Using proteomic approach to identify tumor -associated antigens as biomarkers in hepatocellular carcinoma
Many studies have demonstrated that cancer sera contain antibodies against a unique group of autologous cellular antigens called tumor-associated antigens (TAAs). Intracellular proteins, which are involved in carcinogenesis, can provoke autoantibody responses. Therefore, autoantibodies can be used clinically for cancer detection and for proteomic analysis in identification of TAAs that are potentially involved in malignant transformation. Emerging evidence confirms that each type of cancer can induce unique autoantibody response that reflects the nature of the malignant process in the tissue specimens and serum samples. Liver cancer, especially hepatocellular carcinoma (HCC), is one of the most common tumors in the world. The majority of people with HCC will die within 1 year of its detection. This high case-fatality rate can partially be attributed to lack of diagnostic methods that allow early detection. In the present study, sera from 20 patients with HCC, 30 patients with chronic hepatitis (CH) and 30 patients with liver cirrhosis (LC) as well as 10 sera from normal individuals were used in proteomic approach to identify HCC-related TAAs. Thirty-four immunoreactive protein spots were excised from the two-dimensional electrophoresis (2-DE) gel, digested with trypsin, and subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of 34 immunoreactive protein spots, 28 were identified. Seventeen of 28 identified proteins were not only reactive with serum antibodies in HCC but also with antibodies in pre-HCC conditions, and 11 were only reactive with serum antibodies in HCC but not with antibodies in pre-HCC conditions. In the subsequent analysis, two representative proteins, namely HSP60 and HSP70 were selected as examples for the purpose of validation. The results from immunoassay were consistent with the data from proteomic analysis, supporting our hypothesis that proteins identified with autoantibodies that have been present in pre-cancer conditions may be not appropriate to use as TAA markers in cancer detection. Further study on other identified proteins in this study may help us to define more HCC-specific TAAs as markers in HCC detection. ^ Our previous studies indicated that detection of autoantibodies in cancer can be enhanced by using a mini-array of a panel of TAAs as target antigens. In the present study, we have also integrated our TAA array with protein chip technology to develop a novel TAA chip for further screening 20 HCC serum samples and 16 normal human sera. A total of 19 TAAs including 17 TAAs used in previous studies and 2 TAAs identified in the current study were printed on microscope slides using a robotic arrayer. The results indicate that different HCC serum has different anti-TAA profiles. For example, one HCC serum had immunoreactions with cyclin D, cyclin E, p62, koc, c-myc, survivin, wnt and RalA, and another serum had immunoreactions with p16, cyclin D, cyclin E, koc, c-myc, survivin, wnt and RalA. Of 20 HCC sera tested, six sera showed a much higher intensity value compared to normal controls, indicating that antibody responses to 19 TAAs in some cancer patients were quite robust and not just mildly elevated. Of 16 normal sera screened, only one had weak immunoreactions with cyclin D and wnt protein. In conclusion, this preliminary study demonstrates that it is feasible to develop specific chips in the future for immunodiagnosis of certain type of cancer.^
Looi, Kok Sun, "Using proteomic approach to identify tumor -associated antigens as biomarkers in hepatocellular carcinoma" (2008). ETD Collection for University of Texas, El Paso. AAI3310671.