Vol. 1, 1071-1077. October 1995 Clinical Cancer Research 1071 Advances in Brief Antibody to ras Proteins in Patients with Colon Cancer1 Masazumi Takahashi,2 Wei Chen,3 David R. Byrd, Mary L. Disis, Eric S. Huseby, Huilian Qin, Larry McCahill, Heidi Nelson, Hiroshi Shimada, Kiyotaka Okuno, Masayuki Yasutomi, David J. Peace, and Martin A. Cheever Division of Oncology, Departments of Medicine [M. T., W. C., M. L. D., E. S. H., H. Q., M. A. C.] and Surgery [D. R. B., L. M.], University of Washington, Seattle, Washington 98195; Colon and Rectal Surgery, Mayo Clinic, Rochester, Minnesota 55905 [H. N.]; Division of Hematology/Oncology, Loyola University, Maywood, Illinois 60153 [D. J. P.]; Department of Surgery, Yokohama City University School of Medicine, Yokohama 236, Japan [H. S.]; and Kinki University, Osaka 589, Japan [K. 0., M. Y.J Abstract The current study examined sera from 160 colon cancer patients and 60 normal individuals to determine whether antibody to mutated p21 ran protein was present. Studies focused on the aspartic acid substitution at amino acid position 12 (denoted D12), one of the most common muta- tions in colon adenocarcinoma. IgA antibodies directed against mutated p2! ras-D12 protein were detected in 51 (32%) of 160 colon cancer patients, but only in 1 (2.5%) of 40 normal individuals. The greater incidence of antibody in cancer patients provides presumptive evidence that immu- nization to the ras proteins occurred as a result of the malignancy. Examination of sera for antibody reactivity to wild-type p21 ras protein (denoted p2! ras-G12) as well as p2! ran proteins bearing the D12, V12, S12, or L61 muta- tions showed that antibody detected was largely to normal segments of the p21 ras protein. Epitope mapping, using peptide neutralization assays with mutated or normal ras peptides as competitors, demonstrated that in 10 (67%) of 15 sera examined the antibody reactivity to p2! ras-G12 pro- tein was neutralized by peptides near the carboxyl terminus of p21 ras protein, but not by peptides spanning the specific point mutation region. Antibody reactivities correlated with peripheral blood lymphocyte count, but did not correlate with patient age, sex, histology, stage, tumor locus, lymph node metastasis, or serum carcinoembryonic antigen. Introduction The ras oncogenes are cancer-related genes that become activated by specific point mutations and can be identified in a Received 5/4/95; revised 7/10/95; accepted 7/13/95. This work was supported by Grant CA54561 from the National Cancer Institute, Department of Health and Human Services. 2 Present address: Department of Surgery, Yokohama City University School of Medicine, 3-9 Fukuura Kanazawa-ku, Yokohama 236, Japan. To whom requests for reprints should be addressed, at Division of Oncology, University of Washington, Box 356527, Seattle, WA 98195- 6527. wide variety of human malignancies (I, 2). Three ras genes have been defined in the human genome, H-ras-1, K-ras-2, and N-ras (3). These genes encode a highly conserved group of Mr 21,000 proteins, denoted as p21 ras. Activation of the ras proto-oncogene occurs most commonly at codon 12 or codon 61 and results in corresponding single amino acid substitutions within the p2l protein. The activating amino acid substitutions impair the GTPase activity of the ras protein and generate constitutively activated signaling complexes with transforming activity (4, 5). Mutated ras proteins are implicated in malignant transformation of many human cancers including approximately 45% of colon adenocarcinomas. The most common specific amino acid substitutions replacing glycine at amino acid posi- tion 12 of the wild-type p21 ras protein in colon adenocarci- noma are aspartic acid (13%), valine (15%), cysteine (6%), or serine (4%; Ref. 6). Only a few cases with leucine replacing glutamine at amino acid position 61 have been reported. Mutated p2l ras proteins are not expressed by normal tissue and thus represent cancer-specific proteins. Mice immu- nized to whole mutated ras proteins are able to mount a T-cell response that is specific for the mutated segment of the molecule (7, 8). Conversely, immunizing animals with peptides that span the mutated segment can result in the generation of both helper/ inducer and cytolytic T-cell responses that recognize mutated protein (9, 10). Recently, it has been demonstrated that T cells from normal individuals can recognize peptides that span the mutated segment of the molecule (11-13). Furthermore, mem- ory T cells, isolated from a patient with a follicular thyroid carcinoma, were found to specifically recognize a ras peptide with a leucine substitution at residue 61 (14), a common muta- tion in that tumor. Studies in our laboratory examining periph- eral blood lymphocytes from patients with pancreatic cancer showed that CD4 T-celI immunity to the mutated segment of ras protein was present in some patients with pancreatic cancer. Studies focused on the aspartic acid substitution (denoted D12) as the most common mutation. Patients were found in whom T cells responded to both ras D12 peptides and p21 ras-D12 protein (15). Existent specific T-celI reactivity to mutated ras peptides and proteins imply that the patient’s own tumor acts as a source of immunizing protein and that mutated ras proteins can elicit immune responses in humans. Often, helper/inducer T cells and antibodies can respond to the same protein. The current study examined whether antibod- ies specific to ras proteins exist in sera of patients with colon cancer. Sera from 160 patients with colon cancer and 60 normal donors were examined to evaluate antibody reactivity to p21 ras proteins using ELISA. Studies focused on the aspartic acid substitution (D12). IgA antibodies directed against p21 ras-Dl2 protein could be detected in colon cancer patients to a substan- tially greater extent than in normal individuals. However, the majority of antibody detected was to the normal but not the mutated portion of p21 ras proteins. The greater incidence of antibody in cancer patients provides evidence that immunization to the ras proteins occurred as a result of the malignancy. Antibody correlated to peripheral blood lymphocyte counts.