The Clinical Significance Of HBsAg Expression Characteirstics In Hepatocellular Carcinoma

BackgroundWith a rising incidence, hepatocellular carcinoma (HCC) is one of the most popularcancers worldwide. Hepatitis B infection is the most important cause of HCC. Expressionof hepatitis B surface antigen (HBsAg) in HCC has been documented since beginning ofHBV research. It is generally accepted that positive rate of HBsAg in HCC is significantlylower than pericarcinous tissue due to genetic or epigenetic inactivation of HBsAgexpression. And this may contibutes to the immune evasion of HCC cells.However,wehypothesize that the posttranslational modifacation or teriary structure of HBsAg betweenthose from cancer and pericarcinous tissue may be different.And this,at least inpart,contributes to the diminished immunogenicity of HCC cells.Thus,the identification ofa monoclonal antibody selectively binding HCC derived HBsAg will benefit the earlydiagnosis of primary or recurrent HCC.More importantly,tumor HBsAg specific antibodybinding the membrane of HCC cells will be of great interest for targeting immuno-orchemo-therapy of HBV related HCC.PurposeIn order to verify the above hypothesis, we plan to prepare HBsAg monoclonalantibodies, and use flow cytometry, ELISA and immunohistochemistry to detect bindingcharacteristics of these antibodies, in order to find the antibodies with specific recognitionof HBsAg expressed on HCC cell membrane. Using these antibodies, we also want tocompare the different characteristics of HBsAg between HCC tissues and adjacent tissues,and with clinical data, to find the correlation between the different HBsAg expressioncharacteristics and prognosis of HCC.Materials and methods1. Preparation and assessment of monoclonal antibody we use HBsAg to immunize Balb/c mice and use hybridoma technique to preparemonoclonal antibody, and assessed the monoclonal antibody by ELISA, flow cytometry,and immunohistochemistry.2. Screening antibody expression characteristics in HCC specimensWith14hybridoma supernatants, we examined tumor and pericarcinous tissue fromresected HCC specimens with immunohistochemistry and flow cytometryrespectively,.The pattern of HBsAg staining of each antibody in human specimens wereanalyzed.3. Analysis of clinicopathological prognosis of HCCFrom January2007to June2009,42HCC specimens were retrieved from consecutivepatients who had HBV-related HCC underwent hepatectomy at PLA GeneralHospital.Accompany with complete clinical data, each specimen includes canceroustissues and pericarcinous tissue. Ascites purified antibodies were selected and eachspecimen was immunohistochemistrically stained to compare the difference of antibody’sHBsAg detection rate. All patients were divided into groups according to tumor size, AFPlevels, presence of portal vein cancer embolus, tumor differentiation, HBV-DNA levels,single/multiple foci, pericarcinous tissue GGHⅠ expression levels and pericarcinoustissue GGH Ⅱ expression levels, and analyzed by survival analysis to comparerecurrence and survival time. Using COX regression model, we identified the risk factorsassociated with cancer recurrence and survival.Results1. Preparation of monoclonal antibodyWe obtained14monoclonal hybridoma cells and5purified antibodies from ascites.2. Screening antibody characteristicsAccording to the results of immunohistochemistrical staining, we divided antibodiesinto four types: typeⅠ, pericarcinous tissues staining stronger than cancerous tissues; Ⅱ,pericarcinous tissues staining equal to cancerous tissues; Ⅲ, pericarcinous liver cells andHCC cell nuclear staining;4, HCC cell membrane staining. Flow cytometry analysis forHepG2.215cell showed that there were three kinds of antibodies expressed positive results. Representative ascites purified antibodies, No.4in typeⅠand No.2in typeⅡ, wereselected for further investigation.3. HBsAg detection positive rate of the two kinds of antibodiesIn cancerous tissues, the HBsAg detection positive rate of NO.2and NO.4antibodieswas19.04%and16.66%respectively; in pericarcinous tissues, the HBsAg detectionpositive rate of the No.2and No.4antibodies was73.81%and71.42%, respectively.Comparing HBsAg detection positive rate between cancerous tissues and pericarcinoustissues, there was no significant difference between the two clone of antibodies.4. Survival AnalysisLarger tumor size, higher AFP level, portal vein cancer embolus, higher2AbGGHⅡand4AbGGHⅠ levels were associated significantly with decreased recurrence andsurvival time.（P<0.05）5. COX regression modelPortal vein cancer embolus, AFP level,2AbGGH Ⅱ and4AbGGH Ⅱ expressionlevels were independent risk factors for HCC recurrence. Tumor size, AFP level, portalvein cancer embolus were independent risk factors for HCC overall survival time aftersurgery.