Characterization of monoclonal antibody 436 recognizing the Arg-Pro-Ala-Pro sequence of the polymorphic epithelial mucin (PEM) protein core in breast carcinoma cells.

Epithelial mucins have obtained increasing clinical relevance since they were found in the serum of cancer patients and were shown to be elevated in metastatic disease. We report here the characterization of the monoclonal antibody (MAb) 436 which recognises the protein core of the polymorphic epithelial mucin (PEM) of the human breast. MAb 436 was generated by immunizing Balb/c mice with membrane-enriched fractions prepared from metastatic lesions in the axillary lymph nodes. The antigenic determinant recognized by the MAb 436 is expressed on the surface of breast cancer cells and was measured by ELISA on all of 50 cytosol preparations of primary breast tumors. Immunohistochemistry showed 98% of primary and 100% of metastatic breast cancer lesions to be positive with the 436 antigenic determinant expressed both in the cytoplasm and at the plasma membrane level of the tumor cells. Moreover, the antigen was expressed in a homogeneous fashion (80-100% of the total number of tumor cells) in more than 60% of the tumors. Reactivity with normal tissues was rare and scattered and restricted to glandular structures particularly at the luminal border level except for the distal and collecting tubules of adult and fetal kidney, where a cytoplasmic 436 antigen distribution was observed. Other cancers proved positive but the reactivity was always variable and heterogeneous. The antigen recognized by MAb 436 appears in Western Blotting as a M(r) of more than 200,000 daltons protein resolved in two bands. Epitope mapping experiments using overlapping octapeptides in the repeat unit of the PEM identified in the RPAP (Arg-Pro-Ala-Pro) sequence the binding site of the 436 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

OBJECTIVES--To determine whether variations in the expression of tumour related antigens can predict the origin of tumours. DESIGN--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. SETTING--Histopathology department of teaching hospital. SUBJECTS--100 pathology sections of metastatic adenocarcinoma and their related primary tumours. MAIN OUTCOME MEASURES--Concordance of reactivity between primary and metastatic tumours. Reactivity profiles of tumour sites. RESULTS--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. CONCLUSION--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.     

Expression of monoclonal-antibody-defined antigens in fractions isolated from human breast carcinomas and patients' serum

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these smaples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.     

Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads

The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3␣weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens. Received: 30 June 1998 / Accepted: 5 October 1998     

CA 15.3 as a tumour marker in breast cancer

CA 15.3 is an antigenic determinant associated with human mammary carcinomas. Two murine monoclonal antibodies have been raised against the determinants, and an immunoradiometric assay (IRMA-Kit, Centocor, USA) has been developed to determine the antigen levels in plasma of cancer patients. Based on the 99% confidence limit of healthy women, plasma values above 30 U/ml are considered abnormal. Plasma samples from 357 women were examined in the present study. Healthy females (n = 84) ranged below the cut-off level between less than 10 and 29 U/ml. Higher values were found in 12.5% of benign breast diseases and in 23.6% of breast cancer patients, including all stages. Depending on the stage of the disease, there were elevated levels in 11% of operable breast cancer patients preoperatively, in 7% of the cases with no evidence of disease after primary treatment and in 63.5% of patients with disseminated mammary carcinoma. In metastasized breast cancer the frequency and the degree of abnormal titers were closely related to the extent of the metastatic disease. Follow-up examinations of 63 patients under cytotoxic therapy showed CA 15.3 changes correlating well with the clinical course in up to 90% of the antigen positive cases. The present data indicate that CA 15.3 may be useful in the surveillance of breast cancer patients. However in our study one third of the patients with metastatic breast cancer did not show any increase in CA 15.3 and must be regarded as antigen negative.     

Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins.

Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope® sialyl-Tn-KLH cancer vaccine in active specific immunotherapy

Patients with metastatic breast, colorectal or ovarian cancers received active specific immunotherapy (ASI) with Theratope® sialyl-Tn-KLH (keyhole limpet hemocyanin) cancer vaccine emulsified in Detox? adjuvant. The median log₂ anti-STn IgG titer generated by ASI, estimated by enzyme-linked immunosorbent assay with solid-phase ovine submaxillary mucin, was 5.322 (range = 0?–?9.322). Following ASI, 51 patients who generated titers higher than the median value for anti-STn⁺ mucin IgG survived longer than 46 patients who generated lower titers below the median. 38 of the patients were phenotyped for CD69 prior to ASI. The patients with lower numbers of CD69⁺ peripheral blood lymphocytes prior to immunotherapy (pre-ASI) also had low serum CA27.29 cancer antigen (MUC-1) levels, and had longer times to disease progression and improved survival following ASI. Elevated pre-ASI serum CA27.29 tumor antigen levels were associated with higher numbers of CD69⁺ PBL, with decreased anti-STn antibody production and decreased survival following ASI. The data are compatible with the hypothesis that elevated serum MUC-1 mucin is specifically immunosuppressive.     

A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen. cDNA cloning, expression of the transfected gene and over-expression in breast cancer tissue

A monoclonal antibody, H23, that specifically recognizes a breast-tumor-associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850-bp cDNA insert, shows that they are composed of 60-bp (G + C)-rich tandem repeating units. The coding strand was determined and codes for a proline-rich 20-amino-acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60-bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60-bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragment produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over-expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.     

Cellular and humoral immune responses to MUC1 mucin and tandem-repeat peptides in ovarian cancer patients and controls

The objective of this study was to demonstrate the presence of proliferative T cell responses to human polymorphic epithelial mucin (MUC1) and its tandem-repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and to correlate these cellular responses to a humoral response to MUC1. PBMC were obtained from 6 healthy women, from 13 women in the third trimester of pregnancy and from 21 ovarian cancer patients. Only 1 of the 6 healthy women showed a weak primary proliferative response (stimulation index, SI<2) to a 20-mer MUC1 tandem-repeat peptide in the presence of interleukin-2 (IL-2). In PBMC from 5/13 pregnant women (38%) a weak response could be induced by the 20-mer and/or 60-mer tandem-repeat peptides (SI ≤ 3.0) and in PBMC from 8/15 ovarian cancer patients (53%) 20-mer and/or 60-mer MUC1 tandem-repeat peptides induced primary responses (SI ≤ 5.4). MUC1 mucin purified from a breast tumor cell line and/or from urine of a healthy donor had a relatively strong stimulating effect (SI ≤ 19) on PBMC from 4 of 16 ovarian cancer patients (25%). In contrast, in PBMC of 9 ovarian cancer patients stimulated by the addition of a Candida albicans extract, MUC1 mucin strongly inhibited proliferation. This inhibition could partially be abrogated by the addition of IL-2. MUC1 (CA 15.3 assay) and free circulating MUC1 IgG and IgM antibodies (PEM.CIg assay) were determined in the plasma of all subjects. The MUC1 and the free circulating MUC1 IgG antibody plasma levels were significantly higher in the ovarian cancer patients than in the healthy women. Although no significant correlations were found between MUC1 mucin, MUC1 Ab plasma levels and the individual proliferative responses to the MUC1 antigens, an association may exist between them, since all three are significantly higher in the ovarian cancer patients than in the healthy women. Received: 27 August 1998 / Accepted: 10 December 1998     

Soluble ICAM-1 in breast cancer: clinical significance and biological implications

OBJECTIVES: In previous experiments, we demonstrated a decreased expression of intercellular adhesion molecule I (ICAM-1) on both tumour cells and antigen-presenting cells derived from patients with breast cancer, resulting in an abrogation of antigen presentation and tumour cell lysis. Recently, increased levels of a soluble isoform of ICAM-1 (sICAM-1) have been detected in the sera of breast cancer patients. The present investigation was performed in order to investigate the biological relevance of serum concentrations and the effects of sICAM-1 in patients with breast cancer. PATIENTS AND METHODS: sICAM-1 was determined using a sandwich enzyme immunoassay on sera from 88 patients with various stages of breast cancer and correlated with clinical parameters. The effect of sICAM-1 present in the sera of patients with breast cancer upon unspecific and anti-Her-21/neu antibody-mediated cytotoxicity (ADCC), as well as upon antigen presentation, was determined using a 51Cr-release assay and [3H]thymidine-uptake of T cells after co-incubation with tetanus-toxoid-pulsed antigen-presenting cells. RESULTS: In patients with early breast cancer, serum levels of sICAM-1 were significantly lower compared to patients with metastatic disease, but did not correlate with usual clinical parameters. In patients with metastatic breast cancer, a significant correlation of sICAM-1 with tumour markers CEA and CA 15-3 was observed. No influence of sICAM-1 upon unspecific cytotoxicity, ADCC, or the ability to present antigen was observed. DISCUSSION: The origin of sICAM-1 in the sera of patients with breast cancer remains unknown. In contrast to its membrane-bound isoform, sICAM-1 was increased in the sera of patients with various stages of breast cancer, but its presence did not influence unspecific cytotoxicity, ADCC, or antigen-induced T cell proliferation.     

Clinical evaluation of CA 15.3 in the post-operative follow-up of breast cancer patients

In 265 patients operated for breast carcinoma the monoclonal antibody serum test CA 15.3 was predictive of metastatic diffusion of the disease. Its level increased in cases of distant metastasis with no significant difference between multiple and single sites (p = 0.014). The concentration of the marker was higher in 21 (23.8%) patients without nodal involvement and in 19 (27.5%) with nodal involvement (p = 0.193). Our study suggests that CA 15.3 may be an aid in the follow-up of patients with metastatic diffusion of breast cancer.     

Immunoscintigraphy of colorectal cancer using111In-labeled monoclonal antibody to mucin

A murine monoclonal antibody MLS102 recognizes sialosyl-Tn antigen in mucin and immunohistochemically reacts with more than 80% of colorectal cancer tissues. The purpose of this study was to assess the usefulness of this monoclonal antibody for the immunoscintigraphy of colorectal cancer. Planar and SPECT images were obtained on day 2 or day 3 after injection of 2 mg and 74 MBq¹¹¹In-labeled MLS102 antibody into 17 patients with colorectal cancer. Nine of 11 primary tumors and 4 of 6 locally recurrent tumors were detected. Positive images were obtained in all tumors larger than 4.5×2.7 cm. Three tumors of less than 2.5 cm and 1 recurrent tumor, which was missed by other imaging modalities, were negative. There were no adverse reactions. Human anti-(mouse Ig) antibody developed in 4 patients. Although improvement of detectability for smaller tumors needs to be pursued, the antibody MLS102 is potentially promising for use in immunoscintigraphy of colorectal cancer.     

HER2阳性乳腺癌中CD44v6的表达与曲妥珠单抗耐药的关系

目的:探讨曲妥珠单抗在治疗HER2阳性的转移性乳腺癌时,产生耐药性与CD44v6表达的相关性。方法:共66例HER2阳性转移性乳腺癌患者入组。接受曲妥珠单抗治疗的患者37例,其中18例获得了治疗前和治疗后转移性癌组织。采用免疫组化S-P法对治疗前、治疗后的不同乳腺组织进行CD44v6表达的研究。结果:CD44v6在经曲妥珠单抗治疗产生耐药的活检组织中阳性表达程度明显高于治疗前。结论:CD44v6的表达与曲妥珠单抗耐药相关。     

Circulating CA 15.3 levels in breast cancer. Our present experience

CA 15.3 is an antigen expressed by human breast carcinoma cells, and defined by two monoclonal antibodies, 115D8 and DF3. We used IRMA to determine the circulating serum levels of CA 15.3 in 1178 subjects with breast cancer, non-breast malignancies, benign diseases and controls. A threshold level of 40 U/ml was established with 140 healthy controls and 650 patients with benign diseases (respectively 0% subjects and 1.5% patients had abnormal antigen levels). Elevated CA 15.3 was found in 12 of 184 patients with malignancies different from breast cancer (6.5%), either epithelial carcinomas with distant metastases, mainly in the liver, or primary liver tumors. Breast cancer patients (n = 204) were analysed by prior therapy, UICC stage and WHO response to therapy. Eight of 134 (5.9%) patients with stage II or III breast cancer at presentation and no evidence of disease (NED) had elevated CA 15.3. All of 22 patients with stage IV breast cancer not responding to therapy (SD and PD) had antigen levels greater than 40 U/ml, as did 10 of 34 (29.4%) stage IV patients in objective response (CR + PR). Three of 14 pretreatment patients had abnormal marker levels, and they later proved to have distant metastases. Serum CA 15.3 values were statistically different (p less than 0.01) in NED (20.6 +/- 11.2 U/ml), CR + PR (33.5 +/- 24.0 U/ml), stable disease (98.8 +/- 50.4 U/ml) and progressive disease (greater than 200 U/ml) breast cancer patients. Our results suggest that circulating CA 15.3 antigen levels agree with the stage of breast cancer and with the response to therapy.     

HER2阳性乳腺癌中CD44v6的表达与曲妥珠单抗耐药的关系

目的：探讨曲妥珠单抗在治疗HER2阳性的转移性乳腺癌时,产生耐药性与CD44v6表达的相关性。方法：共66例HER2阳性转移性乳腺癌患者入组。接受曲妥珠单抗治疗的患者37例,其中18例获得了治疗前和治疗后转移性癌组织。采用免疫组化S-P法对治疗前、治疗后的不同乳腺组织进行CD44v6表达的研究。结果：CD44v6在经曲妥珠单抗治疗产生耐药的活检组织中阳性表达程度明显高于治疗前。结论：CD44v6的表达与曲妥珠单抗耐药相关。     

A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity

The Tn antigen (GalNAc α-O-Ser/Thr) is heterogeneously synthesized by a variety of tumors and contains an epitope defined by lectins and antibodies as a cluster of αGalNAc carbohydrates synthesized within a peptide sequence, which is rich in serine and/or threonine. The Tn antigen has been utilized as a target in vaccine experiments and also used as a biomarker for prognosis of different cancer forms. In this paper, we present a new monoclonal antibody, GOD3-2C4, with the clear hallmarks of an anti-Tn antibody. It was generated through somatic cell hybridization after immunization of a mouse with a tumor cell line and a Tn carrying mucin. The antibody recognizes synthetic Tn antigen and binds breast, colon, lung, ovarian and pancreas cancer. The GOD3-2C4 antibody has antibody-dependent cellular cytotoxicity activity against Jurkat cells in vitro, and for the first time, it can be shown that an anti-Tn antibody has a significant in vivo effect on a human cancer cell line grown as a xenograft in severe combined immunodeficiency mice.     

The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine

 The identification and cloning of several tumour antigens together with an improvement in the understanding of the mechanisms involved in antigen presentation and immune recognition has opened up the possibility of using active specific immunotherapy as a treatment for certain cancers. This review discusses the tumour-associated MUC1 gene product of the polymorphic epithelial mucin (PEM), as a potential target molecule for cancer treatment. PEM is both over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule. Furthermore, immune responses specific for PEM have been detected in cancer patients. Both syngeneic and transgenic murine model systems have been developed in order to compare the efficacy and toxicity of various PEM-based immunogens in tumour rejection studies, and to further improve the understanding of antigen presentation and the mechanisms underlying tumour rejection. Such models also allow the examination of MUC1-based immunogens as a treatment for existing tumours. Clinical trials in progress using immunogens based on the MUC1 gene product are briefly discussed. Received: 23 November 1995 / Accepted: 4 January 1996     

Chromogenic in situ hybridization analysis of HER-2/neu status in cytological samples of breast carcinoma

The current use of humanized monoclonal antibody trastuzumab for the treatment of patients with metastatic breast cancer has made evaluation of HER-2/neu status an important clinical issue. Chromogenic in situ hybridization (CISH), in which the DNA probe is detected with an immunohistochemistry (IHC)-like peroxidase reaction, has been recently developed for the assessment of HER-2/neu status in formalin-fixed breast cancer specimens. We have applied the technique of dual-colour CISH using HER-2/neu and chromosome 17 centromere probes in 27 cytological smears, and these cytological samples were obtained from scrapings of fresh breast tumours. We also investigated HER-2/neu amplification and protein overexpression in the corresponding surgical tissues by CISH and IHC using the monoclonal antibody CB11. Of the 27 cytological cases, HER-2/neu gene amplification was observed in nine cases that were positive cases (2+ and 3+) for IHC. Among the 13 IHC positive cases (2+ and 3+), four of them showed no gene amplification. Identical results for the CISH technique were obtained in the matched surgical samples. The scrape samples from fresh breast tumour offer a monolayer cell population that is especially suitable for CISH. This study has shown that the cytological smear might be a good alternative for the CISH test.     

Detection of a soluble serum antigen by a new monoclonal antibody ED8 in patients with breast cancer

A murine hybridoma, secreting monoclonal antibody ED8, was generated by immunization with the breast cancer cell line H466B. ED8 reacts with a 300 kDa antigen present on H466B cell surface. The antigen can be detected in serum from breast cancer patients, using ELISA and immunoblotting.