Detection of minimal residual breast cancer in bone marrow.
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Bone Marrow↗
Biomedical subjects
R L Ceriani
Publications and source records attributed to R L Ceriani.
5-Iododeoxyuridine increases the efficacy of the radioimmunotherapy of human tumors growing in nude mice.
Recently, there has been much interest in the use of radionuclide conjugated monoclonal antibodies for the treatment of human malignancies. One way to potentially maximize the therapeutic effectiveness of radioimmunotherapy would be to sensitize tumor cells to the radiation dose delivered by the antibody. Since radioimmunotherapy can potentially treat disseminated disease, including micrometastasis, we chose to study a halogenated pyrimidine radiosensitizer, a class of compounds that affect nonhypoxic cells. 5-Iododeoxyuridine, administered with pyrimidine metabolism modulators, increased the therapeutic effectiveness of radioimmunotherapy, resulting in individual cures of human tumors growing in BALB/c nu/nu (nude) mice. 5-Iododeoxyuridine was administered with N-(phosphonacetyl)-L-aspartic acid and 5-fluoro-deoxycytidine plus tetrahydrouridine. This drug treatment was combined with radioimmunotherapy using 131I conjugated to a monoclonal antibody, Mc5. Mc5 binds to a mucin component of the human milk fat globule. This antigen is expressed on the surface of MX-1 cells, the transplantable human tumor used in this study. Tumor-bearing mice treated with both the drug protocol and 131I-Mc5 (540 microCi, 10 microCi/micrograms) showed a regression in average tumor volume. The average tumor volume was reduced below the initial size at treatment for 50 days; two of five cures were obtained. Neither cures nor regressions were observed with either the drug or antibody treatments alone. Our results indicate the potential for increasing the therapeutic effectiveness of radioimmunotherapy of human solid tumors with halogenated pyrimidines.
Animals↗
A Mr 46,000 human milk fat globule protein that is highly expressed in human breast tumors contains factor VIII-like domains.
The human milk fat globule has proved to be a good source of antigenic material for production of antibodies against surface components of breast epithelial cells. Monoclonal antibodies against one of the major components of the human milk fat globule, which identify a glycoprotein with an apparent molecular weight of 46,000, have been found to be useful for both breast cancer diagnosis and therapy. In order to characterize this Mr 46,000 glycoprotein, specific monoclonal antibodies were used to select complementary DNAs from a lambda gt11 expression library from lactating breast. The largest complementary DNA insert (BA46-1) was 1270 base pairs and encoded 217 amino acids. A single 2.2-kilobase RNA was specifically detected in a variety of carcinoma cell lines, using this complementary DNA probe, and it was overexpressed in some carcinoma lines. The mRNA levels correlated with the level of expression of the antigen in these cell lines as detected by Western blot analysis. Sequence analysis revealed strong homology of the Mr 46,000 glycoprotein with serum factors VIII and V, in the region implicated in phospholipid binding.
Amino Acid Sequence↗
Breast epithelial mucin serum clearance.
The breast epithelial mucin is one of the currently used serum breast cancer markers. Immunoassays detect elevations of this marker in breast cancer relapse; however, values obtained do not usually correlate with breast tumor load and false negatives are frequent. The causes for such results were investigated in an immunodeficient mouse grafted with transplantable human breast tumors. Increases in serum breast epithelial mucin values are only found after a significant threshold of tumor load is reached and only when a highly increased release of the marker is created by radioimmunotherapy. This indicates that substantial amounts of the marker must be released to overcome clearance. Further, a hepatic clearance mechanism is demonstrated since carbon tetrachloride toxicity increases breast epithelial mucin levels in tumor bearing mice.
Animals↗
Evidence of extensive phospholipid fatty acid methylation during the assumed selective methylation of plasma free fatty acids by diazomethane.
We compared a conventional method (Method I) for measuring plasma free fatty acid (FFA) concentrations with two more rapid procedures (Method II and Method III). Method I required total lipid extraction, separation of FFA by thin-layer chromatography, methylation, and gas-liquid chromatographic analysis of the fatty acid (FA) methyl esters. Method II was a colorimetric procedure. Method III relied upon diazomethane's presumed ability to selectively methylate FFA even in the presence of FA esters. The three methods were compared using plasma from fasted and from fed nude mice, tumor-bearing mice (MX-1 and ZR-75-1 human mammary carcinomas), and controls. Method II, was less reliable than Method I, but both gave similar mean values for plasma FFA levels in fasted mice. Both Methods I and II also showed similar lowering of plasma FFA levels after feeding previously fasted mice. Method III consistently gave values that were far greater than those obtained using Methods I and II. Moreover, highly significant differences between fasted and fed mice were obscured by direct methylation of plasma FFA with diazomethane (Method III). The excess FA methyl esters formed in Method III were derived from plasma phospholipids, but not from plasma triacylglycerols. After feeding fasted mice, plasma free palmitic acid and oleic acid levels fell (Method I); by contrast, the excess "FFA" formed by methylation of plasma phospholipid FA increased two-fold and fourteen-fold, respectively. Caution is therefore advised in the use of direct methylating agents when measuring total and individual plasma FFA levels.
Animals↗
Cloning and sequencing of a complementary DNA encoding a Mr 70,000 human breast epithelial mucin-associated antigen.
The human milk fat globule (HMFG) membrane contains several glycoproteins that have been referred to as breast differentiation antigens and that are expressed in normal breast, breast tumors, breast tumor-derived cell lines, and are found in breast cancer patient serum. These antigens include a high molecular weight mucin and several smaller components including Mr 150,000; 70,000; and 46,000 glycoproteins. We have used 2 monoclonal antibodies (McR2 and Mc13) that bind the Mr 70,000 component of HMFG to immunoscreen a lambda gt11 expression library prepared from human lactating breast tissue. We report here the sequence of a complementary DNA clone (BA70-1) that codes for a peptide that binds both McR2 and Mc13 but not monoclonal antibodies to the breast mucin or other components of HMFG.A 1.8-kilobase RNA was detected in 9 of 9 breast tumor cell lines using 32P-labeled BA70-1 as probe. The BA70-1 RNA was highly expressed in 6 of 9 cells lines of breast and several other carcinomas lines compared with a lymphoblastoid cell line (Raji). The BA70-1 complementary DNA sequence has no extensive homology with previously reported sequences including the high-molecular weight mucin complementary DNA. Since the Mr 70,000 molecule appears to be associated with the breast mucin by disulfide bonds, its study could help elucidate the structure of this latter complex and how it is organized in the cell membrane, and prove useful in diagnosis and therapy of breast cancer.
Antibodies, Monoclonal↗
Effect of dietary fat and monoclonal antibody therapy on the growth of human mammary adenocarcinoma MX-1 grafted in athymic mice.
A diet containing 11% per weight fish oils (Max-EPA) reduced the rate of growth of a transplantable human breast tumor (MX-1) grafted into immunodeficient mice (BALB/c, nu/nu) when compared to MX-1 tumors in mice fed polyunsaturated (corn oil) and saturated (lard) fatty acids; however, there was no difference between the corn oil and lard animal groups. Treatment with a cocktail of monoclonal antibodies (MoAbs) decreased the rat of growth of MX-1 in the corn oil fed animals but not in those fed lard or Max-EPA, but the rate of tumor growth of the Max-EPA treated group, either MoAb treated or not, was slower than that of the corn oil and lard groups.
Adenocarcinoma↗
Diagnostic ability of different human milk fat globule antigens in breast cancer.
Human mammary epithelial antigens (HME-Ags) are released into the circulation by breast tumors and not by normal breast tissue (Proc. Natl. Acad. Sci. USA 74: 582-586, 1977). This characteristic made them valuable, together with other breast cancer related antigens later identified, to develop immunoassays useful in the follow-up of breast cancer. Assays for these antigens in serum have less than complete sensitivity and partial specificity, and as a result of this have not been totally successful in studying the relapsing breast cancer patient. In the present work, correlations are made among 3 assays available for breast cancer disease follow-up. They detect HME-Ags, CEA, and the heavy molecular weight mucin of the human milk fat globule (HMFG). Values for sensitivity and specificity for the 3 assays were obtained from approximately 300 samples of patients whose clinical diagnosis at the time of blood drawing was rigorously established. A small but definite advantage in sensitivity is demonstrated for the HME-Ags assay over the other two. A similar advantage is also demonstrated in the sequential follow-up of breast cancer patients, where HME-Ags respond more rapidly in most instances to changes in tumor burden. Further, the ability of increases in levels of these assays to predict relapse was studied in 15 patients who relapsed. HME-Ags demonstrated a predictive value of 73%, while CEA and the heavy molecular weight mucin remained at 47%. The present study exemplifies the search for novel antigens (Ags) with maximal ability to detect breast cancer relapse and with improved sensitivity to monitor tumor burden changes. Here, assays for different antigens to be compared are tested in the same serum samples obtained from carefully staged patients. The results suggest a role as breast cancer markers for antigens of lower molecular weight than the epithelial mucin-like components studied previously.
Antigens, Neoplasm↗
Breast epithelial antigen levels and breast tumor antigen content.
The relationship between the primary tumor expression of a breast epithelial antigen, called non-penetrating glycoprotein (NPGP) or breast epithelial mucin, and the same patient's serum level of this antigen at the time of relapse was studied in 23 cases. The expression of NPGP on breast tumors was measured by immunoperoxidase staining using monoclonal antibody Mc5, and quantitated by a histopathological index created for this purpose. Serum levels were measured by a competitive RIA using the same monoclonal antibody. An inverse correlation between these parameters was found, such that tumors having high NPGP levels in serum had a low index, while low NPGP serum levels had a high index. These results show that cellular events in breast tumors could participate in determining NPGP serum levels in breast cancer.
Antibodies, Monoclonal↗
Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule.
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.
Animals↗
Breast epithelial antigens in the circulation of breast cancer patients.
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Adult↗
Human milk-fat globule membrane derived mucin is a disulfide-linked heteromer.
The human milk-fat globule membrane (HMFG) contains a number of antigens also expressed on breast tumors. The dominant antigens are a mucin of MW greater than 400,000 and a group of antigens of MW 67,000-70,000. The mucin was separated with monoclonal antibodies against HMFG and a MW 70,000 doublet was found to be associated with the mucin under non-reducing conditions, the latter identifiable by another set of monoclonal antibodies. Immunoprecipitation of the mucin and analysis of the precipitated material using Western blots and identification of transferred material with monoclonal antibodies demonstrated that the MW 70,000 protein and the mucin were co-precipitated and linked by reducible disulfide bonds. Treatment with detergents, protein-dissociating agents and glycosidases did not release the component from the mucin. Mucins in HMFG membrane and breast tumor cells may be associated therefore to disulfide-linked linker proteins similar to those described for intestinal and gastric mucins, that would co-purify with the mucin under non-reducing conditions.
Antibodies, Monoclonal↗
Experimental therapy of human breast tumors with 131I-labeled monoclonal antibodies prepared against the human milk fat globule.
Breast tumors are susceptible to attack by unconjugated anti-human milk fat globule monoclonal antibodies (MoAbs) and most particularly by their mixture (cocktail) (Cancer Res., 47: 532-540, 1987). In the present study the same MoAbs (Mc1, Mc3, Mc5, and Mc8) labeled with 131I, either singly or in cocktails, were used for a similar purpose. Biodistribution studies showed that a transplantable human breast tumor line (MX-1) implanted in BALB/c nude mice (nu/nu) had the maximum incorporation of injected 131I-MoAbs at day 4 while levels in circulation and in normal tissue declined steadily from day 1. Also, these studies showed that the amount of radiolabeled Mc3 MoAb incorporated by MX-1 tumors was greater than that for cocktail of MoAbs and MoAb Mc5. Tumor destruction by injected 131I-MoAb cocktail was shown in therapy experiments to be dose dependent. A single injection (1500 [corrected] microCi/mouse) of 131I-MoAb Mc3, or of cocktail, produced large breast tumor volume diminution and inhibition of growth for up to 30 days while a similar dose of 131I-labeled control IgG had no effect. A second dose of 1500 [corrected] microCi 131I-MoAb of Mc3 or of cocktail, injected at an appropriate interval, again diminished tumor mass significantly and inhibited its growth for another 20 days. In control experiments, non-breast tumors (colon) were marginally affected by the 131I-MoAbs. These results show that the systemic injection of radioiodinated MoAbs against human milk fat globule destroy the epithelial cells of human breast tumors and control their growth for an appreciable length of time. Radioiodoconjugated MoAbs proved to be more effective than unconjugated MoAbs in reducing breast tumor mass and also in inhibiting growth for longer periods of time at immunoglobulin doses 100 to 200 times lower. Further exploration of their role in breast cancer treatment seems warranted by these results.
Animals↗
Localization of human breast tumors grafted in nude mice with a monoclonal antibody directed against a defined cell surface antigen of human mammary epithelial cells.
A mouse monoclonal antibody (BLMRL-HMFG-Mc5) prepared against a defined cell surface antigen of human mammary epithelial cells, non-penetrating glycoprotein (NPGP), was used in imaging and distribution studies in athymic nude mice grafted with human breast tumors. For in vivo tissue distribution studies, 125I-labeled monoclonal antibody was injected into nude mice carrying simulated metastases of human tumors (breast and colon carcinomas). After 22-24 hr the amount of radioactivity per gram of tissue was 3-4 times higher in the breast tumor than in liver, brain, lung, muscle, or spleen. In contrast, colon carcinoma tissue, grafted and treated likewise, did not show higher accumulation of radioactivity relative to other tissues. At 4 days, the incorporation in breast tumors remained almost as high, while the circulating radioactive tracer and the incorporation in tissues other than breast had fallen significantly. In tumor imaging studies, breast tumor masses as small as 4 mm in diameter were clearly localized on a whole body scan using 131I-labeled BLMRL-HMFG-Mc5 antibodies with a High-Purity germanium gamma camera. Normalization of 131I-distribution to that of 99mTc-pertechnetate increased the specificity of this imaging methodology. The quantitative density of 131I-label was 2-3 fold higher over the breast tumor than over comparable areas of the mouse. No positive localization images were obtained for similar implants of colon and lung carcinomas or melanomas after injections of 131I-labeled BLMRL-HMFG-Mc5. Localization of human breast tumors in this model can be achieved with 131I-labeled anti-breast epithelial monoclonal antibodies.
Animals↗
Significance of breast carcinoma-associated antigens as a monitor of tumor burden: characterization by monoclonal antibodies.
Use of monoclonal antibodies (Mc 3 and Mc 8) prepared against human mammary-epithelial antigens of human mild fat globule membranes has enabled characterization of breast carcinoma (BC) associated antigens (BCAA), antibodies, and circulating immune complexes (CIC). For this study, BC patients were grouped on the basis of measurable tumor burden: Group I patients with no evidence of disease at sampling time; Group II patients with tumor burden less than or equal to 5 g; and Group III patients with known regional or distal metastases. In an in vitro simulation of tumor burden change, selected BC patients' sera were admixed with Mc 3 and Mc 8 at optimal concentration. CIC reduction (dissociation) for Groups I and III and increment (formation) for Group II were noted. Unlike Group I sera, Groups II and III sera required 4- to 16-fold dilution of Mc 3 and 4-fold more concentrated Mc 8 to achieve maximal CIC changes. Serum BCAA isolated by use of both Mc 3 and Mc 8 immunobead procedures showed apparent Mr 33,000 monomer, 66,000 dimer, and 95,000 trimer. When BCAA were added to BC patients' sera, autologous combinations resulted in small (7.7S) CIC for Groups I and III, and medium (9 to 12S) CIC for Group II. Conversely, allogenic combinations resulted in mainly small CIC for Group I, and intermediate CIC for Group II and Group III. Evaluation of circulating BCAA concentration by use of a three-step radioligand technique demonstrated significant discrimination between BC patients' sera (mean = 105 ng/ml) and normal control sera (less than or equal to 20 ng/ml). BCAA were found to be elevated in 31 of 46 (67%) Group I (mean = 70 ng/ml), 41 of 43 (95%) Group II (mean = 197 ng/ml), and 30 of 46 (65%) Group III (mean = 50 ng/ml) patients' sera, as compared to "background" levels in malignant melanoma and normal controls. Benign breast disease sera showed moderate BCAA increases (mean = 48 ng/ml) in 20 of 35 (57%) patients. Furthermore, serial sample determination of BCAA in 36 selected BC patients confirmed the above pattern, indicating that this assay can be used with some restriction to monitor tumor burden. Whereas in early breast carcinoma increase in BCAA concentration was concurrent with or antedated clinical objective evidence of tumor burden increase, significant decreased BCAA concentration was observed with tumor burden reduction. Overall, increased BCAA levels were associated with limited tumor burden (Group II) while decreased BCAA levels were observed with no evidence of disease (Group I) and known regional or distal metastatic advanced disease (Group III) during patients' follow-up.
Antibodies, Monoclonal↗
Experimental immunotherapy of human breast carcinomas implanted in nude mice with a mixture of monoclonal antibodies against human milk fat globule components.
Immunological therapy of BALB/c nude mice (nu/nu) implanted with human breast tumors, estrogen receptor negative MX-1 and estrogen receptor-positive MCF-7, was carried out with four monoclonal antibodies (MoAbs) raised against human milk fat globule membrane glycoproteins also present on normal breast epithelial cells. MoAbs injected singly or as a partial mixture arrested growth of the tumors but to a lesser extent than a mixture ("cocktail") of all four MoAbs. Two model systems were developed in order to examine the capabilities of the four MoAbs to arrest human mammary tumor growth. In the first model the ability of these MoAbs to arrest tumor growth during a 6- to 8-week period was tested by injection of the MoAbs immediately before and after implantation (passive immunization) and thereafter every other day. In the second model the effect of these MoAbs on established and growing tumors was tested. Using the cocktail in the passive immunization protocol, human mammary tumor growth in nu/nu mice was arrested either completely or averaging to one-tenth the size of the controls for those mice in which the tumors had taken. Other human carcinomas, colon and lung, under the same protocol, were not affected. Injection of cocktail every 2 days into nu/nu mice with established and growing human breast tumors (both estrogen receptor positive and negative) produced arrests of tumor growth of 44.1, 45.2, 49.8% of their controls after 7 to 8 days of treatment. Previously, it has been established that human mammary tumors are heterogeneous in expression of the human milk fat globule antigens recognized by our antibodies to the extent that some cells may have large amounts and others no detectable amount of a particular antigen. Those MX-1 tumors treated for a prolonged time with the cocktail of MoAbs that survived and continued to grow could be the result of the preferential multiplication of those cells in the heterogeneous population which had low or no antigen content. The breast tumors that did grow in the nu/nu mice after 8 weeks of injection of the cocktail revealed by immunoperoxidase staining a 90% reduction in the antigen content as recognized by these MoAbs when compared with untreated tumors. These results attest to the effectiveness of unconjugated anti-human milk fat globule MoAbs to arrest human breast tumor growth in nu/nu mice, and they also suggest that to best arrest tumor growth the use of a mixture of MoAbs should be considered.
Animals↗
Second international workshop on monoclonal antibodies and breast cancer. San Francisco, November 20-21, 1986.
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Antibodies, Monoclonal↗
Role of circulating human mammary epithelial antigens (HME-Ags) as serum markers for breast cancer.
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Alkaline Phosphatase↗