cefoxitin and Breast-Neoplasms

Almost 40 years ago, researchers found out that the Thomsen-Friedenreich (TF) and the Thomsen nouvelle (Tn) antigens could be detected in carcinoma, but not in healthy tissue. A short time after that it became clear that TF and Tn are precursor molecules of the MN-blood group antigens. In normal tissue TF and Tn are coated by glycosyl structures, thereby forming the glycoproteins which are known to account for the MN-blood group, but in malignant tissue these molecules are uncovered.TF, which has an additional Galectin-residue compared to Tn, is correlated with a more favourable prognosis for patients. On the contrary, patients with Tn-bearing tissues have a worse prognosis for overall and progression-free survival. It is known that TF and Tn are involved in the adhesion of tumour cells to the endothelium via a mechanism recruiting Galectin-3 and MUC-1, which is the first step in metastasis formation. Furthermore, it became clear that this pathway can be blocked by a growing number of molecules, thereby creating ways of therapeutical intervention.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Adhesion; Female; Humans; Neoplasm Invasiveness

Physical and chemical nature of the T and Tn pancarcinoma [CA] glycopeptide epitopes [EPs], which are immediate precursors of the blood group MN EPs, and their role in CA pathogenesis and in clinical disease are discussed. T/Tn are immuno-occluded in non-CA diseased and in healthy tissues. Well-differentiated CAs usually express a higher proportion of T than Tn EPs, while Tn predominates in poorly differentiated primary CAs. Measurement of density of T and Tn EP expression on primary breast CA permits disease prognostication. CA-T and -Tn are cell adhesion molecules involved not only in invasion but also in metastasis. Immunological methods readily detect in vivo autoimmune responses to CA-T and -Tn EPs in about 90% of all CA patients from incipience and throughout. Everyone has preexisting anti-T and anti-Tn antibodies [Abs] induced by the intestinal flora. T/anti-T immunoassays are highly efficient in detection of incipient and clinically overt CAs and, importantly, predicted CA in 77% of the patients, months to many years before their biopsy/X-ray turned positive; there were no false immune predictions of CA. Since 1974, we use human O MN red cell-derived T/Tn glycoprotein vaccine plus adjuvants successfully in safe, specific, effective, long-term, active immunotherapy against recurrence of advanced breast CA pTNM Stages IV, III, and II.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Biopsy; Breast Neoplasms; Cell Adhesion; Epitopes; Erythrocyte Membrane; Female; Humans; Hypersensitivity, Delayed; Immunotherapy; Leukemia; Lymphoma; Recurrence

This interpretive review attempts to dovetail advanced work by different groups of investigators on blood group and carcinoma (CA) glycoconjugates that have terminal, immunoreactive Tn epitopes (GalNAc alpha-O-Ser/Thr), and on the interaction of those structures with complementary antibodies and lectins. Fenlon et al. (1987) and Leathem and Brooks (1987) found a positive correlation between primary breast CA aggressiveness and its affinity for Helix pomatia (HPA) lectin. This phenomenon was used successfully to accurately predict, in studies on 305 breast CA patients, early or late CA recurrence and patient survival time. The innate specificity of the large HPA combining groove (aside from its avid reactivity with appropriately spaced GalNAc alpha-O-) remains obscure, despite careful investigation for more than a decade (Baker et al., 1983). Leathem and Brooks presumed that HPA recognizes a hitherto "undefined biological marker" that indicates a breast CA's aggressiveness. Our own work has shown that the chemically fully defined Tn epitope, as measured with human polyclonal and murine monoclonal anti-Tn antibodies, occurs in immunoreactive form in approximately 90% of all breast and lung adenoCAs studied. Tn is occluded and non-reactive in healthy and non-CA-diseased tissues. We found that CA-associated Tn is an adhesion molecule in attachment to healthy cells; an increase in its density on breast CA cell membranes parallels greater aggressiveness of breast tumors in both humans and mice (the only species studied). Thus, Tn may be all or a major part of the postulated "as yet undefined biological marker" associated with high breast CA aggressiveness. Besides being helpful in the elucidation of some aspects of breast CA pathogenesis, these findings on primary breast CA have clinical implications in that they should facilitate stratification of breast CA patients for adjuvant treatment.

Topics: Antibodies, Neoplasm; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Epitopes; Female; Humans

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT

Topics: Adjuvants, Immunologic; Adult; Aged; Animals; Antibodies, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cancer Vaccines; Female; Glycopeptides; Humans; Immunogenicity, Vaccine; Injections, Intramuscular; Jurkat Cells; Middle Aged; Neoplasm Recurrence, Local; Treatment Outcome; Vaccination; Vaccines, Synthetic

For nearly 20 yrs, we used T/Tn antigen vaccine in safe, specific, effective, long-term intradermal vaccination against recurrence of advanced breast carcinoma. Treatment is ad infinitum. All 18 breast carcinoma patients treated, pTNM Stages IV (6), III (6), and II (6), survived > 5 yrs postoperatively; 10 survived > 10 to > 18 yrs; of the latter, three patients each are Stages III and IV. Five additional 5 yr survivors have not yet reached 10 yrs. The probability that our survival results are due to chance, with NCI "1991 Standard PDQ Data" as control, for all three stages taken together is: 5-yr survival: p < 1 x 10(-8); 10-yr survival: p < 1 x 10(-5). There were no untoward side effects. The vaccination area presented as a delayed-type hypersensitivity reaction, but at variance with the PPD reaction, with significant inflammation, increase of helper T lymphocytes and decrease of the T suppressor/cytotoxic cell ratio.

Topics: Adult; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Combined Modality Therapy; Female; Humans; Hypersensitivity, Delayed; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Pilot Projects; Survival Rate; Treatment Outcome; Vaccination; Vaccines

We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3-10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.

Topics: Adjuvants, Immunologic; Antibodies, Neoplasm; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carcinoma; Complement System Proteins; Cytotoxicity, Immunologic; Female; Glycoconjugates; Haptens; Humans; Immunization; Ovarian Neoplasms; Tumor Cells, Cultured

Breast cancer is one of the most serious and deadly cancers in women worldwide, with distant metastases being the leading cause of death. Tn antigen, a tumor-associated carbohydrate antigen, was frequently detected in breast cancer, but its exact role in breast cancer metastasis has not been well elucidated. Here we investigated the impact of Tn antigen expression on breast cancer metastasis and its underlying mechanisms. The expression of Tn antigen was induced in two breast cancer cell lines by deleting T-synthase or Cosmc, both of which are required for normal O-glycosylation. It showed that Tn-expressing cancer cells promoted epithelial-mesenchymal transition (EMT) and metastatic features as compared to Tn(-) control cells both in vitro and in vivo. Mechanistically, we found that cancer susceptibility candidate 4 (CASC4), a heavily O-glycosylated protein, was significantly downregulated in both Tn(+) cells. Overexpression of CASC4 suppressed Tn-induced activation of EMT and cancer metastasis via inhibition of Cdc42 signaling. Furthermore, we confirmed that O-glycosylation is essential for the functional role of CASC4 because defective O-glycosylated CASC4 (mutant CASC4, which lacks nine O-glycosylation sites) exerted marginal metastatic-suppressing effects in comparison with WT CASC4. Collectively, these data suggest that Tn-mediated aberrant O-glycosylation contributes to breast cancer metastasis via impairment of CASC4 expression and function.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Melanoma, Cutaneous Malignant; Membrane Proteins; Molecular Chaperones

The high mortality of breast cancer (BC) is associated with the strong metastatic properties of primary breast tumor cells. The present study was conducted in order to clarify the effect of Cosmc on the growth and metastasis of BC cell lines of different molecular types, which may be implicated in the regulation of Tn and T glycans.. BC cell lines with different molecular types were transduced with shRNA targeting Cosmc or, Cosmc overexpression plasmid in order to explore the role of Cosmc in cell proliferation, migration, invasion, and apoptosis. The protein levels of Tn, T, Cosmc, proliferation-related factors (Ki67 and PCNA) and apoptosis-related factors (Bax and Bad) in BC cell lines were determined by Western blot analyses. Finally, the role of Cosmc was substantiated through in vivo experiments.. Cosmc was down-regulated in different subtypes of BC cell lines compared with normal control cells. Overexpression of Cosmc suppressed the proliferation, migration, and invasion, yet promoted the apoptosis of BC cells, as reflected by in vitro experiments. Additionally, in vivo tumor xenografts in nude mice showed that ectopic overexpression of Cosmc inhibited the tumor growth of BC cells. Consequently, the levels of proliferation-related factors and Tn antigen were decreased, while those of apoptosis-related factors and T antigen were increased in BC cells. This observation was confirmed in vivo in xenograft tumors.. Collectively, up-regulation of Cosmc potentially impedes BC growth and metastasis by modulating the balance between Tn and T glycans.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Apoptosis; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Molecular Chaperones; Neoplasm Invasiveness; Neoplasm Metastasis; Polysaccharides; Signal Transduction; Tumor Burden

The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Antineoplastic Agents, Immunological; Breast Neoplasms; Humans; Immunoglobulin M; Immunotoxins; Male; MCF-7 Cells; Mice; Mice, Inbred C57BL; Mice, SCID; Polysaccharides; Xenograft Model Antitumor Assays

Carbohydrate-protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn)-MGL and MUC1(STn)-MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carbohydrates; Cell Line; CHO Cells; Cricetulus; Female; Humans; Lectins, C-Type; Mucin-1; Optical Tweezers; Polysaccharides

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cancer Vaccines; Female; Humans; Immunization; MCF-7 Cells; Mice, Inbred C57BL; Mice, Transgenic; Mucin-1; Peptide Fragments; Tetanus Toxoid; Vaccines, Synthetic

Failure in O-glycan chain extension exposing Tn antigen (GalNAc-O-Ser/Thr) is clinically associated with cancer metastasis. This study provides evidence of a functional role for aberrant GalNAc-glycans in cancer cell capture from blood flow and/or adhesion to endothelium. Adhesion of breast cancer cells to human umbilical vein endothelial cell monolayers was modelled under sweeping flow. Adhesion of metastatic, GalNAc glycan-rich, MCF7 and ZR 75 1 cells to endothelium increased over timepoints up to 1.5 hour, after which it plateaued. Adhesion was significantly inhibited (p < 0.001) when cell surface GalNAc-glycans were masked, an effect not seen in GalNAc glycan-poor, non-metastatic BT 474 cells. Masking irrelevant galactose- and mannose-glycans had no inhibitory effect. Imaging of cells post-adhesion over a 24 hour time course using confocal and scanning electron microscopy revealed that up to 6 hours post-adhesion, motile, rounded cancer cells featuring lamellipodia-like processes crawled on an intact endothelial monolayer. From 6-12 hours post-adhesion, cancer cells became stationary, adopted a smooth, circular flattened morphology, and endothelial cells retracted from around them leaving cleared zones in which the cancer cells proceeded to form colonies through cell division.

Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Adhesion; Cell Membrane; Endothelial Cells; Female; Human Umbilical Vein Endothelial Cells; Humans; Lectins; MCF-7 Cells; Neoplasm Metastasis; Polysaccharides

The serum levels of IgG antibodies reactive to glycoconjugates (TF, Tn and αGal) were found to be associated with prognosis of gastrointestinal cancer patients.. To study the relation between the levels of serum antibodies to TF-pAA, Tn-PAA and αGal-PAA polyacrylamide-based glycoconjugates and survival in breast cancer.. The preoperative level of IgG antibodies was analysed in the serum of patients (n = 59) using ELISA with polyacrylamide-glycoconjugates namely, TF-pAA (amide-type), and ethanolamide-conjugates Tn-PAA and αGal-PAA. Survival rate and hazard ratio (HR) were assessed by the Kaplan - Meier method and Cox univariate analysis in different pathomorphological groups.. Significantly better survival was observed in patients with an increased level of anti-TF-pAA antibodies both for all patients in total and groups in stages II-III; N1-2 and G3 (p = 0.008-0.021, HR = 0.18-0.23, mean survival time in months 164-186 vs 69-121). A trend to worse survival was observed in increased level of anti-Tn IgG (stages II-III) and anti-αGal IgG (G3): p = 0.075, HR = 2.49 and p = 0.066, HR = 3.27, respectively.. The method for the determination of circulating anti-TF-pAA IgG may be a useful supplement in long-term prognostic assessment of patients with breast cancer.

Topics: Adult; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Female; Glycoconjugates; Humans; Immunoglobulin G; Middle Aged; Prognosis; Trisaccharides

Tn antigen (GalNAc-α-O-Ser/Thr), a mucin-type O-linked glycan, is a well-established cell surface marker for tumors and its elevated levels have been correlated with cancer progression and prognosis. There are also reports that Tn is elevated in inflammatory tissues. However, the molecular mechanism for its elevated levels in cancer and inflammation is unclear. In the current studies, we have explored the possibility that cytokines may be one of the common regulatory molecules for elevated Tn levels in both cancer and inflammation. We showed that the Tn level is elevated by the conditioned media of HrasG12V-transformed-BEAS-2B cells. Similarly, the conditioned media obtained from LPS-stimulated monocytes also elevated Tn levels in primary human gingival fibroblasts, suggesting the involvement of cytokines and/or other soluble factors. Indeed, purified inflammatory cytokines such as TNF-α and IL-6 up-regulated Tn levels in gingival fibroblasts. Furthermore, TNF-α was shown to down-regulate the COSMC gene as evidenced by reduced levels of the COSMC mRNA and protein, as well as hypermethylation of the CpG islands of the COSMC gene promoter. Since Cosmc, a chaperone for T-synthase, is known to negatively regulate Tn levels, our results suggest elevated Tn levels in cancer and inflammation may be commonly regulated by the cytokine-Cosmc signaling axis.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Bronchi; Cell Line; CpG Islands; Culture Media, Conditioned; Disease Progression; DNA Methylation; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, ras; Gingiva; Humans; Inflammation; Interleukin-6; Male; Molecular Chaperones; Prognosis; Promoter Regions, Genetic; Prostatic Neoplasms; Signal Transduction; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Lectins, C-Type; Mucin-1; Protein Binding

In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast; Breast Neoplasms; Cadmium Compounds; Carcinoma, Ductal, Breast; Cell Line, Tumor; Female; Fluorescence; Fluorescent Antibody Technique; Horseradish Peroxidase; Humans; Luminescence; Quantum Dots; Selenium Compounds; Spectrum Analysis; Staining and Labeling; Tissue Array Analysis; Water

The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Female; Glycopeptides; Humans; Male; Mice; Middle Aged; Neoplasm Staging; Neoplasms; Protein Binding

The Tn antigen (GalNAc alpha-O-Ser/Thr) as defined by the binding of the lectin, helix pomatia agglutinin (HPA) or anti-Tn monoclonal antibodies, is known to be exposed in a majority of cancers, and it has also been shown to correlate positively with the metastatic capacity in breast carcinoma. The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1), which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin-embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4) did to some extent overlap with the presence of IgA1 in the tumours, but differences were seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence of Tn positive IgA in serum. On average 51% of the tumour cells in the individual breast cancer tumour sections showed staining for IgA1. The overall amount of staining in the invasive part of the tumour with the anti Tn antibody was 67%, and 93% with HPA. The intra-expression or uptake of IgA1 in breast cancer makes it a new potential carrier of the tumour associated and immunogenic Tn antigen.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Line, Tumor; Female; Glycosylation; Humans; Immunoglobulin A; Immunohistochemistry; Lectins; Mice; Neoplasm Invasiveness

The tumor-associated carbohydrate antigen/hapten Thomsen-nouveau (Tn; a-D-GalpNAc-ONH2) was conjugated to a zwitterionic capsular polysaccharide, PS A1, from commensal anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 for the development of an entirely carbohydrate cancer vaccine construct and probed for immunogenicity. This communication discloses that murine anti-Tn IgG3 antibodies both bind to and recognize human tumor cells that display the Tn hapten. Furthermore, the sera from immunization of mice with Tn-PS A1 contain cytokine interleukin 17 (IL-17A), which is known to possess anti-tumor function and represents a striking difference to an IL-2, and IL-6 profile obtained with anti-PS A1 sera.

Topics: Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Bacteroides fragilis; Breast Neoplasms; Cancer Vaccines; Carbohydrates; Cell Line, Tumor; Female; Humans; Immune Sera; Immunity; Immunization; Immunoglobulin G; Interleukin-17; Mice; Mice, Inbred C57BL; Polysaccharides, Bacterial

Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcγR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigen-specific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated γ chain (FcR-γ)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-γ chain-expressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcγR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Neoplasm; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antigens, Tumor-Associated, Carbohydrate; Antineoplastic Agents, Alkylating; Breast Neoplasms; Cell Line, Tumor; Combined Modality Therapy; Cyclophosphamide; Cystadenocarcinoma, Serous; Female; Humans; Immunization, Passive; Immunological Synapses; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neutrophils; Ovarian Neoplasms; Receptors, IgG; Trastuzumab; Tumor Microenvironment

The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Bacteriophages; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Fluorescent Antibody Technique; Glycopeptides; Humans; Immunoglobulin Variable Region; Peptide Library; Sequence Homology, Amino Acid; Single-Chain Antibodies; Surface Plasmon Resonance

Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research.

Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Colonic Neoplasms; Disaccharides; Epitopes; Female; Forssman Antigen; Humans; Lectins

VVA-B4 lectin was used to investigate the differences in Tn antigen expression in tissues of different types of human breast cancer (benign lesions, carcinoma in situ, invasive carcinoma) and in normal tissues neighboring lobular carcinoma. Locations in which Tn antigen was expressed were identified using the avidin-biotin-peroxidase labeling system. Tissues collected during cosmetic procedures and classified as normal were completely negative, except for one case. Benign proliferative changes including fibroadenoma, apocrine and cylindrical metaplasia showed a very weak positive reaction, although strongly positive cells were also observed. The reaction in non-invasive cases of atypical hyperplasia was diversified depending on site. Intralobular hyperplasia was characterized by a particularly high percentage of labeled cells. A majority (up to 80%) of ductal and lobular carcinoma in situ showed very strong or moderate staining. In invasive cancers, there were conspicuous differences between stage of cancer development and tendency towards a decrease in intensely labeled cell count in the most advanced stages. In normal tissues in the direct neighborhood of carcinoma in situ, the cytoplasm of 40% of cells was strongly labeled. However, the findings for normal tissues in the close vicinity of invasive cancer were the most surprising, since there was either no or only very weak positive reaction. It can be concluded that glycosylation modifications during carcinogenesis, as demonstrated by the presence of Tn epitope, develop very early, before any destructive changes in proliferation/apoptosis or cell differentiation become discernible.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Female; Fibroadenoma; Humans; Hyperplasia; Immunohistochemistry; Lectins; Precancerous Conditions

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cancer Vaccines; Cell Extracts; Cell Line, Tumor; Escherichia coli; Female; Glycosylation; Humans; Immunotherapy; Mucin-6; Mucins; N-Acetylgalactosaminyltransferases; Polypeptide N-acetylgalactosaminyltransferase; Recombinant Proteins

The effects of N- and O-glycosylation inhibitors on the expression of membrane proteins (MUC1 and some integrins) were evaluated in human endometrial (Ishikawa) and breast (MCF-7) cancer cells. Subconfluent cells were treated with 1-3 mg% concentration of tunicamycin and 2-10 mM of benzyl-N-acetyl-alpha-galactosaminide for 1-2 days, and used for flow cytometry, immunohistochemical staining, adhesion test and Western blotting. Benzyl-N-acetyl-alpha-galactosaminide inhibits MUC1 expression on the surface of breast more than endometrial cancer cells. Tunicamycin reduces MUC1 concentration on the cellular surface more than benzylglycoside, and greatly reduces glycosylation of glycoproteins, causing an increase in cell adhesion in both types of cancer cells. The expression of alpha2beta1 integrins on the surface of these cells was weak and decreased after treatment with inhibitors. Two different glycoforms of MUC1 proteins in endometrial cells and three in breast cancer cells were expressed and their molecular weights were reduced after treatment with glycosylation inhibitors. It was confirmed with lectin detection of carbohydrate epitopes (Tn and T) in MUC1 proteins. These observations show that glycosylation inhibitors altered the N- and O-glycan patterns in a sufficient manner, and positively modified the biological features of cancer cells.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Collagen Type I; Endometrial Neoplasms; Female; Flow Cytometry; Galactose; Glycosylation; Humans; Integrin alpha2beta1; Membrane Proteins; Mucin-1; Mucins; Protein Binding; Tunicamycin

Aberrant carbohydrate expression frequently occurs in breast cancer and may endow cells with metastatic potential. Here we first studied the relationship between expression of Vicia villosa agglutinin (lectin) (VVA)-binding carbohydrates and aggressive breast cancer. We then investigated the molecular characteristics of these glycoproteins and compared them with those of glycoproteins recognized by the mouse anti-Tn monoclonal antibody (MAb) HB-Tn1. Histochemical studies of samples from 322 cases of invasive ductal carcinoma demonstrated that VVA-binding carbohydrate expression correlated with tumor stage, lymphatic invasion, and lymph node metastasis (p=0.0385, p=0.0019, and p=0.0430. respectively). Western blotting analysis of frozen materials from 39 cases, under denaturing and reducing conditions, revealed that the major cancer cell-specific VVA-binding proteins were molecules of about 30, 33, and >200 kDa. Cases expressing the approximately 33 kDa molecule had significant lymphatic invasion more frequently than did cases not expressing this molecule (p=0.0076). Binding of VVA to the approximately 30 and approximately 33 kDa molecules was completely lost by preincubation of VVA with 1 mM Tn antigen (N-acetylgalactosamine alpha1-O-serine). The VVA-binding molecules appeared to react with VU-3C6 anti-MUC1 MAb. Expression of HB-Tn1 in breast cancer cells showed significant correlation with expression of VVA-binding carbohydrate(s) (p<0.0001) but HB-Tn1 reactivity was not clearly related to breast cancer aggressiveness. Because anti-Tn MAbs bound to Tn antigen clusters, we concluded that atypical MUC1 bearing the noncluster form of Tn antigen is implicated in aggressive growth of primary breast cancer cells, particularly in lymphatic metastasis.

Topics: Adult; Aged; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carbohydrates; Cell Proliferation; Epitopes; Female; Glycoproteins; Humans; Lymphatic Metastasis; Middle Aged; Mucin-1; Plant Lectins

To study the humoral immune response to tumor-associated carbohydrate epitopes (TF, Tn and alphaGal) in patients with breast cancer and healthy donors, the putative impact of the chemotherapy and to evaluate if the level of antibody to these epitopes might be beneficial or detrimental for the patients with breast cancer.. The humoral immune response to TF, Tn and alphaGal was studied in 133 patients with breast cancer, including the patients at stage II-III (n = 44) before and after neoadjuvant chemotherapy (10 patients received cyclophosphamide/methotrexate/fluorouracyl (CMF) chemotherapy regimens, 34 patients received cyclophosphamide/doxorubicin/fluorouracil (CAF)), and in controls (healthy donors and patients with fibroadenoma). Fully synthetic carbohydrate hapten-polyacrylamide conjugates were used as antigens in ELISA for anti-carbohydrate antibody determination. The correlation analysis between the level of anti-carbohydrate antibodies and the stage of cancer, histological grade, expression of TF and alphaGal epitopes in tumor tissue, patient's survival was performed.. The level of anti-carbohydrate antibodies varied between individuals with no significant correlation between IgG immune response to the three epitopes. Lower levels of antibodies were observed at advanced stages of cancer. Neoadjuvant chemotherapy stimulated antibody production to Tn and alphaGal epitopes (increase > 50%) in about one third of patients. Immunosuppression, decrease in antibody levels, was observed only in 4.5-13.6% of cases. High levels of TF-antigen specific IgG antibody before surgery were associated with a better survival time of stage II breast cancer patients.. The widely used regimens of neoadjuvant chemotherapy (such as CMF, CAF) can stimulate the immune response to tumor-associated carbohydrate epitopes in some patients. The high levels of anti-TF antibody before surgery are associated with a better survival of stage II breast cancer patients. This may indicate that the selection of immunopotentiating regimens of neoadjuvant chemotherapy might be beneficial for the host.

Topics: Adult; Aged; Aged, 80 and over; alpha-Galactosidase; Antibodies, Neoplasm; Antibody Formation; Antigens, Tumor-Associated, Carbohydrate; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cyclophosphamide; Doxorubicin; Epitopes; Female; Fibroadenoma; Fluorouracil; Haptens; Humans; Methotrexate; Middle Aged; Neoadjuvant Therapy; Neoplasm Staging; Survival Rate; Thromboplastin

An incomplete elongation of O-glycan saccharide chains in mucins have been found in epithelial cancers, leading to the expression of shorter carbohydrate structures, such as the Tn antigen (GalNAc-O-Ser/Thr). This antigen is one of the most specific human cancer-associated structures and is capable of inducing effective immune responses against cancer cells. We aimed to investigate the causes of the expression of Tn antigen in the Tn-rich MCF-7 breast cancer cell line focusing on the first step of the O-glycosylation process. Interestingly, amino acid sequences derived from "non-mammary" apomucins (MUC5B and MUC6) were very good acceptor substrates for ppGalNAc-Ts, which are the enzymes catalyzing the Tn antigen synthesis. MUC6 peptide glycosylation with MCF-7 microsome extracts as source of ppGalNAc-T activity yielded 95% conversion of the peptide into MUC6-Tn. In addition, the MUC6-Tn glycopeptide was a poor acceptor substrate for core 1 beta3Gal-T, the next enzyme involved in the saccharide chain biosynthesis, yielding only 5% conversion of MUC6-Tn into MUC6-TF. These results indicate that non-mammary apomucin expression could be responsible, at least in part, for Tn antigen expression in MCF-7 breast cancer cells due to a combined action on glycosyltransferases: an increase of ppGalNAc-T activity and a decrease of core 1 beta3Gal-T activity. Our hypothesis is supported by experiments in vivo showing that (a) native MUC6 glycoproteins express the Tn antigen in MCF-7 cells and (b) Tn antigen expression is increased after transfection with a construct encoding for a MUC6 recombinant protein into the low Tn-expressing breast cancer cell T47D. These results open new horizons in breast cancer glycoimmunology, stressing the potential role of non-mammary apomucins.

Topics: Amino Acid Sequence; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Cell Line, Tumor; Glycopeptides; Glycosylation; Humans; Molecular Sequence Data; Mucin-6; Mucins; Polysaccharides; Transfection

MUC1 is a transmembrane molecule characterized by a repeated sequence of 20 amino acid (TAP PAHGVTSAPDTRPAPGS). Abnormal overexpression of MUC1 in cancer cells is thought to contribute to their aggressive growth, but molecular mechanisms associated with this effect are still unclear. Our current study aimed to clarify whether MUC1 expression as recognized by VU-3C6 anti-MUC1 mouse IgG monoclonal antibody (MAb) with a dominant epitope of 12 amino acids: GVTSAPDTRPAP, correlated with aggressive properties of human breast cancer. Immunohistochemical studies of 309 samples of formalin-fixed and paraffin-embedded materials showed no statistical correlation between MUC1 expression and clinicopathological parameters, as well as several breast cancer aggressiveness-related markers. Expression or nonexpression of MUC1 in 50 frozen samples, as determined by Western blotting, demonstrated no correlation with aggressive properties of breast cancer. However, samples with one MUC1-positive band more often had lymphatic vessel invasion and lymph node metastasis than those with more than two or three MUC1-positive bands (p<0.014 and p<0.043, respectively). Because VU-3C6 MAb recognizes MUC1 with short branches of O-glycosylated core carbohydrates, we used immunohistological methods to examine Tn antigen (precursor antigen: GalNAcalpha-O-Ser/Thr), Thomsen-Friedenreich (T) antigen, and sialyl-Tn antigen (STn) antigen. We found a strong correlation between expression of MUC1 and Tn antigen (p<0.0006), and samples with Tn antigen expression had more lymphatic metastasis than those with no such expression (p<0.08). We concluded that the lack of polymorphic MUC1 expression with Tn antigen is one characteristic related to aggressive breast cancer.

Topics: Antigens; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Glycoproteins; Humans; Immunohistochemistry; Japan; Lymphatic Metastasis; Middle Aged; Mucin-1; Mucins; Neoplasm Invasiveness; Polymorphism, Genetic; Tandem Repeat Sequences

Novel biomarkers would facilitate early and accurate diagnosis of breast cancer. The Thomsen-Freidenreich (TF) and Tn antigens are aberrantly glycosylated carbohydrate cancer-associated antigens found in approximately 80% of adenocarcinomas. Both TF and Tn are expressed on cell-surface glycoproteins and glycolipids. Nipple aspirate fluid (NAF) is concentrated in secreted proteins and lipids from cells that give rise to cancer. The objective of this study was to determine if NAF from breasts with cancer contains elevated levels of TF and Tn compared with NAF from normal breasts. A sensitive and specific antigen capture immunoassay for TF and Tn detection in NAF was developed for this purpose.. Fifty NAF samples, 25 from breasts with cancer and 25 from normal breasts, were examined. Antigen capture immunoassays were done on the samples using monoclonal antibodies that specifically recognized either TF or Tn antigen in NAF. These antibodies captured serially diluted NAF samples, and the concentration of TF or Tn was determined by comparing absorbance values against a standard curve generated from standard sources of TF or Tn.. TF and Tn were detected in 19 of 25 and 20 of 25 NAF samples from breasts with cancer, respectively, compared with 0 of 25 and 1 of 25 NAF samples from breasts without cancer (P < 0.001 for both TF and Tn). In 92% of the cancerous breast NAF samples tested, either TF or Tn was found.. Simultaneous measurement of TF and Tn in NAF may facilitate the noninvasive detection of breast cancer and warrants further study.

Topics: Adult; Aged; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Biopsy, Needle; Body Fluids; Breast; Breast Neoplasms; Carbohydrates; Enzyme-Linked Immunosorbent Assay; Female; Glycosylation; Humans; Immunoassay; Mass Screening; Middle Aged; Postmenopause; Premenopause; Recurrence

Three cancer cell lines (MCF-7, HBL-100, MDA-MB 231) and subnormal breast epithelial cell line MCF-10A were labeled with FITC-conjugated VVA-B4 lectin, specific for D-GalNAcalpha-O-ser/thr, matching the structure of Tn antigen sugar residues, and with RTIC-conjugated PNA lectin, specific for DGalbeta1-3GalNAc-O-ser/thr, corresponding to the structure of T antigen. Simultaneous expression of Tn and T antigens on the same cells (but in widely differing proportions) led to their large heterogeneity and occurrence of numerous cell subpopulations within each of the studied cell lines. This observation proved that the changes leading to the formation of Tn antigen are not caused by an irreversible genetic mutation of beta1-3-galactosyltransferase. Expression of Tn antigen on MCF-10A cells with normal (or subnormal) karyotype suggests that the process of malignant transformation of the cell begins with the changes in molecular structure of glycoconjugates.

Topics: Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Binding Sites; Breast Neoplasms; Female; Humans; Microscopy, Confocal; Peanut Agglutinin; Plant Lectins; Tumor Cells, Cultured

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O:-linked to a CD4(+) T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.

Topics: Animals; Antibodies, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Antineoplastic Agents; Binding Sites, Antibody; Breast Neoplasms; Cancer Vaccines; Carbohydrate Sequence; CD4-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Female; Glycopeptides; Humans; Immunotherapy, Active; Injections, Intraperitoneal; Jurkat Cells; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasm Transplantation; Poliovirus; Tumor Cells, Cultured

In the present work we demonstrate that the cancer-associated O-glycosylated Tn antigen (GalNAc-O-Ser/Thr) is expressed by the cestode Echinococcus granulosus. This antigen was detected in both larval and adult worm extracts, with the highest specific activity observed in the adult excretion/secretion preparation. Histochemical analysis showed that Tn is preferentially expressed in the parenchyma in both parasite stages and the external part of tegument in adult worms. A similar pattern was observed for sialyl-Tn, a related O-linked antigen. Tn glycoproteins from protoscoleces were resolved by SDS-PAGE in two main components of 43 and 49 kDa. After purification, this material was reactive with lectins which bind GlcNAc/sialic acid, GalNAc, and T antigen. In a preliminary evaluation, high levels of Tn antigen were detected in serum samples from patients with hydatid cyst, suggesting that the measure of Tn in serum could be a biomarker of this disease, although extensive work is necessary in order to determine the clinical usefulness of this assay. The results reported here, the first evidence of O-glycosylation pathways in E. granulosus and the presence of Tn antigen in cestodes, suggest that the evaluation of O-glycosylated antigens might give new insights in the host-parasite relationship.

Topics: Adult; Animals; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Breast Neoplasms; Chromatography, Affinity; Dogs; Echinococcosis; Echinococcus; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique; Glycosylation; Humans; Lectins; Male

The possibility that tumor-associated antigens T and Tn act as adhesion molecules between normal and malignant breast epithelial cells at the early stages of recognition in the metastatic pathway was examined in vitro. The adhesive specificity of the antigens was assessed by means of in vitro adhesion tests between a carcinomatous breast cancer cell line (ZR75-30) and a normal epithelial breast cell line (HLB100) using both monoclonal antibodies and lectins specific as well as nonspecific for each antigen. Adhesion assay was performed using monolayers of the normal cell line prepared on plastic culture plates and the tumor cell line labeled with a fluorescent dye as a probe. The adhesion between the two cell types occurred with significant specificity via T and Tn antigens (P<0.001), and was temperature-dependent. The results suggest that at the early stages of recognition by tumor cells in the metastatic process, T and Tn antigens play a role as adhesion molecules between the tumor cells and adjacent normal cells.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast; Breast Neoplasms; Cell Adhesion; Cell Adhesion Molecules; Cell Aggregation; Cell Line; Coculture Techniques; Epithelial Cells; Female; Fluorescent Dyes; Humans; Immunohistochemistry; Kinetics; Neoplasm Metastasis; Peanut Agglutinin; Temperature; Tumor Cells, Cultured

Expressions of the carcinoembryonic Tn antigen studied with VVA-B4 and GSI-A4 lectins with the monoclonal antibody 83D4 and of N-acetyllactosamine residues with ECA and LSL lectins, were examined in 54 malignant or benign human breast tumors. Positive membrane labelling with lectins and 83D4 MAb occured in benign cases indicating that modification of glycoconjugates may precede the cytologic anomalies. In fibroadenoma, fibrocystic dystrophy, ductal hyperplasia and grade I invasive ductal carcinomas, the binding sites for all lectins and 83D4 MAb were essentially on the cell membrane with labelling of both apical and basolateral compartments. In grade II and III, the labelling involved the cytoplasm, and cell heterogeneity appeared. The disappearance of reactivity observed for a large proportion of cells at grade III may be due either to the loss of glycosyl-transferase, or to the lack of synthesis of the protein back-bone. Invasive lobular carcinomas showed labelling both on apical membrane and the outermost part of the cytoplasm with a distinct cell polarity. Lectin receptors are present at the surface of metastatic cells, possibly related to their involvement in adhesion.

Topics: Amino Sugars; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Female; Fibroadenoma; Glycosyltransferases; Humans; Hyperplasia; Immunohistochemistry; Lectins

Expression of carcinoembryonic Tn antigen studied with VVA-B4 and GSI-A4 lectins with the monoclonal antibody 83D4 and of T antigen with LDL and PNA lectins with the monoclonal antibody ZCMO4, were examined in 54 malignant or benign human breast tumors and for MCF-7, T47D and MCF-10A cell lines of human breast tumors origin. For breast tissues, positive membrane labelling with D-GalNAc alpha-O-ser/thr (Tn-antigen) specific lectins and 83D4 MAb occurred in benign cases indicating that modification of glycoconjugates may precede the cytologic anomalies. In fibroadenoma, fibrocystic dystrophy, ductal hyperplasia and grade I invasive ductal carcinomas, the binding sites for lectins and 83D4 MAb were essentially on the cell membrane with labelling of both apical and basolateral compartments. In grade II and III, the labelling involved the cytoplasma, and cell heterogeneity appeared. The disappearance of reactivity observed for a large proportion of cells at grade III may be due either to the loss of glycosyltransferase, or to the lack of synthesis of the protein back-bone. Invasive lobular carcinomas showed labelling both on apical membrane and the outermost part of the cytoplasm with a distinct cell polarity. Lectin receptors are present at the surface of metastatic cells, possibly related to their involvement in adhesion. In all cases, T or sialosyl-T antigens are present at the surface of tumors cells. All cell lines from breast tumors cultured in vitro were labelled with lectins and monoclonal antibodies. The simultaneous presence of Tn and T antigens on the cells, indicates that the expression of Tn antigen is due to a partial but non total deficiency in the beta-1- > 3 galactosyltransferase involved in T-antigen synthesis.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Fibroadenoma; Fibrocystic Breast Disease; Glycoconjugates; Humans; Hyperplasia; Lectins; Middle Aged; Neuraminidase; Tumor Cells, Cultured

T and Tn are pancarcinoma epitopes (EPs) which can be immunodetected in about 90% of adenocarcinomas (CAs). To study the location of T and Tn EPs and their relations to adhesion plaques on CA cells, immunogold-silver staining method was employed at scanning electron microscope (SEM) level. Human breast CA cells grown on coverslips were fixed in paraformaldehyde and glutaraldehyde, reacted with cocktails of monoclonal antibodies against T or Tn EPs, followed by incubation with 10 nm gold conjugated goat anti-mouse immuno-globulins. The positive gold particle labelling was amplified with silver enhancement solution, and the specimens were then routinely critical point dried, sputter-coated with palladium and observed under SEM. The studies show that T and Tn EPs are not randomly distributed on CA cell surface; they are aggregated at the adhesion plaque area. These results confirm that T and Tn EPs play roles in CA cell adhesion, and suggest that they may represent the initial points of contact by immuno-effectors and therefore can be utilized in immunointervention and anti-adhesion therapy against CA.

Topics: Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carcinoma; Cell Adhesion; Female; Humans; Immunohistochemistry; Microscopy, Electron, Scanning; Tumor Cells, Cultured

Human polyclonal, monospecific anti-T and -Tn antibodies were found to be reactive in ELISA tests with human ovarian (IGROV-1, OVCAR-3 and SKOV-3), breast (SKBr-3 and T47D)- and oral (KB)-carcinoma cell lines, but less so or non-reactive with normal epithelia and fibroblasts. The direct binding radioimmunoassay, using 125I-labeled human antibodies, to the IGROV-1 cancer cells was inhibited by homologous unlabeled antibodies of the same concentration, but not by the respective immunodominant haptenic monosaccharides (Gal for T and GalNAc for Tn). Rodent ascitic monoclonal anti-T (Ca3114 and Ca3741) and anti-Tn (Ca3250, Ca3268 and Ca3638) antibodies were also reactive with the ovarian- and breast-cancer cells, as measured by FACS and ELISA tests, but to a lower extent than the polyclonal human antibodies. Both the monoclonal anti-T (Ca3741) and anti-Tn (Ca3250 and Ca3638) antibody-binding reactivities were significantly inhibited by the haptenic free monosaccharides. Addition of the above MAbs to IGROV-1 ovarian-cancer or T47D breast-cancer cells cultured in vitro resulted in significant cytological change and inhibition of the viability of the tumor cells, but not of normal epithelial breast cells. This effect on viability was shown to be complement-independent, yet it was profoundly influenced by the concentration of the serum added to the assay medium. In vivo biodistribution of the anti-T (Ca3114) and anti-Tn (Ca3638) MAbs administered i.p. to athymic IGROV-1 tumor-bearing CD1 female nude mice revealed higher 125I-labeled antibody accumulation in the tumor xenografts and in their lung tissues, as compared with other organs of the same mice tested. The above results thus suggest the feasibility of utilizing these antibodies in immunotherapy and drug targeting.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Breast Neoplasms; Carcinoma; CCAAT-Enhancer-Binding Proteins; Cell Survival; Coculture Techniques; DNA-Binding Proteins; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Nuclear Proteins; Ovarian Neoplasms; Tumor Cells, Cultured

The fine specificities of MAbs generated using novel synthetic clustered STn and Tn glycopeptides as immunogens were compared with the anti-TAG-72 antibodies B72.3 and CC49. Hapten inhibition experiments demonstrated the specificity of several of the MAbs for STn and Tn expressed on ovine submaxillary mucin and tumor derived MUC-1 mucin. Amongst the STn specific MAbs only the B195.3 MAb shows absolute dependence on the presence of sialic acid and specificity to the simple disaccharide NANAA alpha2-6-GalNAc. Identification of tumor associated carbohydrate epitopes in cluster and monomer configurations are possible using MAbs detecting the defined structure specificities described herein.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Ascitic Fluid; Biomarkers, Tumor; Breast Neoplasms; Carbohydrate Conformation; Carbohydrate Sequence; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Glycopeptides; Glycosylation; Haptens; Mice; Molecular Sequence Data; Mucin-1; Mucins; N-Acetylneuraminic Acid; Radioimmunoassay; Sheep; Submandibular Gland

We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Tumor-Associated, Carbohydrate; Base Sequence; Breast Neoplasms; Carcinoma; Cloning, Molecular; Gene Amplification; Humans; Hybridomas; Immunoglobulin Fragments; Immunoglobulin kappa-Chains; Immunoglobulin M; Immunoglobulin Variable Region; Mice; Models, Molecular; Molecular Sequence Data; Recombinant Fusion Proteins; Sequence Alignment; Sequence Analysis, DNA; Tumor Cells, Cultured

The precursors of the blood group N and M-immunodominant structures, Tn and T (Thomsen-Friedenreich) epitopes (EPs) occur in approximately 90% of carcinomas (CAs) but are masked in benign-diseased and healthy tissues. We determined quantitatively on 55 primary invasive ductal breast CAs, stages I to IV, the prognostic value of extent of Tn and T EP expression over an observation period exceeding 5 years postoperatively. Classical, established pathological and histological prognostic characteristic indicators associated with survival were subdivided by standard criteria into favorable and unfavorable categories. Tissue sections were reacted with monoclonal anti-Tn and -T antibodies, followed by the streptavidin-biotin-peroxidase-DAB procedure; counterstain was methyl green. Tn and T EPs were then quantitated by computerized image analysis. Of the 55 CAs, 51 clearly expressed Tn and T, and four had traces. Strong Tn EP expression was statistically significantly associated with shortened 5-year disease-free interval, increasing pTNM stages, positive lymph node status, and increasing combined histological grades. T EPs were usually well expressed but showed no significant association with prognostic factors. Our results suggest that quantitative immunohistochemistry-image analysis of Tn EPs of primary breast CAs may add new parameters to prognostication.

Topics: Adult; Aged; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Middle Aged; Prognosis

The composition of tumor-infiltrating lymphocytes (TIL) often reflects the host's immune response to the tumor. To study the relationship of TIL and carcinoma-associated T/Tn antigens in breast carcinoma, a straightforward concurrent immunoenzyme staining procedure was developed. Fresh tissue was directly fixed in a zinc-based fixative to preserve lymphocyte markers and then routinely embedded in paraffin. The TIL subtypes in the sections were identified in the first immunostaining cycle by reaction with a monoclonal antibody (MAb) to lymphocyte markers CD3, CD4, CD8, CD19, or CD56, followed by a modified avidin-biotin procedure and diaminobenzidine tetrahydrochloride-H2O2 for color development. This was followed by paraformaldehyde fixation to block antibody crossreactivity. The T and Tn antigens on carcinoma cells were then demonstrated in a second staining cycle by reaction with an MAb against T or Tn antigen, followed by an indirect immunoalkaline phosphatase procedure and corresponding substrate systems for color development. The distinguishable brown color for TIL and blue or red color for T or Tn antigen enabled us to identify the TIL subsets and to describe their relations with T/Tn antigen expression in situ. This approach may contribute to better understanding of the patients' immune defenses against their tumor and aid in prognostication.

Topics: Antigens, CD; Antigens, Tumor-Associated, Carbohydrate; Biomarkers; Breast Neoplasms; Carbohydrate Sequence; Female; Humans; Immunoenzyme Techniques; Lymphocytes, Tumor-Infiltrating; Molecular Sequence Data

The expression of complex carbohydrates recognised by Helix pomatia lectin (HPA, nominal monosaccharide binding specificity alpha-GalNAc) has been shown to predict unfavourable prognosis in breast and other cancers. It has been suggested that the prognostic significance of HPA binding may be through recognition of either Tn epitope (alpha-GalNAc-O-serine/threonine) or blood group A antigen (terminal alpha-1-->3GalNAc attached to the basic H-antigen, Fuc-alpha-1-->2-Gal-beta-1-->4(or 3) GlcNAc-->R). In this study, the expression of glycoproteins terminating in alpha-GalNAc residues was investigated immunohistochemically using HPA and two monoclonal antibodies--BRIC 66 (anti-alpha-GalNAc) and BRIC 111 (anti-Tn). In paraffin sections, 74/87 (85%) of breast cancers expressed HPA-binding ligands, while 28/87 (32%) were positive for BRIC 66 binding and 25/87 (29%) expressed Tn. Distribution of staining patterns were distinctive and different with the three markers. BRIC 66, BRIC 111 and HPA binding to glycoproteins derived from breast cancer homogenates and to blood group A and Tn positive glycoproteins in Western blots confirmed the immunohistochemistry data. The results suggest that the prognostic significance of HPA binding in breast cancer is unlikely to be simply through recognition of blood group A antigen or Tn epitope on cancer cells. Breast cancers may express a complex profile of related but distinct glycans sharing similar terminal immunodominant sugar GalNAc, which may be implicated in aggressive biological behaviour.

Topics: ABO Blood-Group System; Acetylgalactosamine; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Endothelium, Vascular; Epitopes; Female; Follow-Up Studies; Glycoproteins; Humans; Immunohistochemistry; Lectins; Middle Aged; Sensitivity and Specificity

Since 1974, and as of March, 1993, we have used T/Tn antigen vaccine in safe, specific, effective, long-term intradermal vaccination against recurrence of advanced breast carcinoma (CA). Staging is by the pathologic TNM system. Treatment is ad infinitum. Of 19 consecutive breast carcinoma patients vaccinated, six Stage IV, six Stage III, and seven Stage II all survived > 5 years postoperatively. Three Stage III, three Stage IV, and five Stage II patients (i.e., 11) survived > 10 to > 18 years. Five others are alive but have not reached 10 years; three of them have no evidence of disease (NED). Three patients died of CA before reaching 10 years. An additional three breast CA patients are being treated for > 2 years, but, < 5 years postoperatively, they are NED. The vaccination are presented as a delayed-type hypersensitivity reaction with significant inflammation with increase of helper T lymphocytes and decrease of T suppressor/cytotoxic cell ratio.

Topics: Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Breast Neoplasms; Female; Humans; Hypersensitivity, Delayed; Injections, Intradermal; Neoplasm Staging; Recurrence; Retrospective Studies; Survival Rate; Vaccines

To determine whether the monoclonal antibody (MoAb) 83D4, previously shown to be highly specific for carcinoma cells, can be used as an immunocytological marker to discriminate between benign and malignant cells in serous effusions; and to test for a correlation between expression of the antigen reacting with MoAb 83D4 on effusion cells and the amount of soluble 83D4 antigen in effusion fluids.. Thirty three pleural and 23 peritoneal effusions from 56 cancer patients with metastatic disease were tested for the presence of Tn associated 83D4 antigen by immunocytochemical staining, and for the presence of soluble antigen in supernatants. The patients had undergone various chemotherapy and radiation therapy protocols.. As a result of the various types of treatment, the cytological characteristics of the cells were often modified and the antigenic epitopes may have been altered. Positive staining for 83D4 MoAb was obtained in 36 (97%) of the 37 malignant effusions, eight (73%) of 11 suspect effusions, and three (38%) of the eight apparently benign effusions (free of malignant cells). In these latter cases, cytological reassessment showed a few suspect cells in two cases. 83D4 soluble antigen was detected in 30 of 37 malignant effusions (81%), five of 11 suspected infusions (46%), and five of eight apparently benign effusions (63%).. Immunocytochemical staining with anti-83D4 antibody is useful for differentiating reactive or atypical mesothelial cells from epithelial cells, especially in breast cancer effusions.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Ascitic Fluid; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Ovarian Neoplasms; Pleural Effusion, Malignant; Solubility

Pathogenetic aspects of pancarcinoma T/Tn autoantigens were investigated; they are present in approximately 90% of all carcinomas from incipience and throughout. T/Tn are occluded in noncarcinoma (non-CA) diseased and healthy tissues. By serological and immunohistochemical methods, we found that well-differentiated carcinomata express a higher proportion of T than Tn, while in poorly differentiated carcinomata, Tn predominates over T. Tn density of primary carcinomas correlates positively with aggressiveness, early clinical relapse, and early death. Delayed-type skin hypersensitivity to T (DTHR-T) and solid-phase anti-T antibody immunoassay (SPIA-T), respectively, detected 85% of 461 and 88% of 222 carcinoma patients; < 7% of over 450 benign diseased and healthy subjects reacted positively in these assays. T/anti-T assays are highly effective in detecting incipient (TisN0M0 and T1N0M0) carcinomas: DTHR-T-85% of 41, and SPIA-T-96% of 26 patients. Positive anti-T tests predicted CA in 74% of 47 patients months to years before their biopsy/X-ray turned positive.

Topics: Adenocarcinoma; Antibodies; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Autoantigens; Breast Neoplasms; Carcinoma; Carcinoma, Ductal, Breast; Case-Control Studies; Cohort Studies; Humans; Immunoglobulin M; Isoantigens; Prognosis

Topics: Acetylgalactosamine; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Epitopes; Glycoproteins; Immunohistochemistry; Neoplasm Metastasis

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.

Topics: Adenocarcinoma; Amino Acids; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carbohydrate Sequence; Epitopes; Female; Glycoproteins; Glycosylation; Humans; Lectins; Membrane Glycoproteins; Mice; Milk, Human; Molecular Sequence Data; Molecular Weight; Mucin-1; Mucins; Neoplasm Proteins; Neuraminidase; Protein Binding; Protein Processing, Post-Translational; Subcellular Fractions; Tumor Cells, Cultured

Topics: Antigens, Tumor-Associated, Carbohydrate; Biopsy; Breast Neoplasms; Carcinoma; Humans; Immunotherapy, Active; Time Factors

The effects of cell surface sugar chains combined with certain gene amplifications of breast cancers on the prognosis of patients were studied and the relationships between the sugar chains of cancer cells and amplifications of the proto-oncogenes c-myc, int-2 and c-erb B-2, evaluated. One hundred and fifty three human breast carcinoma tissues were investigated by an immunohistochemical technique using the avidinbiotin-peroxidase method with 1 lectin (HPA; Helix Pomatia) and 4 monoclonal antibodies (B-72-3, St-439, anti-Tn and anti-T). The positive rates of HPA, St-439, B-72-3, anti-Tn and anti-T were 43 per cent (63/153), 52 per cent (80/153), 53 per cent (81/153), 64 per cent (98/153) and 89 per cent (136/153), respectively. Patients whose cancers had positive HPA staining were found to have a lower survival rate than those with negative HPA staining (p less than 0.05), whereas those whose cancers had positive St-439 staining showed a better prognosis than those with negative St-439 staining (p less than 0.01). The positive rate of HPA was related to the gene amplification of c-myc proto-oncogene (p less than 0.01), whereas the negative rate of St-439 was correlated with the gene amplification of c-erb B-2 (p less than 0.01). These data indicate the prognostic value of HPA and St-439 and also the relationships between the gene amplifications and carbohydrate structures in breast cancer cells.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; Female; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Amplification; Humans; Immunohistochemistry; Lectins; Membrane Glycoproteins; Prognosis; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2

The serine/threonine O-linked carbohydrates GalNAc alpha and Gal beta 1-3GalNAc alpha, referred to as Tn and T antigens, respectively, appear to be more prevalent in some human carcinomas than in surrounding tissues. Tn/T antigens may represent incomplete synthesis of O-linked oligosaccharides, due to decreased activity of specific glycosyltransferases, or alternatively, increased glycosidases activity in tumors which may expose these internal O-linked oligosaccharide sequences. To explore these possibilities, we measured UDP-Gal:GalNAc alpha-R beta 1-3 galactosyltransferase (beta 3Gal-T) and Gal beta 1-3GalNAc alpha-R beta 1-3 galactosidase in a series of human breast tumors. In addition, glycoproteins extracted from the tumors were separated by SDS-PAGE and stained with the lectins HPA (GalNAc alpha-R reactive) and PNA (Gal beta-3GalNAc alpha-R reactive). The relative levels of HPA- to PNA-reactive glycoproteins in the carcinomas correlated inversely with beta 3Gal-T activities. The results suggest that Tn antigen expression in human breast carcinoma is due in part to low beta 3Gal-T activity, a situation similar to that observed previously in haematopoietic cells of individuals with a condition called Tn syndrome.

Topics: Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Breast Neoplasms; Carbohydrate Sequence; Electrophoresis, Polyacrylamide Gel; Female; Glycoproteins; Humans; Kinetics; Lectins; Molecular Sequence Data; N-Acetyllactosamine Synthase; Oligosaccharides; Tumor Cells, Cultured

Two monoclonal antibodies (MoAbs), BRIC 66 (IgM) and BRIC 111 (IgG1), were produced by immunizing mice with ovarian cyst blood group A1 glycoprotein and Tn red cells (RBCs), respectively. Their specificities were determined by inhibitions using Tn sialoglycoproteins (SGPs), mucins (armadillo [ASG] and ovine [OSG] submaxillary glycoproteins), and monosaccharides. BRIC 66 agglutinated both Tn and group A RBCs and reacted immunohistochemically with both the vascular endothelium and tumor cells from a group A adenocarcinoma, BRIC 66 was inhibited by N-acetylgalactosamine (GalNAc), Tn SGPs, and mucins on both hemagglutination inhibition tests and radioimmunoassay. BRIC 111 agglutinated Tn RBCs only, and it specifically stained tumor cells from a group O patient's breast carcinoma and a group A patient's adenocarcinoma. In hemagglutination inhibition tests, BRIC 111 was readily inhibited by Tn SGPs, only partially inhibited by GalNAc, and not inhibited by mucins. In a sensitive radioimmunoassay, BRIC 111 was inhibitable by GalNAc. Tn SGP was 2000-fold more effective as an inhibitor than the mucins (ASG and desialized OSG), which contain a high content of terminal alpha-GalNAc-O-serine (threonine) residues. It is postulated that BRIC 66 is specific for terminal alpha-GalNAc units in carbohydrate chains. The exclusive reaction of BRIC 111 with Tn SGP indicates a combining site larger than GalNAc alpha-1, which probably includes amino acid residues in juxtaposition to GalNAc in Tn SGP. In view of its specific agglutination of Tn RBCs, BRIC 111 is a useful reagent for the examination of polyagglutinable RBCs.

Topics: ABO Blood-Group System; Absorption; Acetylgalactosamine; Adenocarcinoma; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Erythrocytes; Female; Glycoproteins; Hemagglutination; Humans; Immunoenzyme Techniques; Mucins; N-Acetylneuraminic Acid; Neuraminidase; Ovarian Cysts; Oxidation-Reduction; Periodic Acid; Sialic Acids; Sialoglycoproteins

In contrast to healthy and noncarcinoma-diseased tissues, greater than 80% of all carcinomas (CAs) tested express immunoreactive O-(2-acetamido-2-deoxy-alpha-D-galacto-pyranosyl)-(1----3)-serine/threon ine [alpha-D-GalpNAc-(1----3)-Ser/Thr] in their glycoproteins. CA cells shed, into the tumor's environment, Tn, which is involved in cancer pathogenesis as adhesion molecule and as autoimmunogen. An increase in density of Tn on primary CA frequently parallels augmented CA aggressiveness. Tn-Active glycoproteins of culture-grown human breast CA DU 4475 cells were isolated from cytoplasm and from spent growth medium, and erythrocyte (RBC) Tn antigen was prepared by (1----3)-beta-D-galactosidase treatment of isolated human O RBC MN glycoprotein-derived Thomsen-Friedenreich (T) antigen. Immunochemical, serological, physical, and chemical analyses showed close resemblance of CA- and RBC-derived Tn antigens. The preponderant carbohydrate in both Tn glycoproteins is the alpha-D-GalpNAc residue, and the antigens have a qualitatively and quantitatively similar amino acid composition. Highly specific rodent monoclonal (Mo) anti-Tn antibodies (Abs) were elicited with Tn RBC and normal O RBC-derived Tn antigen, and compared with CA-anti-Tn MoAbs unwittingly evoked by others. A sensitive enzyme immunoassay (EIA) with Tn antigen as solid phase was developed. In this system, highly purified, "naturally occurring" anti-Tn antibodies, which all humans possess, were more sensitive in quantitating breast CA Tn structures than the anti-Tn MoAbs induced by Tn RBCs, and by RBC- and CA-derived Tn-active antigens. The sensitivity of anti-Tn MoAbs was higher in detecting RBC-Tn.

Topics: Amino Acids; Antibodies; Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carbohydrates; Cell Line; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Immunoenzyme Techniques

Topics: Adenocarcinoma; Agglutination Tests; Antibodies; Antibody Specificity; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; Cell Line; Cell Migration Inhibition; Cytotoxicity, Immunologic; Disaccharides; Epitopes; Erythrocytes; Female; Humans; Hypersensitivity, Delayed; Killer Cells, Natural; Leukocytes; Skin Tests

Tn antigen is the immediate precursor of the carcinoma (CA)-associated T antigen; both are masked in non-CA tissues. Tn antigen was detected by absorption of human anti-Tn antibody in 46 of 50 primary breast CAs and in all 6 metastases originating from Tn-positive primary CAs. Thirteen of 25 (52%) anaplastic CAs, but only 2 of 15 (13%) well differentiated CAs had more Tn than T; 1 anaplastic CA had neither antigen. Eighteen of 20 benign breast lesions had no Tn; the 2 positive lesions were premalignant. All 19 breast CAs, studied immunohistochemically, reacted strongly with human polyclonal anti-Tn; benign or normal glandular tissues had minimal or no reactivity. Among live cancer cell lines, the most malignant sublines had more Tn than T on their cell surfaces. Preliminary studies with rodent monoclonal anti-Tn and anti-T antibodies gave immunohistochemical reactivity patterns similar to those of the polyclonal antibodies, but the former were less sensitive in absorption tests. Tn is a CA marker that promises to be useful in tumor detection.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Blood Group Antigens; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; Cell Line; Female; Hemagglutination Tests; Histocytochemistry; Humans; Immunochemistry; Immunosorbent Techniques; Lymphoma; Mammary Neoplasms, Experimental; Mice; Neoplasm Metastasis; Neoplasm Recurrence, Local; Rats

Primary and metastatic carcinomas are epithelial in origin and comprise by far the largest group of malignant tumors in humans. In most of these tumors, T and Tn antigens, whose epitopes have been synthesized, are uncovered and immunoreactive. In all other tissues T and Tn antigens are masked and not accessible to the immune system; they are generally precursors in normal complex carbohydrate chains. Thus, carcinomas have antigens recognized as foreign by the patients' immune system. The expression of T and Tn antigens has pathogenic and clinical consequences, and the antigens themselves are powerful histological markers in carcinoma diagnosis and frequently in prognosis. Most patients distinguish their carcinoma from all other cells, as shown by strong autoimmune responses to T antigen. These responses are readily measured by assays, and they allow detection of carcinomas with greater sensitivity and specificity frequently earlier than previously possible. Moreover, the extent of T and Tn expression often correlates with carcinoma differentiation; on a molecular level, clustered T- and Tn-active structures on carcinoma cell surfaces may be involved in invasion.

Topics: Antibody Formation; Antigens; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Antigens, Viral, Tumor; Autoantigens; Breast Neoplasms; Cell Adhesion; Humans; Immunity, Cellular; Lung Neoplasms; Neoplasm Metastasis; Neoplasms

Topics: Animals; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Breast Neoplasms; Carcinoma; Female; Humans; Mammary Neoplasms, Experimental; Rats