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We report on the identification of autoantigens commonly recognized by sera from patients with breast cancer. We selected ten sera from patients with invasive ductal carcinoma (IDC) of the breast with high titer IgG autoantibodies for biopanning of a T7 phage breast cancer cDNA display library. A high throughput method involved the assembly of 938 T7 phages encoding potential breast cancer autoantigens. Microarrays of positive phages were probed with sera from 90 patients with breast cancer [15 patients with ductal carcinoma in situ (DCIS) and 75 patients with IDC of the breast], with 51 non-cancer control sera and with sera from 21 patients with systemic autoimmune diseases. A 12-phage breast cancer predictor group was constructed with phage inserts recognized by sera from patients with breast cancer and not by non-cancer or autoimmune control sera (P < 0.0001). Several autoantigens including annexin XI-A, the p80 subunit of the Ku antigen, ribosomal protein S6, and other unknown autoantigens could significantly discriminate between breast cancer and non-cancer control sera. Biopanning with three different sera led to the cloning of partial cDNA sequences identical to annexin XI-A. IgG autoantibodies reacting with the amino acid 41-74 sequence of annexin XI-A were found in 19% of all women with breast cancer but in 60% of sera from women with DCIS of the breast. In addition, partial sequences identical to annexin XI-A, nucleolar protein interacting with the forkhead-associated (FHA) domain of pKi-67, the KIAA1671 gene product, ribosomal protein S6, cyclin K, elongation factor-2, Grb2-associated protein 2, and other unknown proteins could distinguish DCIS from IDC of the breast and appear to be potential biomarkers for the diagnosis of breast cancer.

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The failure to identify biomarkers of clinical significance for cancer diagnosis and prognosis generated a great deal of skepticism in regard to the usefulness of autoantibody-based methods. SEREX was a major advancement in immunoscreening that resulted in the identification of a large group of autoantigens recognized by cancer sera. However, few SEREX-defined autoantigens have proven to have definitive diagnostic value in clinical practice. Often, the identified antigens are patient-specific rather than tumor-specific and many tumor-associated antigens are rare in expression libraries made from non-autologous cells. Since autoantibodies are part of the normal immune response, it can be difficult to single out tumor-associated antibodies from the scores of irrelevant patient-specific responses. In our view, any practical approach for identifying cancer-related autoantigens must include an integral strategy for demonstrating tumor relevance early in the screening process. Care must also be taken not to exclude potentially important autoantibodies by pre-screening manipulations to patient sera. We have introduced substantial modifications in SEREX, designed to minimize confounding effects of unrelated autoantibodies and to eliminate steps that preclude the identification of cancer-related autoantigens commonly recognized by cancer sera. In addition, we incorporate methodology to identify candidate antigens that have potential diagnostic or prognostic value prior to their molecular cloning and characterization.