[CANCER RESEARCH 53. 3233-3236. July 15. 1993] Advances in Brief Identification of a Cell-Surface Antigen (LEA.135) Associated with Favorable Prognosis in Human Breast Cancer1 S. Ashraf Imam,2 Esperanza F. Esteban, Ren S. Chen, Robert D. Cardiff, and Clive R. Taylor Department of Pathology and Comprehensive Cancer Center ¡S.A. I.. E. F. E.. C. K. TJ, University of Southern California School of Medicine, Los Angeles, California 90033: and Department of Pathology [R. S. C., R. D. C.J, University of California Davis Medical Center, Sacramento, California 95817 Abstract The present study was undertaken with a rationale that loss of certain "normal tissue" antigens might have prognostic significance, reflecting inactivation of the corresponding genes during neoplastic progression. An attempt was made to identify such antigens by means of generating mon oclonal antibodies using a tolerization/immunization procedure. A mono clonal antibody generated by immunization of BALB/c mice with normal breast tissue extract, following prior tolerization with mammary carci noma cells, recognized a cell-surface glycoprotein, luminal epithelial an tigen, with an apparent molecular weight of 135,000 (LEA.135). The pat tern of expression on LEA.135 was determined by immunohistochemical- staining techniques on frozen and formalin-fixed and paraffin-embedded tissue sections. LEA.135 was demonstrable on the apical plasma mem brane of normal and nonneoplastic epithelial cells in breast and other tissues. Studies have shown that LEA.135 is distinct from receptors for epidermal growth factor and from known antigens associated with epi thelial cells, including the family of keratins. In a retrospective study, with a follow-up ranging from 5 to 15 years, patients whose breast tumor cells expressed LEA.135 had a superior overall survival rate (78 0.139% at >5 years; P = 0.025). Furthermore, in patients with histologically poorly differentiated tumors, 1,KV. US-positive cases had a better prognosis (80 0.179% at >5 years; P = 0.013) compared with LEA.135-negative cases. In addition, in patients with aneuploid tumors, LEA.135-positive cases again showed an improved survival (90 0.001% at >5 years; P = 0.039) compared with those that were with LEA.135 negative. The results suggest that the expression of LEA.135 provides a useful indication of clinical outcome in patients with breast carcinomas. Introduction Loss of heterozygosity for genes on chromosome Iq, lip, 13q, and 17p in human mammary carcinoma cells has been documented (1-5). However, the reduction to homo- or hemizygosity has not been iden tified at a molecular level. In the current study, an attempt was made to identify any products of gene(s) that may become inactivated in invasive carcinoma cells. In order to achieve the stated goal, the procedure of tolerization/immunization (6) was modified to favor the generation of antibodies to cell products associated with normal cells but lacking malignant cells. The tolerance to invasive malignant mam mary epithelial cell lines (MCF.7 and MDA.MB.231) was induced in neonatal mice, prior to subsequent immunization with an extract of normal breast tissue. A monoclonal antibody that exhibited binding activity with a cell-surface glycoprotein present on normal breast cells, but absent in certain cases of primary breast carcinomas, was identified. The corresponding antigen was purified and termed LEA. 135.3 Received 3/23/93; accepted 6/2/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was in part supported by a grant from Jean Cross Memorial Funds 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: LEA.135. luminal epithelial antigen with an apparent M, 135.000; gp, glycoprotein; MFGM. milk fat globule membrane; HFMG, human milk-fat globule; MEC. mammary epithelial cells; OS, overall survival. Based on the above observation, a retrospective investigation was carried out to investigate the possible prognostic value of LEA.135 expression in 40 cases of primary breast carcinomas with 5-15 years of follow-up. The results suggest that patients whose primary tumor cells express LEA.135 have a significantly favorable prognosis. Materials and Methods Comparison of Epitopes. Competitive immunocytochemically steric-in- ference assays were performed using ¡mmunocytological techniques in order to compare the nature of the epitopes recognized by anti-LEA.135 antibody to those reacting with previously reported antibodies to human mammary epi thelial cells. Sections of breast tissue containing normal epithelial cells were incubated first with the unlabeled test antibodies that included epithelial mem brane antigen (7), MFGM-gp70 (8, 9), MFGM-gpl55 (10, 11), HMFG-1 (12), HMFG-2 (13), pan keratin (14), and receptor for epidermal growth factor (15), followed by incubation with predetermined concentration of biotinylated anti- LEA.I35 antibody. The remainder of the staining procedure was as described previously (6). Any change in the intensity of staining with reference to control preparations was recorded. Metabolic Labeling of Cells and Preparation of Cell Lysate. Owing to the absence of anti-LEA.135 antibody's reactivity to the established tumori- genie mammary carcinoma cell lines (e.g., MCF.7, MDA.MB.231, ZR.75.1, HS578T), a model system that consist of nontumorigenic immortalized MEC lines was utilized in this study (16-18). The cell lines designated 184AI and 184B5 were grown as described by Stampfer (17). Briefly, MEC lines were grown as monolayer cultures in 75-mm2 tissue culture flasks and intrinsically labeled when cultures were still subconfluent. The cells were labeled for 24 to 48 h with either 2 mCi of (3H]leucine or galactosamine (110 Ci/mmol) per flask of leucine or galactosamine-free Dulbecco's minimum essential medium, respectively. Following incubation, the cells were washed 3 times and lysed with 0.05 M Tris-HCI buffer. pH 7.5, containing 0.15 M NaCI, 0.5% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, IITIMphenylmethylsulphonyl fluoride, and 0.5 mM chloromethyl-L-(2-phenyl-l-p-toluenesulphosamide) ethyl ketone on ice for 15 min. The lysates were centrifuged at 40,000 x g and 4°Cfor 10 min. The supernatant containing detergent-solubilized materials were subsequently used for immunoprecipitation. Comparison of LEA.135 with Other Antigens Associated with MEC. Competitive immunoprecipitation analyses were performed to ascertain the nature of antigen recognized by anti-LEA.135 antibody in relation to the antigens of other epithelial cells (7-15). Prior to immunoprecipitation of the various cell line extract (see metabolic labeling), each sample (1 mg protein/ ml) was first preabsorbed separately with antibody to LEA.135, epithelial membrane antigen, MFGM-gp70, MFGM-gpl55, HMFG-1, HMFG-2, pan keratin, or receptor for epidermal growth factor, immobilized individually to Sepharose 4B (5 mg antibody/ml of Sepharose 4B) as described previously (19-21). Preabsorption was carried out by mixing and incubating the suspen sion overnight at 4°C.Following an overnight incubation, the suspension was centrifuged at 10,000 x g and 4°Cfor 15 min. The supernatant containing the preabsorbed extract was removed and subsequently subjected to immunopre cipitation with each antibody separately and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography as described pre viously (19-21). The negative control consisted of the extract which was preabsorbed with the antibody that subsequently also served for immunopre cipitation. Patients. Tissue sections were obtained from the University of California (Davis) Medical Center. Sacramento, CA. The following data were obtained by 3233 Research. on December 24, 2015. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from