Heterologous specific antisera against human mammary epithelial antigens (HME-Ags), which are present in the human milk fat globule membrane and breast epithelial cells, were used in a solid-phase radioimmunoassay to determine the presence of these antigens in the sera of patients with disseminated cancer of the breast and other organs. Breast cancer patients carry high levels of HME-Ags in their circulation, while patients with disseminated nonbreast cancer, as well as normal female controls, do not. A similar release of HME-Ags in the circulation was shown by us in a model system. To further corroborate these findings, a three-step procedure for the extraction and identification of HME-Ags from the sera was devised. In this analytical procedure, circulating HME-Ags are recovered on a solid phase carrying their corresponding antibody (anti-HME) and radioiodinated in situ. Later, the labeled HME-Ags are released from the solid phase and characterized by NaDodSO4 gel electrophoresis. With this procedure, HME-Ags were isolated from sera of breast cancer patients but not from sera of nonbreast cancer patients or of normal female controls. The extracted HME-Ags had molecular masses of 150,000, 70,000, and 46,000 daltons. To further support these findings, a monoclonal antibody, BLMRL-HMFG-Mc3, directed to the 46,000-dalton HME-Ag was also used to extract its corresponding antigen from sera. Breast cancer patient sera contained such antigen while the sera of the other patients and controls did not. This highly sensitive methodology offers a specific approach to breast cancer diagnosis as well as further insight into the nature ofcirculating antigens with a view to increasing our understanding of breast cancer biology.Early detection and follow-up of breast cancer by noninvasive methodology has been the aim of many studies. Breast tissue markers such as casein (1) and a-lactalbumin (2) and purported cancer markers such as glycosyltransferases (3, 4), glycolipids (5), and phospholipids (6) have been measured in the circulation by a variety of methodologies, but to date none of them has gained widespread acceptance as a breast cancer marker. The markers with high specificity such as casein and a-lactalbumin (which rely for their synthesis on appropriate levels ofhormonal stimulation) are expressed in few tumors and cancer markers such as glycosyltransferases and phospholipids lack specificity for breast (7,8). In view of this, we propose, the use of human mammary epithelial antigens (HME-Ags) as high-prevalence specific markers for breast cancer. HME-Ags are detected by antibodies prepared against the human milk fat globule (HMFG) membrane (9). This membrane is derived from the apical membrane of breast epithelial cells during the process of milk secretion and envelops the fat of milk. HME-Ags are considered cell surface differentiation antigens localized in breast epithelial plasma membrane (9), be these cells normal, neoplastic, fibroadenomatous, displastic, or obtained from male gynecomastias (10). The prese...

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.

The preparation of monoclonal antibodies (MAbs) against the human milk fat globule membrane with preferential binding to breast carcinoma cells is described. Using BALB/c mouse myeloma cells; inter-specific, intra-strain, and inter-strain hybridomas were isolated that identified three different components of the human milk fat globule of approximately 46,000, and 70,000 daltons and a mucin-like glycoprotein complex (NPGP) ranging from 400,000 to over a million daltons, respectively. Three MAbs (BrE1, BrE2, BrE3) identified the latter component which consists of at least three different size molecules for which the aforementioned MAb's have different binding specificities. MAbs, BrE2 and BrE3, bound to normal breast epithelial cells but to a lesser extent than to tumors and only at the apical surface facing the lumen, while they bound breast carcinomas strongly, and often in the cytoplasm as well as on the surface. Higher concentrations of BrE3 were required to stain normal breast compared to breast tumors. BrE1 also stained breast carcinomas both on the surface and cytoplasmically but did not stain normal breast tissue. The MAb, Mc13, as well as the previously reported MAb McR2, both against the 70,000 dalton component, did not significantly stain either normal or cancerous breast tissue in histological sections but did bind significantly to cultured breast epithelial cells and to the milk fat globule membrane. The MAbs, Mc8 and Mc3, reported previously to be against the 46,000 dalton component, stained histologically only malignant breast tissue but only weakly; however, they bound strongly to intact breast carcinoma cells and breast cell membrane preparations with a radioimmunobinding assay. These MAbs should be useful in characterizing the surface of breast epithelial cells, studying surface alterations in malignancy, and possibly in breast cancer diagnosis and therapy.

August-Copenhagen-Irish (ACI) rats are unique in that the ovaryintact females develop high incidence of mammary cancers induced solely by hormones upon prolonged exposure to high levels of estrogen alone. Studies have also shown that such prolonged exposure to high-dose estrogen results in human-like aneuploid mammary cancers in ovary-intact ACI rats. To determine the role of progesterone in mammary carcinogenesis, six-week-old intact and ovariectomized ACI rats were continuously exposed to low-and high-dose estrogen alone, progesterone alone, low-dose estrogen plus progesterone, and ovariectomized ACI rats with high-dose estrogen plus progesterone. Also, ovariectomized ACI rats were treated with high-dose estrogen plus progesterone plus testosterone to determine the role of the androgen, testosterone, if any, in hormonal mammary carcinogenesis. The results indicate that continuous exposure to high, but not low, concentrations of estrogen alone can induce mammary carcinogenesis in intact but not in ovariectomized rats. Mammary carcinogenesis in ovariectomized ACI rats requires continuous exposure to high concentrations of estrogen and progesterone. The addition of testosterone propionate does not affect tumor incidence in such rats. These results suggest that both ovarian hormones estrogen and progesterone are necessary for mammary carcinogenesis induced solely by hormones in ovariectomized ACI rats. Our results are in agreement with the Women's Health Initiative studies, where treatment of postmenopausal women with estrogen (ERT) alone did not increase the risk of breast cancer, but estrogen and progesterone (HRT) did. I n women, breast cancer is the most prevailing endocrine-related malignancy (1), and epidemiological studies argue that estrogens (E) are central to its etiology (2). Most of what we know regarding the role of ovarian hormones in human breast carcinogenesis is based primarily on epidemiological (2) and animal studies with mice carrying mammary tumor virus or treated with chemical carcinogen (see ref.3) and/or rats treated with chemical carcinogens (4-6). The Women's Health Initiative studies showed increased breast cancer risk in postmenopausal women treated with estrogen and progesterone (HRT) (7) but not in postmenopausal women treated with estrogen (ERT) alone (8). Although E has historically been the focus of breast cancer etiology, it is clear that this E effect must be viewed in context of the entire woman or animal. However, the possible role of progesterone (P), if any, as the other main ovarian hormone in mammary carcinogenesis is essential for the understanding of breast cancer biology and its prevention. Progesterone receptor (PR) depends on the presence of E for its expression (9-11). E has been regarded as an initiator (12) and promoter (13, 14) of breast cancer. P has also been thought as a promoter (15) of breast cancer. E has also been thought to induce mammary carcinogenesis via the mitogenesis-mutagenesiscarcinogenesis route (16-18) through centrosome disturbance via aurora kin...

Multiple epitope expression on the breast epithelial mucin was explored using a panel of monoclonal antibodies (MoAbs) created against milk and breast tissue preparations, against blood group determinants, and against other non-breast epithelial mucins. Since the breast epithelial mucin is now used in both diagnostic and therapeutic modalities for breast cancer, and also because altered or incomplete glycosylation in varying degrees is expected in breast carcinoma tissue, the antigenic target used here was the native mucin and sequential stages of deglycosylation introduced to it by HF treatment. Partial deglycosylation increased exposure of core peptide amino acid sequences increasing MoAb binding generally, while it either decreased or occasionally increased binding of blood group oligosaccharides. Cross reactivity of MoAbs to other mucins was low with the breast epithelial mucin (BEM). The study of the affinity binding constants of some of the anti-BEM peptide MoAbs predicted carbohydrate participation in their epitope structure. The identification of different epitopes on the BEM, investigations on their possible epitopic structure, and the study of MoAb binding during different stages of glycosylation of the molecule leads to knowledge on the contribution of carbohydrates to their epitopes and strengthens the ability to understand their performance in their diverse possible applications in breast cancer diagnosis, prognosis, and therapy.