzed 238 patients with SVR who were treated with interferon in our department. Serum WFA+-M2BP values were measured at pre-treatment (pre-Tx), post-treatment (24 weeks after completion of interferon; post-Tx), the time of HCC diagnosis, and the last clinical visit. Of 238 patients with SVR, HCC developed in 16 (6.8%) patients. The average follow-up period was 9.1 years. The cumulative incidence of HCC was 3.4% at 5 years and 7.5% at 10 years. The median pre-Tx and post-Tx WFA+-M2BP values were 1.69 (range: 0.28 to 12.04 cutoff index (COI)) and 0.80 (range: 0.17 to 5.29 COI), respectively. The WFA+-M2BP values decreased significantly after SVR (P < 0.001). The median post-Tx WFA+-M2BP value in patients who developed HCC was significantly higher than that in patients who did not (P < 0.01). Multivariate analysis disclosed that age (> 60 years), sex (male), pre-Tx platelet count (< 15.0×103/μL), and post-Tx WFA+-M2BP (> 2.0 COI) were associated with the development of HCC after SVR. Conclusion Post-Tx WFA+-M2BP (> 2.0 COI) is associated with the risk for development of HCC among patients with SVR. The WFA+-M2BP values could be a new predictor for HCC after SVR.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world [1]. Chronic hepatitis C virus (HCV) infection is a major cause of HCC. Millions of people are persistently infected with HCV globally [2][3][4] and these individuals are at high risk of developing HCC [5][6][7]. Several studies have demonstrated that interferon (IFN) treatment in chronic hepatitis C patients reduces the risk for progression of liver disease, HCC, liver-related death, and all-cause mortality [8][9][10][11][12][13], especially in patients who exhibit a sustained virological response (SVR). However, some risk for HCC-albeit a small one-remains even after achieving viral eradication [10,[14][15][16][17][18][19]. Several factors have been reported to affect HCC development among patients with SVR.
Recently, an assay for the measurement of Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA + -M2BP) was reported as a novel, noninvasive, and rapid bedside method to assess liver fibrosis [20]. M2BP has been shown to have multibranching and sialylated N-glycans. WFA is considered to recognize the GalNAc residue of N-glycans and O-glycans or the clustered LacNAc (Gal-GlcNAc) structure. Currently, we are analyzing the glycan structures of WFA + -M2BP in detail using MS-based technology [21]. Glycans can reflect the differentiation stage of cells but not necessarily the level of cellular damage, and therefore they can be very effective markers for chronic disease. Several reports performed with proteome analysis have identified Mac-2 binding protein as a potential marker of liver fibrosis progression [22][23][24][25]. Kuno et al. were the first to report that a rapid and simple glycan-based immunoassay for WFA + -M2BP can quantify fibrosis [20,26]. On the other hand, we reported that AFP and WFA + -M2BP values are noninvasive predictive markers for the development of HCC in patients with HCV [27,28]. In this report we evaluated the utility of WFA + -M2BP values to predict the development of HCC in patients who had achieved SVR after IFN treatment.

Patients and Methods Patients
From December 1989 to December 2010, a total of 601 consecutive HCV patients who received IFN treatment and achieved SVR at the National Hospital Organization Nagasaki Medical Center were enrolled in this retrospective study. The diagnosis of chronic HCV infection was based on continuous positivity for both anti-HCV by a second or third-generation enzymelinked immunoadsorbent assay (ELISA) and positivity for serum HCV RNA by polymerase chain reaction (PCR). Before treatment, HCC was definitively ruled out either by ultrasonography (US), dynamic computed tomography (CT), or magnetic resonance imaging (MRI) on enrollment. Exclusion criteria for this study were: (1) positivity for hepatitis B surface antigen; (2) positivity for human immunodeficiency virus; (3) autoimmune hepatitis or primary biliary cirrhosis; (4) a shorter follow-up period (< 12 months) after the completion of IFN treatment; (5) a history of HCC at the time of IFN treatment; (6) development of HCC within 12 months after the completion of IFN treatment; (7) administration of low dose long-term IFN treatment; and (8) absence of properly stored serum samples or insufficient archival material. After the exclusions, 238 patients who achieved SVR were analyzed retrospectively for the risk factors of HCC.
For all patients in our cohort, a blood sample was taken on the days of the administration of IFN treatment (pre-treatment; pre-Tx), 24 weeks after completion of IFN treatment (posttreatment; post-Tx), and on the days of HCC diagnosis and last clinical visit. All separated serum samples were stored at -20°C until use. Medical histories, along with the results of routine tests for blood cell counts, liver biochemistry and HCV viral load/genotype at the time of IFN treatment and thereafter, were retrieved from medical records. Complete blood cell counts and biochemical tests were performed using automated procedures in the clinical pathologic laboratories of our hospital.

Histological evaluation
Liver biopsies were undertaken using fine-needle aspiration (16G or 18G sonopsy) guided by US. Liver tissue specimens were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. The histological assessment was made by two independent pathologists according to the classification of Desmet et al. [29].

Interferon treatment
Among the 238 patients, 123 received IFN monotherapy for 24 weeks, 28 patients received pegylated (PEG-)IFN monotherapy for 48 weeks, and 87 patients received IFN plus ribavirin or PEG-IFN plus ribavirin combination therapy for 48-72 weeks.

HCV RNA and HCV genotypes
The presence of HCV RNA was determined by reverse transcriptase (RT-) PCR using a commercial kit (Amplicor HCV; Roche Diagnostic Systems, Basel, Switzerland). Genotypes of HCV were determined by RT-PCR with genotype-specific primers (HCV RNA core genotype; Roche Diagnostics, Tokyo, Japan) [30,31]. In patients treated before the availability of PCR, the presence of HCV RNA was investigated by using sera stored at -20°C.
Definitions of response to interferon treatment SVR was defined as the absence of detectable HCV RNA at 24 weeks after the end of IFN treatment. There was no relapse of viremia after 24 weeks among the patients who achieved SVR.

Follow-up and diagnosis of hepatocellular carcinoma
All patients were followed up at an interval of 1-12 months by measurement of blood count and liver biochemistry, along with quantitative detection of HCV RNA, AFP, AFP-L3, and DCP. Diagnostic imaging either by US, CT, or MRI was performed at least once per year. A diagnosis of HCC was made based on positive results of typical vascular patterns, as revealed by either contrast-enhanced CT, contrast-enhanced MRI or angiography. Otherwise, the pathological diagnosis was made by fine-needle biopsy of space-occupying lesions detected in the liver.

Ethical considerations
Informed consent to utilize medical records and specimens was obtained from each patient. We obtained the written consent of participants at the time of serum collection. These processes and the study protocol were approved by the Ethical Committee of National Hospital Orga-

Statistical analysis
Continuous variables (AST, ALT, albumin, total bilirubin, γ-GTP, fasting blood sugar, HbA1c, triglyceride, total cholesterol, BMI, platelet counts, AFP, WFA + -M2BP) were dichotomized with respect to the median value or clinically meaningful values in the multivariate analysis. Statistical analysis was performed using a Wilcoxon signed rank test and Mann-Whitney Utest. To estimate the cumulative risk of developing HCC, the Kaplan-Meier method and the log-rank test were used. Cox proportional hazards regression analysis was performed to evaluate risk factors for HCC. The diagnostic performances of WFA + -M2BP and AFP for censored development of HCC were assessed by examining the area under the time-dependent receiver operating characteristic (ROC) curves (AUROC) [32]. Inclusion of variables was assessed using a stepwise selection method. A P value of 0.05 was considered statistically significant. Data analysis was performed with SPSS ver. 22.0 (SPSS, Chicago, IL).

Risk factors for HCC
Univariate analysis demonstrated factors that increase the risk for HCC development after SVR. Cox regression analysis was performed on 20 variables: age, sex, BMI, alcohol intake, fibrosis stage, degree of steatosis, pre-Tx platelet counts, post-Tx platelet counts, albumin, pre-Tx AST, post-Tx AST, pre-Tx ALT, post-Tx ALT, γ-GTP, T.bilirubin, HbA1c, pre-Tx AFP, post-Tx AFP, pre-Tx WFA + -M2BP, post-Tx WFA + -M2BP. Cutoff values for AFP and WFA + -M2BP were determined by time-dependent ROC analysis as 5 ng/ml and 2.0 COI, respectively.  The following seven factors were identified as posing an increased risk for HCC by the univariate analysis: age, fibrosis stage, albumin, pre-Tx platelet count, post-Tx platelet count, post-Tx AFP, and post-Tx WFA + -M2BP (Table 2).
Multivariate analysis was performed on these seven factors, and the following four factors were identified as independent risk factors: age (> 60 years, HR 5.42, 95% CI = 1.59-18.47, P = 0.007), sex (male, HR 4.71, 95% CI = 1.23-17.92, P = 0.023), pre-Tx platelet count (< 15. Predictive value of HCC incidence versus WFA + -M2BP and AFP Table 3 shows the AUROC analyses for prediction of the development of HCC at 3, 5 and 10 years with AFP and WFA + -M2BP. The post-Tx WFA + -M2BP was superior to the post-Tx AFP for predicting the development of HCC at each of 3, 5 and 10 years.

Chronological changes in the WFA + -M2BP and AFP values after IFN treatment
In the 238 patients with SVR, the median values of the chronological change in WFA + -M2BP at pre-Tx and post-Tx were 1.70 (range: 0.28 to 12.04 COI) and 0.80 (range: 0.17 to 5.29 COI). The post-Tx WFA + -M2BP values were significantly decreased relative to the pre-Tx WFA + -M2BP values (P < 0.001).
Next, we analyzed the WFA + -M2BP and AFP values in the 16 patients who developed HCC. Fig 4A shows       time of pre-Tx (P < 0.001). Similarly, the AFP values at the time of last clinical visit were significantly higher than those at the time of post-Tx (P < 0.001) (Fig 4B).
The first main finding of our study was that the post-Tx WFA + -M2BP was selected as a new predictive marker for development of HCC among patients with SVR ( Table 2). The values of WFA + -M2BP for predicting the development of HCC were determined to have a COI of 2.0 by time-dependent receiver operator characteristics (ROC) analysis. The cumulative incidence was significantly higher in the post-Tx WFA + -M2BP > 2.0 COI group. We were able to stratify the patients into different risk groups using the post-Tx WFA + -M2BP values and another simple risk factor, for example, age (Fig 2). Older age has been reported to confer a risk for hepatocellular carcinoma [14], which is an important association in Japan due to the aging of the population. Moreover, the post-Tx WFA + -M2BP values were significant predictor for HCC among patients with F3/4. Cumulative incidence of HCC was significantly higher in patients with higher post-Tx WFA + -M2BP values when patients were stratified by the stage of fibrosis (Fig 3). The post-Tx WFA + -M2BP values are not just a marker for liver fibrosis. Elevation of post-Tx WFA + -M2BP values as a potential risk for hepatocarcinogenesis with advanced fibrosis. And the time-dependent AUROC analysis suggested that WFA + -M2BP is superior to AFP as a predictor for the development of HCC.     The second main finding of our study was that the WFA + -M2BP values were decreased in patients who achieved SVR, even in those who developed HCC. Kuno previously reported that WFA + -M2BP values were decreased by IFN treatment [20]; to this result we added that the WFA + -M2BP values were decreased even in our IFN-treated patients who achieved SVR. However, the post-Tx WFA + -M2BP values were significantly higher in the patients who developed HCC than in those who did not. This finding is particularly important because the post-Tx values of WFA + -M2BP have not been adequately evaluated. Our data are thus the first to demonstrate the distribution of WFA + -M2BP values at post-Tx.
The third main finding of our study was that AFP and WFA + -M2BP values manifested different behaviors between the time of post-Tx and HCC diagnosis in patients who developed HCC. Our previous paper reported a close association between AFP values and the stage of fibrosis [27], whereas another report showed an elevation in AFP values caused by necroinflammation injury and regeneration of the liver [42]. However, WFA + -M2BP values do not always correlate with the grade of hepatic activity as defined by HAI scoring of inflammation [20,28]. A slight elevation of post-Tx AFP values (> 5ng/mL) could indicate substantial risks for the development of HCC [36]. In the 16 patients who developed HCC in our study, AFP values were elevated from post-Tx to the time of HCC development. However, the WFA + -M2BP values decreased after SVR and decreased further at the time of HCC diagnosis (Fig 4). The Mac-2 binding protein is secreted from many cell types, including hepatocytes, and it has been shown to modulate many processes, particularly those related to cell adhesion [22,43,44]. Alterations in the quality and quantity of the Mac-2 binding protein have been observed during the progression of fibrosis [22][23][24]. Hepatic stellate cells are considered the main fibrogenic cell type of the liver [45,46]. Activation of hepatic stellate cells and reversal of hepatic stellate cell activation [47] might be associated with WFA + -M2BP values. WFA + -M2BP has been associated with changes in both the quality and quantity of the Mac-2 binding protein due to changes in glycosylation [20]. From these considerations, we think that the WFA + -M2BP values do not reflect the results of HCC development, but rather a pre-cancer status or hepatocellular carcinogenesis.
One of the limitations of the present study was its retrospective nature. A future prospective analysis will be needed to validate the efficacy of WFA + -M2BP as a predictor of HCC development. Another limitation is that we analyzed a relatively small number of HCC cases after SVR. Multi-center prospective registration of patients with SVR could overcome this deficiency.
Regardless of these limitations, this is the first report to describe the relationship between WFA + -M2BP values and HCC development after SVR. The rapid progress in the development of anti-viral agents [48,49] for hepatitis C suggests that the number of patients who achieve SVR-including elderly patients or patients with advanced fibrosis, who are regarded as being at high risk for HCC-might increase in the near future, especially in Japan. Therefore, the prediction of HCC development in patients with SVR is of increasing clinical relevance.
In conclusion, this study revealed an association between WFA + -M2BP and the risk of HCC development in patients with SVR. The results suggested that the WFA + -M2BP should not be limited to use in fibrosis stage screening but rather could be applied as a new predictor of HCC development after SVR.