Abstract INTRODUCTION Hepatocellular carcinoma (HCC) is one the common malignancies in the world and is the leading cause of cancer death in some Asian area. The most well studied biomarker for HCC is alpha-fetoprotein (AFP), which has, however, suboptimal sensitivity and specificity for HCC. Newer biomarkers, such as des-gamma carboxyprothrombin and lens culinaris agglutinin-reactive AFP (AFP-L3) are still far from perfect, and searching for new biomarkers are needed. The goal of this study is to identify potential biomarkers in the sera of patients with HCC by using 2D-DIGE (two-dimensional fluorescence difference gel electrophoresis). PATIENTS AND METHODS The training set consisted of sera from 10 HCC patients who received surgical resection in National Taiwan University Hospital. The sera samples were obtained at two time points: 1st time point, immediate before operation, and 2nd time point, one month after operation. The validation set included sera from 13 health donors, 50 hepatitis B virus (HBV) carriers, 30 HBV-related liver cirrhosis (HBV-LC) patients and 80 HBV-related HCC (HBV-HCC) patients. The clinicopathological data is retrospectively obtained from chart reviews The sera samples were first depleted of albumin and IgG and then minimally labeled with CyDyeTM (Amersham Biosciences). Proteins extracted from pretreatment sera and post-treatment sera were labeled with Cy3 and Cy5, respectively, mixed with Cy2-labeled internal pooled standard and ran in the same gel. The Cy-dye labeled samples were separated by 2D-PAGE and visualized using a TyphoonTM 9410 imager (Amersham Biosciences). Images were preprocessed to remove areas extraneous to the gel image using ImageQuantTM V5.2 (Amersham Biosciences). Gel analysis was performed using DeCyder TM DIA (Difference In-gel Analysis) V5.0 (Amersham Biosciences). Intra-gel spot detection and inter-gel matching were performed using DIA V5.0 and BVA mode of DeCyder TM software (Amersham Biosciences). Proteins of interest were manually excised and digested with trypsin. The proteins were subjected to nanoLC-MS/MS and identified using MASCOT software (www.matrixscience.com) RESULTS A total of eight over-expressed proteins in the pre-operation sera were identified. To confirm the validity of proteomic results, Western blot was performed by using antibodies to detect sera α 2-macroglobulin (A2M) in the validation groups. Average intensities of A2M progressively increased from normal control, HBV carriers, HBV-LC and HBV-HCC groups. HCC patients who had high pre-operative A2M level had worse survival. Subgroups analysis revealed high serum A2M in HCC patients with normal AFP had poor survival. However, the A2M level didn't affect the outcome in AFP-positive HCC patients. CONCLUSIONS A2M could be a potential biomarker for the diagnosis of HCC and as a prognostic biomarker in patients with normal AFP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1743.