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Hepatocellular carcinoma (HCC) is a common malignancy worldwide. Alpha-fetoprotein (AFP) is still the only serum biomarker widely used in clinical settings. However, approximately 40% of HCC patients exhibit normal AFP levels, including very early HCC and AFP-negative HCC; for these patients, serum AFP is not applicable as a biomarker of early detection. Thus, there is an urgent need to identify novel biomarkers for patients for whom disease cannot be diagnosed early. In this study, we screened and identified novel proteins in AFP-negative HCC and evaluated the feasibility of using autoantibodies to those protein to predict hepatocarcinogenesis. First, we screened and identified differentially expressed proteins between AFP-negative HCC tissue and adjacent non-tumor liver tissue using SWATH-MS proteome technology. In total, 2,506 proteins were identified with a global false discovery rate of 1%, of which 592 proteins were expressed differentially with 175 upregulated and 417 downregulated (adjusted  p -value <0.05, fold-change FC ≥1.5 or ≤0.67) between the tumor and matched benign samples, including 14-3-3 zeta protein. For further serological verification, autoantibodies against 14-3-3 zeta in serum were evaluated using enzyme-linked immunosorbent, Western blotting, and indirect immunofluorescence assays. Five serial serum samples from one patient with AFP-negative HCC showed anti-14-3-3 zeta autoantibody in sera 9 months before the diagnosis of HCC, which gradually increased with an increase in the size of the nodule. Based on these findings, we detected the prevalence of serum anti-14-3-3 zeta autoantibody in liver cirrhosis (LC) patients, which is commonly considered a premalignant liver disease of HCC. We found that the prevalence of autoantibodies against 14-3-3 zeta protein was 16.1% (15/93) in LC patient sera, which was significantly higher than that in patients with chronic hepatitis (0/75,  p  = 0.000) and normal human sera (1/60, 1.7%,  p  = 0.01). Therefore, we suggest that anti-14-3-3 zeta autoantibody might be a biomarker for predicting hepatocarcinogenesis. Further follow-up and research of patients with positive autoantibodies will be continued to confirm the relationship between anti-14-3-3 zeta autoantibody and hepatocarcinogenesis.

Serum Anti-14-3-3 Zeta Autoantibody as a Biomarker for Predicting Hepatocarcinogenesis