In vitro modulation of cellular localization of milk fat globule membrane antigens in human breast carcinomas

Alterations in the cellular localization of cell surface components such as the milk fat globule membrane are a common feature of breast carcinomas and relate to the differentiation of a tumour. This study has examined the potential modulation of such components. A group of carcinomas were cultured with and without insulin and/or hydrocortisone and the site of staining for milk fat globule membrane, as detected by the antibodies HMFG 1, HMFG 2, and NCRC 11, was assessed using light microscopic and electron microscopic immunohistochemistry. Modulation of localization, with a shift from cytoplasmic vesicle labelling to submembraneous vesicles/cell surface labelling and intracytoplasmic luminal labelling, was observed in 5 of 14 moderately differentiated and 8 of 11 poorly differentiated carcinomas. Two well differentiated carcinomas continued to show peripheral labelling; three poorly differentiated carcinomas showed no change from cytoplasmic labelling only; and the other carcinomas exhibited heterogeneous localization, making any change difficult to assess. Insulin was required for any change to be observed and it is suggested that this has an effect on the mechanisms for intracellular transport of membrane and secretory proteins.     

Use of immunohistochemical staining panel for characterisation of ovarian neoplasms.

Eighty five ovarian epithelial and non-epithelial tumours were studied by peroxidase histochemical staining for their reactivity with six monoclonal human milk fat globule (HMFG) antibodies, peanut agglutinin (PNA) lectin, and a monoclonal cytokeratin antibody. HMFG IIIC12 and cytokeratin antibodies distinguished epithelial from non-epithelial tumours. The staining patterns of mucinous and serous tumours were essentially different from each other; poorly differentiated anaplastic carcinomas showed similar antigenic content to that of the serous cystadenocarcinomas. Furthermore, staining with PNA lectin and HMFG antibodies was useful in distinguishing clear cell carcinomas from other malignant epithelial tumours of the ovary.     

The expression of CEA, CA 19-9 and HMFG antigens in ovarian clear-cell and endometrioid carcinomas.

The tumour antigen expression of ovarian and endometrial endometrioid carcinomas, ovarian clear-cell carcinomas as well as endometrial and cervical clear-cell carcinomas were immunohistochemically compared. Of special interest were potential differences between the endometrioid and clear-cell carcinomas of the ovary. The expression of CEA and CA 19-9 tumour antigens in all these tumour types was heterogeneous, with 10-20% of the cases being positive for CEA and 40-75% being positive for CA 19-9. In contrast, HMFG IIIC 12, a monoclonal antibody originally directed against human milk fat globule (HMFG) membrane antigens, invariably detected a corresponding antigen on every case of these tumour types. Another HMFG antibody, SM IF 3, on the other hand, detected antigenic material on all clear-cell tumour types, but only rarely on endometrioid tumours of the ovary or endometrium. While HMFG IIIC 12 detects an antigen present on all ovarian, endometrial and mammary carcinomas, antibody SM IF 3 thus appears to be more restricted in its staining patterns. Our results with both of these antibodies indicate that ovarian clear-cell carcinomas and ovarian endometrioid carcinomas have antigenic differences, which provides further evidence that they belong to different tumour entities.     

Ultrastructural localization of milk fat globule membrane antigens in human breast carcinomas

The localization of milk fat globule membrane components has been assessed using post-fixation immunoelectron microscopy with three different antibodies for a group of breast carcinomas of different type and histological differentiation. For well differentiated carcinomas localization was in relation to the cell membrane, with polarization being evident in a proportion of cases. Moderately differentiated carcinomas showed a combined picture of cell membrane, vesicular, and intracytoplasmic luminal localization. The latter is a feature of infiltrating lobular carcinomas. Poorly differentiated carcinomas exhibited vesicular labelling throughout the cytoplasm, with no cell membrane localization. No labelling was seen over endoplasmic reticulum. It is proposed that carcinomas exhibit defects in intracellular transport of milk fat globule membrane components resulting in failure of expression at the cell surface and accumulation of vesicles within the cytoplasm, the extent of change relating to tumour differentiation.     

Human milk fat globule antigen III D 5, steroid receptors and histopathologic parameters in breast cancer

Immunohistochemical reactivity of mammary carcinomas with the monoclonal human milk fat globule (HMFG) antibody III D 5, and the estrogen receptor (ER) and progesterone receptor (PR) status were compared with the histopathology of primary breast cancer. The reactivity with III D 5 has earlier been shown to be associated with the estrogen receptor status of tumours and with a favourable prognosis. The reactivity of tumours with III D 5, as well as the presence of ER and PR correlated significantly with the histological features of differentiation; histological grade, nuclear grade, tumour necrosis and lymphoid infiltration. Reactivity with III D 5 correlated with all these parameters, while the presence of ER did not correlate with the nuclear grade and that of PR correlated only with the nuclear grade and the lymphoid infiltration of tumours. Reactivity of III D 5 may thus have prognostic and therapeutic implications in the management of breast cancer.     

Detection of surface antigens defined by monoclonal antibodies in primary mucinous breast carcinomas

Summary Three monoclonal antibodies, 67D11, 115H10 and 115C2, raised against human milk fat globule membranes, have been applied to 207 primary mucinous breast carcinomas. The turnouts reacted positively in 18% (67D11), 54% (115H10), and 20% (115C2) of the cases. Thedetected epitopes (MAM-3a (67D11), MAM-3b (115H10), and MAM-3c (115C2)) have formerly been shown to be markers of differentiation in infiltrating ductal carcinomas. In the present group of mucinous breast carcinomas, statistically significant correlation to high risk factors, such as occurrence of primary lymph node metastases, large tumour size, and local invasion of the tumour into overlying skin or deep fascie, were found. Furthermore, the antigen expression was less marked in pure mucinous carcinomas as compared to carcinomas also presenting with non-mucinous tumour areas. Thus, especially the antigen MAM-3b, is more frequently present in mixed tumours, in large tumours, in tumours with local invasion, and in tumours with primary lymph node metastases. However, no association could be demonstrated between expression of MAM-3b and recurrence-free survival.Mucinous carcinomas of the breast apparently differ from other carcinomas not only with respect to morphology, but also in their pattern of antigenic expression in relation to other prognostic factors.     

Quantitation of human milk fat globule (HMFG1) expression in breast carcinoma and its association with survival.

Expression of human milk fat globule (HMFG1) in immunohistochemically stained sections of breast carcinomas was assessed subjectively and objectively from 82 women (age range 41-96 years) to determine its prognostic importance. No correlation was observed between the degree of staining and prognosis even when the subcellular distribution of antigen expression was assessed. The total absence of staining with HMFG1 was possibly associated with a favourable outcome, although this did not quite achieve significance with the small numbers involved. The Quantimet 970 was used for objective semiautomated measurement of immunohistochemical reactions in paraffin wax sections and was found to produce better resolution and to eliminate subjective error.     

Expression of the pS2 peptide in primary breast carcinomas: Comparison of membrane and cytoplasmic staining patterns

The pS2 protein is oestrogen-regulated in breast cancer cell lines. Previous studies have shown a relationship to oestrogen receptor in primary breast carcinomas. This study examined 178 breast carcinomas for pS2 using immunohistochemistry. A high frequency (77 per cent) of positive tumours was found, using a 10 per cent cut-off point to define a positive tumour. There was no relationship with menopausal status or node status, a significant association with differentiation, a weak association with oestrogen receptor, and no association with progesterone receptor or overall survival. Two patterns of cellular localization were observed: cytoplasmic and membrane. The former showed a stronger relationship with oestrogen receptor status, although there were oestrogen receptornegative tumours with marked pS2 staining. Membrane staining showed a stronger relationship with differentiation, with a staining pattern similar to that observed for milk fat globule membrane. The staining patterns observed may support a role for pS2 in a secretory mechanism. However, the expression and function of pS2 in breast carcinomas emain complex, and are not simply related to oestrogen regulation.     

Subcellular localization of HMFG2 in breast carcinomas: an immunohistochemical and immunoelectron microscopic study.

Malignant transformation of human cells is associated with morphological and biochemical alterations. We have studied the distribution and pattern of staining of HMFG2 (human milk fat globulin) in normal breast, benign breast lesions, and 137 primary and metastatic breast carcinomas. Immunohistochemical staining was performed with an antibody to HMFG2 using the indirect peroxidase technique. Three patterns of staining were noted: 1) secretion and luminal staining (in normal breast, most benign breast lesions and some breast carcinomas); 2) plasma membrane staining (in breast carcinomas); 3) intracytoplasmic staining (in breast carcinomas). Immunoelectron microscopy was also performed on normal breast, infiltrating duct, and lobular carcinomas. Immunoelectron microscopy showed localization of the gold particles on the electron dense granules of the HMFG2 protein. These were localized along the surface of the extracytoplasmic lumina in normal breast ducts/acini and breast carcinomas, whereas localization was also noted within the intracytoplasmic lumina in cancer cells only. These results show that there is altered localization of milk fat globulin in breast cancer cells associated with membrane internalization and formation of intracytoplasmic lumina. This contributes to the understanding of the phenotypic alterations associated with malignant transformation in breast cancer.     

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma

Summary The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.     

Modulation of cell surface componts of human breast cancer cells by retinoic acid: Enhanced HLA-DR Expression

The effect of the differentiating agent all-trans-retinoic acid on the expression of components of the milk fat globule membrane and HLA-DR by breast cancer cell lines has been examined. Effects on proliferation were also considered, to determine whether any cell surface changes were related to or independent of proliferation effects. No significant differences were observed in the expression of components detected by the milk fat globule membrane antibodies HMFG1 and HMFG2 over 8 days culture with 10^-7-10^-9 M retinoic acid for the cell lines MCF-7, T-47 D, ZR-75, MDA-MB-231, and BT-20. In contrast, there was enhanced expression of HLA-DR by two oestrogen receptor-positive cell lines T-47 D and ZR-75 and the oestrogen receptor-negative line MDA-MB-231, with differing sensitivities. These effects were independent of inhibition of proliferation, which was only observed for oestrogen receptor-positive cell lines and for different durations of exposure. The finding of enhanced HLA-DR expression after retinoic acid treatment has not previously been reported and is of interest regarding clinical potential for the induction of tumour immunity.     

Similarities of antisera to casein and epithelial membrane antigen

Summary Antisera raised against human milk fat globule membranes and against the casein fraction of human milk have been compared. Using an immunohistochemical stain of tissue sections it has been shown that many of the antigenic determinants detected by the different antisera are identical. A radioimmunoassay for epithelial membrane antigen (EMA) showed that casein preparations are associated with small quantities of EMA. Antisera to casein frequently contained appreciable concentrations of antibodies to EMA and this accounts for the immunohistochemical staining of non-mammary tissues.     

CEA and HMFG in hyperplastic and malignant lesions of the breast

In order to evaluate a possible transition from proliferative lesions of the breast to ductal carcinoma in situ (DCIS), an immunohistochemical retrospective analysis was carried out. Twelve patients with hyperplasia without atypia, 11 patients with hyperplastic lesions with atypia, 21 patients with DCIS and 24 patients with invasive carcinoma were studied. The expression of carcino-embryonic antigen (CEA), a dedifferentiation marker, was investigated, applying one monoclonal antibody (Amersham). The expression of human milk fat globulin (HMFG), a differentiation marker, was studied by means of three monoclonal antibodies. The results reveal that no CEA activity can be demonstrated in normal and hyperplastic breast tissue, either with or without atypia. The monoclonal antibody was positive in 48% of DCIS and 50% of the invasive carcinomas. We failed to observe a correlation between the presence of CEA and the differentiation of the tumor. The application of this antiserum adds no substantial information about the phenotypic alterations during the progression from atypical hyperplasia to DCIS. HMFG was present in benign as well as in malignant breast lesions. Therefore, we conclude that HMFG is not useful for the evaluation of the phenotypic change from atypical hyperplasia to malignancy. However, as pointed out by others, it can be used in a diagnostic panel of different antibodies in the distinction between epithelial and non-epithelial lesions.     

Patterns of reactivity with the monoclonal antibodies HMFG1 and HMFG2 in normal endometrium, endometrial hyperplasia and adenocarcinoma

Using an indirect immunoperoxidase technique the expression of the epitopes in human milk fat globule (HMFG) membranes detected by the monoclonal antibodies HMFG1 and HMFG2 was studied in the normal endometrium and in cases of cystic glandular hyperplasia, glandular hyperplasia with architectural atypia (complex hyperplasia), glandular hyperplasia with cytological atypia (atypical hyperplasia) and invasive adenocarcinoma. Luminal reactivity with HMFG1 was seen in cases of normal endometrium, cystic glandular hyperplasia and glandular hyperplasia with architectural atypia. In contrast most cases of glandular hyperplasia with cytological atypia and invasive adenocarcinoma also showed areas of cytoplasmic reactivity. Reactivity with HMFG2 was scanty.     

Use of antibodies to carcinoembryonic antigen and human milk fat globule to distinguish carcinoma, mesothelioma, and reactive mesothelium.

Antibodies raised against human milk fat globule (HMFG 1 and 2) and carcinoembryonic antigen were used in an immunoperoxidase technique to differentiate mesothelioma, carcinoma, and benign, reactive mesothelium. Sixteen mesotheliomas, 27 lung carcinomas, and 13 specimens of reactive mesothelium were examined. Staining for carcinoembryonic antigen was not seen in reactive mesothelium or mesothelioma but was present in 22 of 27 carcinomas. Mesothelioma and carcinoma usually stained with HMFG 1 and 2; reactive mesothelium did not. These three antibodies may help to distinguish carcinoma, mesothelioma, and reactive mesothelium.     

Lectin binding affinities of human milk fat globule (HMFG) membrane antigens

Six biotinylated lectins with differing specificities and two monoclonal antibodies (III D 5 and III H 2) were used to characterize the sugar-residues in human milk fat globule (HMFG) membrane antigens. Immunoblotting analysis revealed that most of the antigens contain several sugars. However, the molecules exclusively reacting with anti-HMFG III D 5, a monoclonal antibody previously shown to detect antigen(s) positively correlating with the expression of estrogen receptors in mammary and gynaecological carcinomas, could only be stained with peanut agglutinin and Ricinus communis-lectins. One of these antigens, a 42-57 kDa molecule, was shown to have a complexed quaternary structure with galactose determining the antigenic specificity. It is suggested that the production of this glycoprotein in estrogen sensitive tissues results from activation of galactosyl-transferase-enzyme at the same time as the expression of estrogen receptors.     

Immunohistochemical study of mammary and extra-mammary Paget''s disease

Paraffin sections of 13 cases of Paget's disease (six mammary and seven extra-mammary) were investigated with mono- and polyclonal anti-carcinoembryonic antigen (CEA) and with monoclonal anti-human milk fat globule membrane antigen (HMFG). One of these cases was also analysed in cryostat sections with monoclonal anti-cytokeratin and anti-keratin. Three immunohistochemical labelling patterns were identified: (I) All six cases of mammary Paget's disease were positive for anti-HMFG and negative with monoclonal anti-CEA (although they stained to a variable degree with polyclonal anti-CEA). (2) Two cases of extra-mammary Paget's disease (both anal location) were positive with monoclonal anti-CEA and only weakly stained for HMFG suggesting epidermal spread from a colo-rectal carcinoma. (3) The other five cases of extra-mammary Paget's disease were negative or weakly stained for CEA and positive for HMFG. We speculate that this group of cases represents epidermotropic eccrine carcinoma. The immunohistochemical use of monoclonal anti-HMFG and -CEA is helpful in the diagnosis of Paget's disease; moreover it gives information about the origin of the primary tumour.     

Classical and chondroid chordoma. A light-microscopic, histochemical, ultrastructural and immunohistochemical analysis of the various cell types.

The aim of the present investigation was to characterize the various cell types of classical and chondroid chordomas. Eight cases of classical chordoma, 1 case of sacrococcygeal chordoma with chondroid areas and 2 cases of spheno-occipital chondroid chordoma were studied. Ultrastructurally and immunohistochemically (immunoreactivity for cytokeratins, epithelial membrane antigen [EMA], tissue polypeptide antigen [TPA] and human milk fat globule protein [HMFG]) the 3 cell types (physaliferous, epithelial-like, and spindle-shaped) recognized light-microscopically presented features of epithelial differentiation and rather formed a continuous spectrum than being distinct cell types. The chondroid areas of the chondroid chordomas had similar ultrastructural and immunohistochemical properties except for the lack of immunoreactivity for EMA and HMFG. The results of the critical electrolyte concentration technique according to Scott and Dorling indicated that there was no difference in the sulfated glycosaminoglycan content between classical and chondroid chordomas: all the tumors contained chondroitin sulfate. The presence of chondroitin sulfate, immunoreactivity for vimentin and S-100 protein and areas of cartilaginous differentiation in three cases indicate a relationship both to chondromatous tumors and to normal notochord, from which chordoma is believed to originate.     

Reactivity of a monoclonal antibody recognicing an estrogen receptor regulated glycoprotein in relation to lectin histochemistry in breast cancer

Summary We have raised monoclonal antibodies against human milk fat globule membrane antigens and previously shown that one of them, called III D 5, recognises a glycoprotein associated with estrogen receptor activity of breast cancer. In immunoblotting it was shown that the molecule in human milk exclusively stained with III D 5 also binds peanut agglutinin (PNA) and Ricinus communis. In this study we correlate the staining of III D 5 and binding of lectins to tissue sections fixed in formalin and embedded in paraffin. Similar rections were seen only with III D 5 and PNA. Our results suggest that III D 5 and PNA detect overlapping antigenic epitopes in mammary carcinoma. This is in keeping with previous results that PNA or III D 5 reactivity is correlated with estrogen receptor status of breast cancer.     

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.