Serum Mac-2 Binding Protein Glycosylation Isomer to Predict the Severity of Hepatic Fibrosis in Patients with Hepatitis C Virus Infection

Large-scale studies to assess the utility of the Mac-2 binding protein glycosylation isomer (M2BPGi) in predicting hepatic fibrosis in patients with hepatitis C virus (HCV) infection are limited. Serum M2BPGi level determination was performed in 1460 patients with HCV who received liver stiffness measurement (LSM) using transient elastography (TE). The correlation of LSM and grade of hepatic fibrosis as staged by TE with M2BPGi was assessed. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic power of M2BPGi for fibrosis stages of ≥F2, ≥F3, and F4. The selected M2BPGi cutoff values were chosen based on the maximal Youden index, a positive likelihood ratio (LR) ≥ 10, and a negative LR ≤ 0.1. Serum M2BPGi level was highly correlated with LSM (Pearson correlation coefficient: 0.567, p < 0.001) and hepatic fibrosis stage (Spearman’s rank correlation coefficient: 0.772, p < 0.001). The areas under ROC curves (AUROCs) of M2BPGi for ≥F2, ≥F3, and F4 were 0.865 (95% confidence interval [CI]: 0.846–0.884), 0.937 (95 % CI: 0.922–0.952), and 0.962 (95% CI: 0.951–0.972). The maximal Youden indices for ≥F2, ≥F3, and F4 were 1.72, 2.65, and 3.93. By selecting M2BPGi cutoff values with a positive LR ≥ 10 and a negative LR ≤ 0.1, clinicians were able to correctly discriminate F2, F3, and F4 in 69.1%, 77.8%, and 90.1% of patients. In conclusion, serum M2BPGi is a good diagnostic tool to predict the severity of hepatic fibrosis in patients with HCV infection.


Introduction
Chronic hepatitis C virus (HCV) infection remains a significant health problem that affects approximately 0.7% of the world's population [1]. Following chronic HCV infection, persistent hepatic inflammation can induce progressive hepatic fibrosis, which may result in cirrhosis, hepatocellular carcinoma (HCC), and hepatic decompensation [2][3][4]. In contrast, hepatic fibrosis can be halted or even regressed in most patients whose HCV is successfully eradicated with antiviral treatment [5].
The advent of direct-acting antivirals (DAAs) has revolutionized the care of HCV because nearly all patients can achieve viral eradication through a finite and short course of We conducted a retrospective study, enrolling patients with chronic HCV infection who received liver stiffness measurement (LSM) with transient elastography (FibroScan ® , Echosens, Paris, France) at the National Taiwan University Hospital (NTUH) and NTUH Yun-Lin Branch before antiviral treatment and for whom serum samples had been stored between January 2012 and December 2021 and could be used to determine the serum M2BPGi level. All serum samples were stored at −80 • C until analysis. Chronic HCV infection was defined as the presence of detectable HCV antibody (anti-HCV; Abbott HCV EIA 2.0, Abbott Laboratories, Abbott Park, IL, USA) and quantifiable serum HCV RNA (Cobas TaqMan HCV Test v2.0, Roche Diagnostics GmbH, Mannheim, Germany, lower limit of quantification [LLOQ]: 15 IU/mL) for ≥6 months. Patients who had hepatitis B virus (HBV) or human immunodeficiency virus (HIV) coinfection; chronic kidney disease stage 5, which was defined as an estimated glomerular filtration rate (eGFR) <15 mL/min/1.73 m 2 ; decompensated cirrhosis (Child-Pugh B or C); a history of HCC; organ transplantation; or failed or unreliable LSM by TE, or who refused or were unable to provide written informed consent, were excluded from the study.
Serum M2BPGi levels were quantified using a Wisteria floribunda agglutinin (WFA)antibody sandwich immunoassay with an automated HISCL-800 immunoanalyzer (Sysmex Co., Kobe, Japan). The level was expressed as cutoff index (COI) using the following equation: , where NC and PC denote negative and positive controls.

Statistical Analysis
The statistical analyses were performed using the Statistical Program for Social Sciences (SPSS Statistics Version 23.0, IBM Corp., Armonk, NY, USA). When appropriate, the baseline characteristics are shown as median (interquartile range, IQR) and number (percentage). We analyzed the relationship between serum M2BPGi level and LSM with Pearson correlation, and that between serum M2BPGi level and hepatic fibrosis stage (F0-F1, F2, F3, and F4) with Spearman's rank correlation [18,43]. Receiver operating characteristic (ROC) curves were constructed for M2BPGi. The areas under ROC curves (AUROCs) with 95% confidence interval (CI) of M2BPGi are shown according to fibrosis stages of significant hepatic fibrosis (≥F2), advanced hepatic fibrosis (≥F3), and cirrhosis (F4) [44]. We also assessed the AUROCs of M2BPGi in subgroups of interest, including age > 60 years, male sex, MAFLD, HCVRNA > 2,000,000 IU/mL, HCV genotype 1, ALT > 2 folds ULN, and CKD stage 3 or 4. Three selective cutoff values of M2BPGi to predict fibrosis stages of ≥F2, ≥F3, and F4 were chosen: (1) the maximal Youden index with a maximal value of (sensitivity + specificity −1); (2) the index with a negative likelihood ratio (LR) ≤ 0.1; (3) index with a positive LR ≥ 10. For each selective cutoff value, we showed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive LR, negative LR, and accuracy. All statistics were two-tailed and results were considered statistically significant when the p value was <0.05.

Correlation between Serum M2BPGi Level and Hepatic Fibrosis
The serum M2BPGi level was significantly correlated with LSM (Pearson correlation coefficient: 0.567, p < 0.001) ( Figure 2). Figure

AUROC of M2BPGi to Predict the Severity of Hepatic Fibrosis
The  (Table 2).   (Table 2).

Selective M2BPGi Cutoff Values to Predict the Severity of Hepatic Fibrosis
The  (Table 3) (Table 3). When we combined the selected M2BPGi cutoff values with a negative LR ≤0.10 and a positive LR of ≥10, 69.1%, 77.8%, and 90.1% patients could be correctly diagnosed for F2, F3, and F4. M2BPGi, Mac-2 binding protein glycosylation isomer; COI, cutoff index; PPV, positive predictive value; NPV, negative predictive value; LR, likelihood ratio. a Youden index is defined as the value

Discussion
In line with the published reports, we confirmed that serum M2BPGi level was highly correlated with LSM and hepatic fibrosis stage as determined with TE in our large-scale study [19,20,[22][23][24][25]. However, the AUROCs of serum M2BPGi level in predicting patients with fibrosis stages of ≥F2, ≥F3, and F4 varied significantly among various studies. The AUROCs in our study for predicting fibrosis stages of ≥F2, ≥F3, and F4 were 0.865, 0.937, and 0.962, and were superior to the reported AUROCs of 0.6-0.79 for ≥F2, 0.83-0.84 for ≥F3, and 0.76-0.89 for F4 in several studies [19,[22][23][24]. However, the AUROCs in our study were similar to those in a report from Thailand which showed AUROCs of 0.86, 0.93, and 0.96 in predicting fibrosis stages of ≥F2, ≥F3, and F4 [25]. While the AUROCs of M2BPGi in predicting fibrosis stages of ≥F2 and ≥F3 in Kuno et al.'s study were lower than ours, the AUROC in predicting F4 was 0.96, which was identical to our report [19]. In other studies, the divergent AUROCs may be explained by relatively small sample sizes, recruitment of heterogeneous populations, and different cutoff levels of LSM used to define the stage of hepatic fibrosis [19,[22][23][24][25].
We further examined whether some specific patient characteristics, including old age, male sex, coexistence of MAFLD, high HCV load, HCV genotype, elevated serum ALT level, or more advanced CKD stage, might significantly affect the AUROCs. The diagnostic performance of serum M2BPGi in these subgroups remained similar to the overall population, implying that the accuracy of M2BPGi was not compromised in patients with these factors. Based on the excellent diagnostic performance, serum M2BPGi may serve as a good noninvasive mean to assess hepatic fibrosis in patients with HCV infection.
Regarding the selection of optimized cutoff values for serum M2BPGi, we performed a comprehensive analysis by choosing the maximal Youden index with the highest level of sensitivity plus specificity, the cutoff point with a positive likelihood ratio ≥10.0 which provided strong evidence to rule in disease, and the cutoff point with a negative likelihood Diagnostics 2022, 12, 2650 9 of 12 ratio ≤0.10 which provided strong evidence to rule out disease [45,46]. The diagnostic accuracy of serum M2BPGi levels with the maximal Youden indices tended to increase from 81.1% for ≥F2, 86.2% for ≥F3, to 95.7% for F4, and was in line with other noninvasive indices where the discrimination power increased with increasing severity of hepatic fibrosis [47][48][49][50]. In daily practice, it would be more informative and helpful for clinicians to exclude or include a defined stage of hepatic fibrosis. Our report demonstrated that M2BPGi cutoff values of less than 1 [27].
The strengths of our study include (1) a sizable number of patients used for the analysis; (2) a homogeneous population achieved by excluding potential factors that might affect serum M2BPGi levels, such as HCC, organ transplantation, decompensated cirrhosis. HBV or HIV infection, and CKD stage 5; (3) application of TE, which has been demonstrated to be a good reference standard for assessing hepatic fibrosis. However, our study has several limitations. First, this was a retrospective study, which used archived serum samples to determine the serum levels of M2BPGi. Therefore, it remains elusive whether the diagnostic accuracy of serum M2BPGi would be affected using archived samples. Second, we cannot extrapolate our results to patients excluded from the analysis. Third, we did not use liver biopsy, which is seldom performed now with the rapid development and the widespread use of noninvasive means, as the reference standard. Lastly, we cannot discriminate in our study a minority of patients concomitantly presenting with drug-induced liver injury (DILI), autoimmune liver disease, hereditary hemochromatosis, or Wilson's disease, which might affect liver fibrosis.
In conclusion, our large-scale study indicated that serum M2BPGi level was highly correlated with LSM and stage of hepatic fibrosis. The diagnostic performance of serum M2BPGi increased with the increasing severity of hepatic fibrosis. Using the optimized cutoff values with high positive LR and low negative LR for serum M2BPGi, 69.1%, 77.8%, and 90.1% patients with HCV infection could be discriminated according to fibrosis stages of F2, F3, and F4 using this simple serological index. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data for this study, though not available in a public repository, can be made available upon reasonable request.