Combined Serum Biomarkers in Non-Invasive Diagnosis of Non-Alcoholic Steatohepatitis

Non-alcoholic steatoheaptitis (NASH), the critical stage of non-alcoholic fatty liver disease (NAFLD), is of chronic progression and can develop cirrhosis even hepatocellular carcinoma (HCC). However, non-invasive biomarkers for NASH diagnosis remain poorly applied in clinical practice. Our study aims at testing the accuracy of the combination of cytokeratin-18 M30 fragment (CK-18-M30), fibroblast growth factor 21 (FGF-21), interleukin 1 receptor antagonist (IL-1Ra), pigment epithelium-derived factor (PEDF) and osteoprotegerin (OPG) in diagnosing NAFLD and NASH. 179 patients with biopsy-proven NAFLD were enrolled as training group, 91 age- and gender-matched healthy subjects were recruited at the same time as controls. 63 other NAFLD patients were separately collected as validation group. 45 alcoholic fatty liver disease (AFLD) patients, 50 hepatitis B virus (HBV) patients, 52 hepatitis C virus (HCV) patients were also included. Serum biomarker levels were measured by enzyme-linked immunosorbent assay. Serum levels of CK-18-M30, FGF-21, IL-1Ra and PEDF increased, while OPG decreased in a stepwise fashion in controls, non-NASH NAFLD patients and NASH patients (P < 0.01). The area under receiver-operating characteristics curve to diagnose NASH was 0.86 for CK-18-M30, 0.89 for FGF-21, 0.89 for IL-1Ra, 0.89 for PEDF and 0.89 for OPG. CK-18-M30 had 70% negative predictive value (NPV) and 79% positive predictive value (PPV) to diagnose NASH. A 5-step approach measuring CK-18-M30 followed by FGF21, IL-1Ra, PEDF and OPG gradually improved the NPV to 76% and PPV to 85%, which reached 80% and 76% respectively in the validation cohort. Compared to single biomarker, stepwise combination of CK-18-M30, FGF-21, IL-1Ra, PEDF and OPG can further improve the accuracy in diagnosing NASH.