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Alpha fetoprotein (AFP) has been used as a serologic indicator of hepatocellular carcinoma (HCC). We aimed to identify an HCC-specific serum biomarker for diagnosis using a multiplexed proteomic technique in HCC patients with normal AFP levels. A total of 152 patients were included from Guro Hospital, Korea University. Among 267 identified proteins, 28 and 86 proteins showed at least a two-fold elevation or reduction in expression, respectively. Multiple reaction monitoring (MRM) analysis of 41 proteins revealed 10 proteins were differentially expressed in patients with liver cirrhosis and HCC patients with normal AFP. A combination of tripartite motif22 (Trim22), seprase, and bone morphogenetic protein1 had an area under receiver operating characteristic of 0.957 for HCC diagnosis. Real-time PCR and western blot analysis of the paired tumor/non-tumor liver tissue in HCC revealed a reduced expression of Trim22 in the tumor tissue. Also, serum levels of Trim22 were significantly reduced in HCC patients with normal AFP compared to those with liver cirrhosis (p = 0.032). Inhibition of Trim22 increased cellular proliferation in human hepatoma cell lines, whereas overexpression of Trim22 decreased cellular proliferation in hepatoma cell lines. In conclusion, the combination of three serum markers improved the chance of diagnosing HCC. MRM-based quantification of the serum protein in patients with normal AFP provides the potential for early diagnosis of HCC.

Multiplexed Proteomic Approach for Identification of Serum Biomarkers in Hepatocellular Carcinoma Patients with Normal AFP