Polymorphic Epithelial Mucin Research Articles

Polymorphic epithelial mucin (MUC-1)-containing circulating immune complexes in carcinoma patients

Circulating immune complexes (CICs) containing polymorphic epithelial mucin (PEM/MUC-1) were found in sera of 24.5% of 151 primary breast carcinoma patients and 18-21.4% of patients with advanced ovarian (n = 56) and breast carcinomas (n = 61), 37% of patients with benign breast tumours, but in only 2.1% of 96 healthy individuals. The incorporation of PEM into CICs affects the detection of circulating PEM in commercial immunoassays such as the CA 15-3 assay, as suggested by a negative correlation between levels of PEM-containing immune complexes (PEM-CICs) and CA 15-3 values, and confirmed by isolation of PEM from CA 15-3-negative sera containing high levels of PEM-CICs. The amounts of PEM masked by human antibodies correspond to significant values of the CA 15-3 assay when monitoring patients for carcinoma. Most antibodies in PEM-CICs were of IgG class, suggesting their specific nature to the PEM epitopes.

Mechanisms underlying aberrant glycosylation of MUC1 mucin in breast cancer cells.

The product of the MUC1 gene, the polymorphic epithelial mucin (PEM) is aberrantly glycosylated in breast and other carcinomas, resulting in exposure of normally cryptic peptide epitopes. PEM expressed by breast cancer cells contains more sialylated O-glycans and has a lower GlcNAc content than that expressed by normal cells. The exposure of peptide epitopes is thus thought to be due to the sugar side chains being shorter on the tumour-associated mucin. To investigate possible mechanisms underlying the different pattern of glycosylation in breast cancer cells, we analysed the pathways involved in the biosynthesis of O-glycan chains of mucins in normal and cancerous mammary epithelial cells. An immortalized mammary epithelial cells line originating from normal human milk. MTSV1-7, and three human breast cancer cell lines, BT20, MCF-7 and T47D, were studied. Glycosyltransferase activities assembling, elongating and terminating O-glycan core-1 [Gal beta 1-3GalNAc alpha-R] and core-2 [GlcNac beta 1-6 (Gal beta 1-3) GalNAc alpha-R] were present in the normal mammary cell line. Many of the glycosyltransferase activities were also expressed at variable levels in breast cancer cells. However, a sialyltransferase activity (CMP-sialic acid Gal beta 1-3GalNAc alpha 3-sialyltransferase) was increased several fold in all three cancer cell lines. Moreover, mammary cancer cell lines BT20 and T47D have lost the ability to synthesize core-2, as shown by the lack of UDP-GlcNAc: Gal beta 1-3GalNAc (GlcNAc to GalNAc) beta 6-GlcNAc-transferase activity, which corresponded to the absence of the mRNA transcript. However, MCF-7 breast cancer cells expressed this enzyme. Thus, the mechanism for the exposure of peptide epitopes in BT20 and T47D cells is proposed to be the loss of core-2 branching leading to shorter, sialylated O-glycan chains. A different mechanism is proposed for MCF-7 breast cancer cells.

Polymorphic forms of the epithelial mucin, PAS-I (MUC1), in milk of Holstein cows ( Bos taurus)

The polymorphic epithelial mucin, PAS-I (also known as MUC1), in individual milk samples from 119 Holstein cows was resolved into bands on SDS-gels. Mobility indices established for these bands provided evidence of four and possibly five polymorphic forms. Sialic acid, a major component of the oligosaccharide portion of PAS-I, was removed from the mucin by treatment of milk samples with neurominidase. This reduced the mobility of the mucin bands but did not alter their mobility relationships within a sample or among samples. Consideration of evidence from this and other studies indicates that the four or five polymorphic forms correspond to alleles, which are inherited, one each from sire and dam, and co-dominantly expressed. It appears that the Holstein population may carry several more alleles for PAS-I than do Ayrshire, Jersey or Brown Swiss cattle. In addition to these breed differences, some remarkable molecular differences have been noted between MUC1 (PAS-I) of human and mouse suggesting that research regarding molecular evolution of this mucin could provide another approach to understanding relationships among species.

Expression of tumor-associated polymorphic epithelial mucin and carcinoembryonic antigen in gastrointestinal carcinoid tumors. Implications for immunodiagnosis and immunotherapy.

Gastrointestinal neoplastic epithelium of glandular origin commonly produces N-linked and O-linked glycoproteins such as carcinoembryonic antigen (CEA) and tumor-associated polymorphic epithelial mucin (PEM). Antibodies to these glycoproteins increasingly are being used in immunodiagnosis and/or immunotherapy. Although GI carcinoid tumors have a neuroendocrine immunophenotype and generally have an indolent clinical course compared with their adenocarcinomatous counterparts, they also arise from undifferentiated crypt epithelium. The purpose of this study was to determine whether GI carcinoids similarly expressed epitopes for CEA and PEM. Thirty-nine GI carcinoid tumors from various topographic sites were analyzed by immunohistochemistry using the murine monoclonal antibodies B72.3, ACT19, and T84. The former two antibodies recognize epitopes in PEM, whereas the latter is an anti-CEA antibody, which was confirmed using enzyme-linked immunosorbent assays. The majority of carcinoid tumors (74%), particularly jejunoileal carcinoids, reacted for ACT19 antibody, whereas considerably fewer reacted for B72.3 (31%) and T84 (26%). The extent of staining was also greatest with ACT19. The GI mucosa adjacent to or overlying the carcinoid tumors also stained for the various antibodies. The extent and degree of positivity of carcinoid tumors for ACT19 raises the possibility that this antibody may be used in the future for the radioimmunodiagnosis and/or immunotherapy of these tumors, particularly in cases that are multicentric, unresectable, or with metastatic disease.

Autologous human B-cell immune response to pulmonary adenocarcinomatous polymorphic epithelial mucin.

In order to investigate whether a B-cell immune response to the polymorphic epithelial mucin (PEM) is present in adenocarcinomatous patients, peripheral blood lymphocytes from 20 patients with adenocarcinomas from various sites were fused with the mouse-human heteromyeloma cell line SHM-D33. One IgM(K) monoclonal antibody derived from peripheral blood lymphocytes of a patient with a lung adenocarcinoma was generated with binding reactivity for bovine submaxillary mucin (BSM). The latter is one of the PEMs, and it contains a large amount of the tumor-associated sialosyl-Tn epitope. The generated antibody was designated KMD-2. Immunohistological studies on various tissues showed that KMD-2 reacted with 6/6 colorectal adenocarcinomas and 4/6 pulmonary adenocarcinomas. The antibody also displayed a slight degree of cross-reactivity with a limited number of normal tissues, especially those that elaborate mucin. Our data show that an autologous antibody response in the form of IgM to the immunosuppressive PEM is present in some adenocarcinomatous patients. The data also suggest that the KMD-2 (IgM/K) antibody may be of clinical importance for diagnostic purposes of adenocarcinomatous disease.

Quantitation of polymorphic epithelial mucin: a challenge for biochemists and immunologists.

Polymorphic epithelial mucin (PEM) is a heavily glycosylated protein present at the apical surface of glandular epithelial cells which is shed into the lumen of epithelial tissue. In carcinomas cell polarisation is lost, PEM is overexpressed and found on the entire cell surface. High amounts of PEM are shed into the circulation of cancer patients. CA 15.3 is the first commercial assay for the detection of PEM. After roughly one decade of use in clinical practice it is considered valuable for breast cancer therapy monitoring and, in the follow up, for early detection of metastatic disease. The extreme polymorphism of this molecule, with its varying number of multiple epitopes and tremendous variation in glycosylation which can mask catcher/tracer epitopes, impairs its precise measurement. A further impediment is complex formation with autoantibodies, as revealed by a recently developed assay.

The detection of breast carcinoma micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction.

The development of a sensitive method for the detection of breast carcinoma micrometastases in axillary lymph nodes is reported. The method was based on amplification of MUC1 mRNA, which encodes a core protein of polymorphic epithelial mucin, by a reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA, which was extracted from a breast carcinoma cell line (MCF-7), primary breast carcinomas, and axillary lymph nodes, was subjected to analysis of MUC1 mRNA expression by the RT-PCR method. MUC1 mRNA expression was detected by RT-PCR in MCF-7 cells and in all 15 primary breast carcinomas but not in control lymph nodes taken from patients with benign diseases. A serial dilution study revealed that MUC1 RT-PCR was a very sensitive method, detecting one MCF-7 cell per 1,000,000 lymph node cells. The detection sensitivity of MUC1 RT-PCR method was compared with that of immunohistochemical staining of an epithelial marker (polymorphic epithelial mucin). Fifty axillary lymph nodes were obtained from 15 patients with primary breast carcinomas, and metastasis in each lymph node was investigated by both methods. The immunohistochemical method demonstrated metastasis in nine lymph nodes, and MUC1 mRNA was detected in all of them. Of the 41 lymph nodes that were diagnosed to be devoid of metastasis by immunohistochemistry, MUC1 mRNA was expressed by 6 but not by the other 35, indicating the presence of micrometastases in these 6 lymph nodes that could be detected only by the MUC1 RT-PCR method. The MUC1 RT-PCR method is more sensitive than immunohistochemistry for the detection of micrometastases in axillary lymph nodes. This new method would be of practical value in selecting the patients at high risk for relapse from those who are histologically lymph node negative.

The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase.

After ovulation, progesterone stimulates a temporally regulated secretory transformation in human endometrial epithelium. Using a combination of immunohistochemistry, and Western and Northern blotting, we demonstrate that 1) the polymorphic epithelial mucin MUC1 is secreted by human endometrial epithelium; 2) low levels of both mRNA and core protein are present in the preovulatory phase of the menstrual cycle; 3) mRNA levels increase several-fold after ovulation, consistent with transcriptional regulation by progesterone; 4) there is an increase in translation product in postovulatory endometrium; and 5) the tandem repeat domain of the MUC-1 polypeptide is glycosylated in endometrium.

The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase

The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase

Cytotoxic T cells from ovarian malignant tumors can recognize polymorphic epithelial mucin core peptides.

CTL isolated from tumor infiltrating lymphocytes/tumor associated lymphocytes (TAL) infiltrating ovarian tumors have been demonstrated to mediate lysis of tumor targets after in vitro culture. These effector cells are of particular interest as cellular probes to detect and define human tumor Ag and epitopes that stimulate cellular immunity to human tumors. Polymorphic epithelial mucin (PEM) core peptides are potential candidates as tumor specific Ag because of their preferential expression on epithelial tumors. We report here that ovarian CTL-TAL can recognize mucin (Muc-1) core peptide of PEM. Several ovarian CTL-TAL lines were developed that recognized in a non-MHC restricted fashion an Muc-1+ ovarian tumor, but not Muc-1-tumor. To define the specificity of these CTL-TAL and exclude cross-reactivity with other potential Ag, cytotoxicity experiments were performed using as targets EBV-transformed cell lines with an expression construct containing the Muc.1 cDNA. These ovarian CTL-TAL lysed mucin core-peptide transfected cells but not targets transfected with an expression construct containing a mucin frame-shift mutant cDNA as control. In addition, targets pulsed with short synthetic peptides composed of amino acids 1-14 of the Muc 1 core peptide repeat were also lysed by the same CTL-TAL. This lysis was inhibited by the mAb SM3 that recognize an epitope on the mucin core peptide. These results, which are a demonstration of a specific Ag recognized by ovarian CTL-TAL, may be of interest for specific immunotherapy of ovarian cancer.

S20.16 Characterisation of model systems for studying the aberrant glycosylation of the MUC1-1 gene product

The polymorphic epithelial mucin (PEM), coded for by the MUC1 gene is expressed on the luminal surface of many simple epithelial cells. Like other members of the mucin family, PEM contains a large domain which consists of tandemly repeated amino acids. This domain acts as a scaffold for the O-linked carbohydrate. PEM is upregulated in pregnant and lactating human mammary gland and in many adenocarcinomas. In addition, in malignant cells the molecule appears to be aberrantly glycosylated resulting in shorter sugar side-chains leading to the exposure of cryptic peptide epitopes such as that recognised by the antibody, SM3. In order to study the mechanisms underlying this aberrant glycosylation it is necessary to have an in vitro model system. To this end we have developed, by SV40 T antigen immortalisation, a cell line (MTSV1-7) which has many characteristics of normal mammary epithelial cells (Bartek et al., 1991, PNAS 88, 3520-3524). MTSV1-7 does not grow in soft agar or form tumours in nude mice and, unlike breast carcinoma lines, forms ordered structures in collagen gels (Berdichevsky and Taylor-Papadimitriou, 1991, Exp. Cell Res., 194, 267-274). Furthermore, by the criterion of antibody binding, MTSV1-7 appears to glycosylate the mucin like a normal cell. We are now in the process of purifying the MTSV1-7 mucin to confirm its carbohydrate composition by mass spectrometry (in collaboration with Professor Anne Dell). We intend to use this cell line and a breast carcinoma cell line to study the mechanisms responsible for the aberrant glycosylation of tumour cells.

The polymorphic epithelial mucin as a target for immunotherapy.

The polymorphic epithelial mucin as a target for immunotherapy.

Tumor-specific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells transfected with polymorphic epithelial mucin complementary DNA.

Human breast and pancreatic adenocarcinomas are tumors of ductal epithelial cell origin and as such produce and express on their surface polymorphic epithelial cell mucin encoded by the MUC 1 gene. We have previously reported that tumor-specific cytotoxic T cells derived from patients bearing such tumors recognize specific epitopes on the mucin polypeptide core. This recognition was not MHC-restricted because of the highly repetitive sequence of the polypeptide core, which allows simultaneous recognition of many identical epitopes, and cross-linking and aggregation of TCR on mucin-specific T cells. Those studies were performed with limited numbers of tumor cells or allogeneic tumor cell lines. A renewable source of autologous cells presenting this Ag was necessary to further explore mucin-specific immunity. We report here successful establishment and functional analysis of mucin-specific CTL lines and clones derived from breast and pancreatic cancer patients, using either autologous or allogeneic mucin-transfected B cells as Ag. Our results demonstrate that transfection of autologous or allogeneic B cells with mucin confers upon them tumor Ag-presenting ability as well as susceptibility to lysis by mucin-specific CTL. Transfection of APC with this or any other human tumor Ag that may be molecularly defined in the future provides a unique and powerful tool with which to examine the ability of a tumor-associated Ag to stimulate T cell responses.

Development of an anti-idiotypic antibody reactive with an antibody defining the epitope RPAP in the MUC-1 epithelial mucin core.

C595 is a murine IgG3 monoclonal antibody (MAb) raised against human urinary mucin. C595 antibody binds to the protein core of the MUC-1 mucin (the polymorphic epithelial mucin, PEM) which is elevated in tissue and secretions from human breast carcinomas. The antibody defines the tetrameric epitope, RPAP. In the present study, a syngeneic anti-idiotypic MAb, 911, was raised in BALB/c mice against the C595 of C595 to its mucin core antigen. In turn, pre-blocking of C595 with a 20 amino-acid mucin core peptide specifically inhibited binding of 911 antibody to C595. Cross-reactivity of antibody 911 was only observed against the IgM MAb, 789/91, which has the same minimum antibody-binding epitope as C595, namely, RPAP. Syngeneic and xenogeneic anti-sera against 911 antibody displayed increased binding to heptameric peptide sequences containing the motif, RPA(P). The anti-idiotypic antibody 911 therefore appears to recognize a site within or close to the binding site of the C595 antibody and thus carries an internal image of the parental mucin epitope. Consequently, the 911 idiotypic network offers a promising model system to investigate the mechanism of anti-idiotype-induced tumour immunity.

Radioimmunoscintigraphy with technetium-99m-labelled monoclonal antibody, SM3, in gynaecological cancer

Radioimmunoscintigraphy (RIS) with technetium-99m labelled SM3, a monoclonal antibody reacting with a polymorphic epithelial mucin glycoprotein core antigen, is evaluated. No adverse effects or thyroid uptake were observed. Studies in 45 patients (one twice) had a sensitivity for gynaecological malignancy of 100% (35/35) and a specificity of 73% (8/11), giving an overall accuracy of 93% (43/46). These results have led to the routine adoption of 99mTc RIS in the management of patients suspected of or having primary or recurrent gynaecological cancer.

Regulation of sialomucin production in colon carcinoma cells

In this study, we investigated the biochemical basis for the induction of sialomucin production in HT-29 LMM human colon carcinoma cells. Previous studies showed that conditioned medium from organ cultures of normal human colonic connective tissues (NCCM) stimulated the biosynthesis of high molecular weight sialoglycoproteins (M(r) 740,000 and 450,000) by colon carcinoma cells in vitro. Using monoclonal antibodies specific for human milk mucin, we determined that the polypeptide core of these sialomucins was the polymorphic epithelial mucin core peptide coded by the MUC1 gene. Northern analysis showed that cells treated with NCCM expressed a higher level of MUC1 poly(A)+ mRNA than untreated cells. The UDP-GalNAC: polypeptide N-acetylgalactosaminyltransferase activity in microsomal fractions from these cells did not change upon NCCM treatment. The ratios of oligosaccharides with various negative charges secreted by HT-29 LMM cells in the presence of benzyl-alpha-D-N-acetylgalactosaminide were not affected by NCCM treatment indicating that the degree of sialylation of O-linked carbohydrate chains is not influenced by NCCM treatment. [3H]Glucosamine pulse-chase labeling studies using antibodies specific for a COOH-terminal unglycosylated region of the MUC1 mucin core peptides suggested that NCCM treatment of HT-29 LMM cells did not change the rate of MUC1-related sialomucin processing.

Immunohistochemical expression of tumour-associated glycoprotein and polymorphic epithelial mucin in the human endometrium during the menstrual cycle.

An epitope defined by a second generation murine monoclonal antibody (LU-BCRU-G7) produced against a breast tumour-associated fucosylated glycoprotein of M(r) 230,000 was found to exhibit phase specific reactivity in human endometrium during the menstrual cycle. Distribution and cycle related expression of the LU-BCRU-G7 determinant was different from the staining patterns observed with antibodies that react with the polymorphic epithelial mucins, HMFG 1 and NCRC 11, as determined by immunohistochemistry. Initial expression of the LU-BCRU-G7 determinant was associated with the peri-basal region of glandular epithelial cells with maximal staining during the mid-secretory phase. Diffuse cytoplasmic staining was also observed from day 10 to day 26, with a marked increase in the early secretory phase. Reactivity of NCRC 11 also showed maximal expression in the midsecretory phase with no reactivity detected in early and mid-proliferative phases. The HMFG 1 defined epitope exhibited the opposite pattern of expression with maximal reactivity in the proliferative phase and little or no reactivity in the secretory phase. These findings suggest that expression of the LU-BCRU-G7, HMFG 1 and NCRC 11 defined determinants in the human glandular epithelium of the endometrium is differentially hormonally regulated, and may be of value as markers for endometrial function.

Polymorphic epithelial mucin (PEM) epitopes in the estimation of the sequence of neoplastic changes in colon adenocarcinoma.

The relation between the expression of PEM epitopes and the sequence of neoplastic changes in individual patients with colon adenocarcinoma was investigated. Immunohistochemical staining was performed using four monoclonal antibodies to PEM epitopes (139H2, 140C1, 115D8, 115F5) on tissue sections from frankly malignant lesions, adjacent mucosa, colonic mucosa taken 1 cm and 5 cm from the lesion and normal control mucosa. A clear correlation between PEM expression and the site of mucosa sampling was demonstrated. The expression of PEM molecules defined by all studied monoclonal antibodies was highest in colon adenocarcinoma, similar but slightly weaker in adjacent mucosa and colon mucosa taken 1 cm from the frankly malignant lesion, lowest in mucosa 5 cm from the lesion, and virtually absent in control normal colonic mucosa. The immunological changes could be one of the first signs of a premalignant process and the estimation of PEM epitopes could show some cellular abnormalities not detectable by standard pathomorphological criteria.

Use of tumour marker immunoreactivity to identify primary site of metastatic cancer.

To determine whether variations in the expression of tumour related antigens can predict the origin of tumours. Immunohistological study of tumour marker expression in primary adenocarcinomas and respective metastatic deposits. Antibodies to the following tumour markers were used: polymorphic epithelial mucin (NCRC-11 and SM3), carcinoembryonic antigen, carcinoembryonic antigen with non-specific antigen co-specificity, CA125, CA19.9, prostate specific antigens, and thyroglobulin. Histopathology department of teaching hospital. 100 pathology sections of metastatic adenocarcinoma and their related primary tumours. Concordance of reactivity between primary and metastatic tumours. Reactivity profiles of tumour sites. The correct primary site of origin was predicted in 70% (33/47) of tumours in men and 54% (27/43) tumours in women with antibodies SM3, 288, CA19.9, CA125, and PSA (men only). Specificities ranged from 68% for breast tumour to 98% for prostate tumour. Use of tumour markers in patients presenting with metastatic adenocarcinoma of unknown origin can help localise the probable primary sites and reduce the need for extensive and expensive imaging techniques.

IMMUNOHISTOCHEMICAL EVALUATION OF MAM-3 AND MAM-6 ANTIGENS IN SQUAMOUS-CELL CARCINOMA OF THE ORAL CAVITY

MAM-3 and MAM-6 antigens of human milk fat globule membrane designated as human polymorphic epithelial mucin (PEM) were detected immunohistochemically in a total of 86 specimens from 39 cases of oral squamous cell carcinoma (SCC) and 6 cases of normal oral mucosa. The two antigens were identified by MoAbs 67D11 and 115G3 (MAM-3 antigen) and by MoAbs 115D8 and 115F5 (MAM-6 antigen) in paraffin sections. MoAb 115G3 staining was found in well keratinized foci; whereas, MoAb 115D8 and 115F5 staining was very low in keratinized tumor cells. The cytochemical distribution of the antigens was classified into 3 types; (i) cell membrane positive type, (ii) whole cell positive type and (iii) antigen negative type. Reduction and disappearance of the two antigens were correlated to the mode of tumor invasion and suggested that glycocalyx complex in cell membrane properties was changed for malignant transformation.