The potential usefulness of monoclonal antibodies in the determination of histologic types of lung cancer in cytologic preparations

Two monoclonal antibodies (MAbs) were tested for their reactivity with antigens of exfoliated malignant cells in respiratory secretions of lung cancer patients. MAb CE 407 was developed from tissue culture cell line SW 756, derived from human uterine cervical squamous cell carcinoma; MAb BL 99-57 was developed from cell line T-24, derived from human transitional cell bladder cancer. MAb CE 407 reacted preferentially with squamous cell carcinomas (80%) and with some (44%) of the adenocarcinomas of the lung; BL 99-57 reacted with 76% of the adenocarcinomas, but only with 27% of the squamous cell carcinomas of the lung. The reactivity of BL 99-57 was more apparent in well-differentiated adenocarcinomas (89% positive), but less in poorly differentiated adenocarcinomas (65% positive). Neither of these antibodies reacted with antigens of small cell anaplastic carcinoma. These two MAbs may be useful in differentiating histologic types of lung cancer in cases that are difficult to diagnose morphologically and/or in which tissue is not available for study.     

Identification of a family of high molecular weight tumor-associated glycoproteins

The monoclonal antibody (MAb) designated DF3 was prepared against a human breast carcinoma metastatic to liver. This MAb reacts with a high molecular weight glycoprotein detectable in human breast carcinomas and human milk. In contrast, MAb F36/22 was prepared against the MCF-7 breast carcinoma cell line, MAb 115-D8 against human milk fat globule membrane (HMFGM) and MAb Ca1 against the HEp-2 human laryngeal carcinoma cell line. These MAb have similar patterns of reactivity with normal tissues and tumors based upon immunoperoxidase staining. In the present study we have monitored reactivity of these MAb against DF3 antigen purified from human breast carcinoma cell lines (MCF-7, BT-20) and HMFGM. Solid phase immunoassays and immunoblotting demonstrate that MAb DF3, F36/22, 115-D8, and Ca1 all react with the same purified DF3 antigen. Furthermore, immunoblot analysis indicates that the DF3 antigen reactive with these MAb differs structurally in preparations from breast carcinoma cells and HMFGM. We also demonstrate that MAb F36/22 completely inhibits MAb DF3 binding in competitive blocking assays. In contrast, the results indicate that MAb 115-D8 and Ca1 only partially block MAb DF3 reactivity and the extent of this inhibition varies with DF3 antigen purified from breast carcinoma cells and HMFGM. Taken together, these findings with multiple MAb prepared against a variety of immunogens suggest that existence of a family of related but not identical high molecular weight tumor-associated glycoproteins.     

Effects of maturational agents on expression and secretion of two partially characterized high molecular weight milk-related glycoproteins in MCF-7 breast carcinoma cells

We have previously defined a human mammary epithelial antigen using a murine monoclonal antibody (MAb), designated DF3, prepared against a membrane-enriched fraction of a human breast carcinoma. MAb DF3 detects a cell surface antigen with a molecular weight (mw) of approximately 300 Kd and a higher mw species also detectable in human milk. These findings and the demonstration that butyric acid (BA) increases DF3 antigen expression suggested that MAb DF3 reacts with a differentiation antigen detectable in human breast carcinoma cells. The results of the present study demonstrate that MAb DF3 reacts with two mucin-like high mw glycoproteins (330 and 450 Kd) present in MCF-7 breast carcinoma cells. The results also demonstrate that the intracellular content and secretion of DF3 antigen is increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-beta-D-arabinofuranosylcytosine (ara-C). Other known inducers of differentiation including retinoic acid (RA), hexamethylene bisacetamide (HMBA), 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and certain polar solvents decrease DF3 antigen expression. Furthermore, the results demonstrate that DF3 antigen is secreted and that the extent coincides with changes in intracellular content. Finally, actinomycin D and cycloheximide inhibit the increases in DF3 antigen expression following TPA treatment thus suggesting that newly synthesized RNA and protein are required for induction of this antigen. Thus, the monitoring of DF3 antigen expression may provide a marker for studying maturation of human breast cancer cells.     

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.     

Localization of mucinous-like carcinoma associated antigen (MCA) in breast pathology: comparison with carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA)

The topographic distribution of a mucinous-like cancer antigen (MCA) recognized by a monoclonal antibody b-12 (MAb b-12) was assessed in benign (38) and malignant (66) breast tissues. The reactivity of MAb b-12 showed a good selectivity for breast tissues, reacting both with normal tissues and breast cancer. The degree of MCA expression was evaluated in the various groups of breast pathology adopting quantitative criteria of assessment. With the criteria of evaluation adopted, strong staining was observed in 71.4% breast carcinomas. The most positive reaction was demonstrated in mucinous carcinoma. MCA distribution in breast tissue was compared with the distribution of two other antigens, CEA and TPA. Reactivity of MAb b-12 was higher than the reactivity shown by the anti-CEA and anti-TPA antibodies.     

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkins lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

On the clinical usefulness of a few sugar antigens and a galactosyl-transferase

Using human cultured cell lines or lymphocytes, two kinds of murine- and one human-monoclonal antibodies were produced, respectively and their clinical usefulness were investigated, and the possibility of galactosyl-transferase as a new tumor maker was also discussed. (1) A murine monoclonal antibody MSN-1, which was raised against human endometrial cancer cell line and recognized blood type sugar chain Leb, reacted with about 85% of endometrial cancer tissues, indicating that useful clinical information may be obtained by applying MSN-1 to immunohistochemistry and flow cytometry. (2) A new assay system using two murine monoclonal antibodies MA54 and MA61, which were raised against human lung cancer cell line and reacted with mucin sugar residues, revealed 76% positive rate in ovarian cancer patients, especially 82% in mucinous cystadenocarcinoma, indicating the clinical effectiveness as a new tumor maker compensating for the drawbacks of CA-125. (3) Galactosyl-transferase isozyme GT-2 was analyzed by the assay system using a newly produced monoclonal antibody. GT-2 was positive in 74% of ovarian cancers, especially in 89% of meso-nephroid cancer, indicating that GT-2 could be a useful tumor maker in ovarian tumors. (4) Human monoclonal antibody, which recognized "type 1 sugar chain" or iso-paragloboside, reacted about one half of endometrial cancer tissues. The production of human monoclonal antibody may contribute to the cancer imaging and the missile therapy.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. I. Functional characterization by monoclonal antibodies

Three rat monoclonal antibodies (MAb) capable of stimulating interleukin 2 (IL 2) production by a variant subline of EL4 thymoma cells (EL4-6.1) have been produced. The stimulatory capacity of these MAb (designated RL73, RL119, and RL388) was originally found to be dependent on the presence of irradiated peritoneal exudate cells; however, this requirement could be replaced by the cellfree supernatant of the "macrophage-like" cell line P388D1 or by biochemically purified human interleukin 1 (IL 1). A number of other rat MAb directed against cell surface structures did not stimulate IL 1-dependent IL 2 production by EL4-6.1 cells; however, certain MAb directed against Thy-1 as well as the lectin phytohemagglutin did have this capacity. Furthermore, the stimulatory activity of MAb RL73, RL119, and RL388 appeared to be restricted to the EL4-6.1 variant line, because neither the parental EL4 line from which it was derived nor a series of ovalbumin-specific T-T hybrids responded to these MAb. The cell surface antigens recognized by MAb RL73, RL119, and RL388 were present on a wide variety of T cell lines and T-T hybrids, as well as on lines of B cell, macrophage, and fibroblast origin. Interestingly, the MAb reacted with the majority (approximately 85%) of thymocytes but not (or only to a very small extent) with resting T lymphocytes. After stimulation by concanavalin A, however, the three MAb reacted strongly with activated T lymphoblasts. The latter data suggest that MAb RL73, RL119, and RL388 may react with cell surface structures that are normally expressed as a consequence of lymphocyte activation.     

Production and Characterization of Monoclonal Antibodies Specific to Closterovirus-like Particles Associated with Grapevine Leafroll Disease

Three hybridoma lines secreting monoclonal antibodies to a closterovirus-like particle (GLRV 3) associated with grapevine leafroll disease were produced. One of the antibody (MAbt) reacted with one extremity of the filamentous virus particle whereas the other two (MAb2 and 3) reacted with the entire surface of the virus particle. Using MAb2 in ELISA it is possible to detect 46 of the 50 GLRV-3 isolates. In order to detect all the 50 isolates, it is necessary to use MAb1 in ELISA in conjunction with polyclonal antibodies. In immunoblotting, the three MAbs recognized a 43 Kd viral protein.     

Characterization of murine monoclonal antibodies to human interferon-gamma (IFN-gamma) and their application for sandwich enzyme-linked immunosorbent assay (ELISA)

The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-gamma (IFN-gamma), but not human IFN-alpha and IFN-beta. They react weakly with heat-treated IFN-gamma. The three antibodies recognize different epitopes of the IFN-gamma molecule, as evaluated by antibody-binding inhibition experiments. We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA). The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer. When we measured IFN-gamma in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained. This may suggest that human IFN-gamma exists in oligomeric form. Recombinant human IFN-gamma expressed in E. coli is detectable at a concentration of 1 ng/ml in this sandwich ELISA. This assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule as well as measurement of IFN-gamma in human sera and tissue culture fluids.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Immunological characteristics of monoclonal antibodies against human carcinoembryonic antigen (CEA)

Four hybridomas secreting monoclonal antibodies (MAbs) of the IgG1 subclass against human carcinoembryonic antigen (CEA) were obtained from fusion of P3-NS1/1-Ag4 myeloma cells with splenic cells from mice immunized with purified CEA. None of the MAbs showed cross-reactivity to perchloric acid extractable antigens from the normal human colon by an inhibition radioimmunoassay. However, MAb C27 showed the highest affinity to CEA. The intensity of immunofluorescence staining of human colorectal cancer cells with MAb C27 correlates well to the cellular CEA content of cancer cells. LS174T showed the highest intensity of fluorescence (95%) while COLO320DM and COLO320HRS were the lowest (0.5%). None of the normal human organs - colon, lungs, liver, spleen or kidneys-showed positive staining by immunoperoxidase anti-peroxidase (PA) techniques, while tissues from colorectal carcinoma (CRC), gastric carcinoma, hepatoma and lung cancer gave a positive rate of 100% (30/30), 96.6% (28/29), 32.1% (9/28) and 82.1% (69/84) respectively. Results suggest that MAb C27 can be used in immunodetection and radiolocalization of colorectal carcinoma.     

Differential expression of cell surface antigens of canine keratinocytes defined by monoclonal antibodies

Nine monoclonal antibodies (MAb) directed against cell surface antigens of canine keratinocytes define distinct keratinocyte subpopulations owing to the differential expression of these antigens during the process of differentiation and depending on the tissue location of the cells. There was distinct antigenic heterogeneity between the different layers of stratified squamous epithelium and between stratified squamous epithelial of different tissue origin. Two MAb reacted only with antigens expressed by esophageal mucosa. Three MAb bound to antigens on keratinocytes of the suprabasilar and granular layers of stratified squamous epithelia, and they crossreacted with the transitional epithelial cells of the urinary tract. Two MAb reacted with antigens only expressed on differentiated cells, superficially located in the stratified squamous epithelium. The use of these MAb as markers for keratinocytes in studies on the characterization and differentiation of keratinocytes, as well as in tumor diagnosis and allograft transplantation, is discussed.     

Monoclonal antibodies that distinguish non-small cell from small cell lung cancer

Two murine IgG2Ak monoclonal antibodies (703D4, 704A 1) were produced and characterized after immunization with a human large cell lung cancer line (NCI-H 157). These antibodies detect different epitopes on 31 kilodalton [35S]methionine incorporating protein(s). Radiobinding and immunohistochemical studies show these antibodies bind to most (11/13) human non-small cell lung cancer (adenocarcinoma, epidermoid, and large cell), but not to small cell lung cancer (0/11) tumors tested. The epitopes these antibodies recognized are also expressed on human melanomas (7/8), two other tumors (osteogenic sarcoma, renal cell carcinoma), but not on many other human tumors (breast, colon, neuroblastoma, lymphoid), and not on a panel of normal adult human tissues. Because the antigen(s) are preserved after fixation and because of their ability to distinguish lung cancer types from each other and normal tissues, they should be of clinical, as well as of biologic interest.     

Inhibition of human tumor growth by IgG2A monoclonal antibodies correlates with antibody density on tumor cells

Eight monoclonal antibodies (MAb) of IgG2a isotype that were produced against human melanomas were tested for tumor growth-inhibiting properties in nude mice injected with human melanoma cells of various origins. Four of the eight MAb inhibited growth of these tumors, and all four of these antibodies reacted in antibody-dependent macrophage-mediated cytotoxicity (ADMC) assays in vitro. The MAb that were inactive in vivo also did not react in these assays in vitro. The number of antibody-binding sites per cell on the tumor cell surface was significantly higher for tumoricidal MAb as compared to unreactive MAb. On the other hand, the percentage of tumor cells binding the MAb and the binding affinity to these cells were the same for the two groups of MAb. Also, tumoricidal and nontumoricidal MAb bound with similar affinity and antibody density to Fc receptors on macrophages. The importance of the number of antibody sites on the tumor cell surface for tumor destruction by MAb was confirmed by the demonstration of tumoricidal effects of mixtures of MAb that were by themselves not tumoricidal. MAb binding to different molecules on melanoma cells were complementary in ADMC, whereas MAb directed to the same molecule but to different epitopes were not.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Identification of antibodies against HAI-1 and integrin alpha6beta4 as immunohistochemical markers of human villous cytotrophoblast.

Syncytiotrophoblast and invasive extravillous trophoblast arise from a common stem cell, namely villous cytotrophoblast, but have very different characteristics. The study of the differentiation process relies on the availability of suitable markers for these different cell types of developing placenta. In this work, we have produced monoclonal antibodies that are specific to human villous cytotrophoblast. Monoclonal antibody (MAb) MG2 was specific to villous cytotrophoblast across gestation, and recognizes hepatocyte growth factor activator inhibitor type 1. MAb MD10 stained villous cytotrophoblast across gestation and also some endothelial cells, particularly in the second or third trimester. MAb MD10 recognizes human integrin alpha6beta4. As a test for specificity, the novel MAbs were also used for staining of frozen tissue from human colon carcinoma. The results show that the two antibodies can be used as tools to study human villous cytotrophoblasts and also human tumors. The MG2 antibody seems most specific and promising for the study of various aspects of human villous cytotrophoblast.     

Patterns of MUC1 tissue expression defined by an anti-MUC1 cytoplasmic tail monoclonal antibody in breast cancer.

Our aim was to determine the pattern of expression of MUC1 mucin cytoplasmic tail (MUC1 CT) in breast carcinoma. A total of 98 invasive breast adenocarcinoma tumor samples were assayed by immunohistochemical (IHC) analysis. The pattern of reaction was classified as membrane, cytoplasmic, or mixed. Subcellular fractions were prepared after SDS-PAGE and Western blotting. The antibodies employed were anti-MUC1 CT (CT2 monoclonal antibody, MAb) and C595 MAb against the extracellular MUC1 core protein. With the CT2 MAb, IHC showed a high percentage of positive staining in 93% of specimens, with membrane staining the most common pattern observed. C595 MAb was reactive in 73% of specimens. Similar percentages of membrane and cytoplasmic staining were found, mainly in a mixed pattern. Western blotting showed different bands. With the CT2 MAb, the membrane fraction showed the most intense reaction; a strong band of reaction was detected at approximately <30 kD. With the C595 MAb, in most cases a double band at 200 kD was found. In breast epithelium, the pattern of MUC1 CT expression may constitute an indicator of MUC1 production because it does not depend on glycosylation. The pattern and extension of MUC1 CT positivity do not vary according to the histopathological subtype of the tumor.