Method for identifying autoantigens relevant to cancer

F Madrid MD^a,b, H Alansari^a,b, N Tang^a,b, RL Karvonen^c

^aDepartment of Internal Medicine, Wayne State University, Detroit, MI, United States, ^bKarmanos Cancer Institute, Wayne State University, Detroit, MI, United States, ^cScience Institute for Biomedical Research, Birmingham, MI, United States

AIM: To minimize the confounding effect of unrelated antibodies and non-specific reactions by introducing substantial modifications in the existing immunoscreening methodology to identify autoantigens relevant to cancer. METHODS: Potential cloning sera were selected from patients with established pathologic diagnosis of breast cancer and known outcome. Immunoblots of tumor cell proteins were probed with a wide selection of sera representative of breast cancer. Sera exhibiting high titer (1: 500 dilution) IgG antibodies were pre-selected, and those sera showing antibodies significantly associated with diagnosis or exhibiting IgG bands of identical molecular mass were used for immunoscreening a T7 phage cDNA display library constructed with non-autologous cancer cells. Ten sera from patients with invasive ductal carcinoma (IDC) of the breast were selected to immunoscreen the T7 phage cDNA library. Absorption steps with host cells with or without vector antigens and biopanning with pools of "non-cancer" sera to eliminate irrelevant clones were omitted. The guidance of immunoreactivity rather than random selection identified positive clones in each round of biopanning. This approach was validated by constructing an autoantigen microarray with the phage inserts cloned by immunoscreening. The microarray was probed with a training set of sera from patients with established diagnoses of breast cancer and known outcome, with non-cancer control sera and with sera from patients with autoimmune diseases. The microarray was then probed with an independent set of sera from patients with cancer and with a different set of subjects without cancer. After identifying informative phages, the corresponding cDNA inserts were amplified by PCR to determine the size of the clones and cDNA sequences were obtained. RESULTS: We assembled a collection of 938 T7 phages encoding potential breast cancer autoantigens and identified a panel of breast cancer autoantigens that were significantly recognized by multiple sera from patients with IDC and ductal carcinoma in situ (DCIS) of the breast, but were non-reactive with non-cancer control sera or with sera from patients with autoimmune disorders, suggesting that they are potentially excellent biomarkers for the diagnosis of breast cancer. Some of the cloned autoantigens reacted exclusively or predominantly with sera from patients with either DCIS or IDC of the breast, suggesting that this method may also reveal antigen heterogeneity of breast cancer. Some of these phage inserts showed complete identity with partial cDNA sequences encoding for known proteins, while other cloned sequences did not show any homology in the GenBank database. CONCLUSIONS: We propose that the use of this modified immunoscreening approach may allow the consistent identification of autoantigens relevant to cancer that could be used as biomarkers with diagnostic significance.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 797 (Manifestations of cancer).