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Thomsen-Friedenreich antigen (TF; Galbeta1-3GalNAcalpha1-) is expressed on many human carcinomas. Evidence suggests that TF-carrying tumor cells specifically bind asialoglycoprotein receptors on hepatocytes resulting in metastasis formation in the liver. We used an animal model to examine the feasibility of preventing metastasis formation by an antibody to TF. Treatment of Colon 26 cells with neuraminidase led to the exposure of TF, and consequently to a higher frequency of liver metastases in syngeneic Balb/c mice. This could be prevented by an antibody to TF (A78-G/A7), but not by a control antibody. The results may open up a new strategy for the prophylaxis of metastatic spread to the liver.

BACKGROUND Controversial findings have been reported regarding the expression of the Thomsen-Friedenreich (TF) antigen in colorectal neoplasms when different monoclonal antibodies (MoAbs) have been used. Moreover, there is no information available regarding the carrier protein(s) of this antigen. METHODS Forty-five colorectal adenomas and 48 carcinomas were studied by avidin-biotin complex-peroxidase immunohistochemistry. The immunohistochemistry employed the MoAb BW835, which was reactive to a carrier specific and site specific TF antigen on MUC1 mucin, as well as reference antibodies directed to MUC1 (HMFG2) or MUC2 core peptides (4F1) and directed to TF antigen irrespective of its carrier (A78-G/A7, peanut agglutinin). To evaluate the coexpression of different epitopes by the same antigen, sandwich enzyme-linked immunoadsorbent assays were performed. RESULTS Although MUC1 peptide antigen and MUC1-bound TF antigen were not detectable in normal or transitional mucosa surrounding colorectal neoplasms, expression of these antigens in adenomas accompanied the development of high grade dysplasia. By contrast, MUC2 expression detected by the MoAb 4F1 was inversely correlated with the progression of the adenoma-carcinoma sequence. In well- and moderately differentiated colorectal carcinomas, the neo-expressed TF antigen is predominantly bound to MUC1. This feature could be demonstrated by antigen coexpression using peptide and the TF antigen specific MoAbs. However, in mucinous carcinomas exhibiting a weak MUC1 peptide expression in most specimens, the presence of TF antigen on the MUC2 peptide core cannot be ruled out. CONCLUSIONS TF antigen is strongly coexpressed with MUC1 mucin peptide core in the colorectal adenoma-carcinoma sequence, resulting in well- and moderately differentiated carcinomas. Only in mucinous carcinomas may it be coexpressed with MUC2 antigen. Cancer 1998;82:1019-27. © 1998 American Cancer Society.

Aim. To study whether alterations in the sialylation of antibodies (Ab) specific to the Thomsen-Friedenreich (TF) glycotope have a diagnostic and prognostic potential in gastric cancer.Methods. Serum samples were taken from patients with gastric carcinoma(n=142)and controls(n=61). The level of TF-specific antibodies and their sialylation was detected using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specificSambucus nigra agglutinin(SNA).Results. The level of TF-specific IgM was significantly decreased in cancer compared with controls(P≤0.001). Cancer patients showed a higher level of SNA binding to anti-TF IgM and IgA(P≤0.001)irrespective of disease stage, tumor morphology, and gender. Changes in the SNA/Ab index demonstrated moderate sensitivity (66–71%) and specificity (60–73%) for stomach cancer. The best diagnostic accuracy (100%) was achieved in 29% patients with high SNA binding and low anti-TF IgM level. This subset of patients demonstrated the poorest survival.Conclusion. Our findings are the first evidence that the increased sialylation of TF-specific Abs combined with a low level of anti-TF IgM is strongly linked to gastric cancer and patients survival, which can be used as a novel biomarker for cancer detection and prognosis.

In many tissues, the TF (Thomsen-Friedenreich) blood group antigen (Galβ1-3GalNAcα-) behaves as an onco-foetal carbohydrate antigen, showing increased expression in malignancy and hyperplasia. Dietary lectins which bind the TF antigen have marked effects on proliferation of epithelial cells without cytotoxicity. This led us to speculate that anti-TF antibodies, including those that naturally occur in humans, might have similar effects. Five anti-TF antibodies, TF2 (human), TF5 (human), 5A8 (mouse), 8D8 (mouse) and BM22 (mouse), but not TF1 (human) or 49H.9 (mouse), showed marked dose-dependent stimulation (95–192%) of [3H]thymidine incorporation by HT29 human colon cancer cells. Similar stimulation of proliferation of HT29 cells by these monoclonal antibodies (MAbs) was found when cell count assessment was used. Antibody-stimulated proliferation was inhibited by co-incubation with glycoproteins expressing Galβ1-3GalNAcα- (asialo glycophorin or [Galβ1-3GalNAcα-O-p-aminophenyl]n-human serum albumin). A proliferative effect of these antibodies was also demonstrated on human colon cancer cell lines LS174T and HT29-MTX but not on Caco-2 cells. Although immunoblotting showed similar binding patterns of all the antibodies on HT29 cell membrane extracts, there was little correlation between cell surface binding assessed by immunofluorescence and proliferative response, and internalization of the biotinylated antibody TF5 was demonstrated by confocal microscopy. Our results provide further evidence that cell surface glycoproteins which express TF antigen may play an important role in the regulation of cell proliferation and also suggest that human anti-TF antibodies may have proliferative effects on cells which express TF antigen. Int. J. Cancer 73:424–431, 1997. © 1997 Wiley-Liss, Inc.

Epithelial mucins are present at the apical membranes of gastrointestinal epithelial cells or in their secretions. In this study, we examined the occurrence of peptide epitopes of the mucins MUC1 and MUC3 and of three mucin-associated glycotopes (TF, Tn, and s-Tn) in a series of colorectal tissue samples (normal colon, adenomas with different grades of dysplasia, carcinoma in situ, and invasive carcinomas). A new monoclonal antibody to a conformation-dependent peptide epitope of MUC1 was employed, which does not react with the fully glycosylated mucin as found in normal gastrointestinal mucosa. We found that adenomas acquired the ability to expose Tn, s-Tn, TF and MUC1 epitopes, and this correlated with increasing malignant potential. The secretory mucin, MUC3, revealed a different pattern: it was detectable in all sections, with maximum expression in adenomas and decrease in carcinomas. Most importantly, normal mucosa and benign lesions showed supra-nuclear and/or apical distribution of these antigens, but malignant lesions and lesions with a very high risk of malignancy revealed diffuse cytoplasmic and basolateral membrane localization. The immunohistological response to a combination of MUC1-related antibodies may assist in assessing the malignant potential and status of lesions of the colon.

Aberrant glycosylation is a hallmark of cancer cells. The blood group precursors T (Thomsen-Friedenreich) and Tn epitopes are shielded in healthy and benign-diseased tissues but uncovered in approx. 90% of carcinomas. T and Tn glycoproteins are specific, autoimmunogenic pancarcinoma antigens. These antigens may also be found in neoplastic blood cells (and on LTV-II infected T lymphocytes). Fundamental chemical and physical aspects of these glycoproteins of primary carcinomas are discussed first. Tn and T epitopes are cell and tissue adhesion molecules, essential in invasion by and metastasis of carcinoma which includes adherent and proliferative phases. These properties are then delineated next, followed by consideration of pathophysiological and clinical aspects of these antigens. Immunohistochemical studies of the extent of Tn antigen expression in primary breast carcinoma demonstrate it highly significant correlation with clinicopathological tumor stages, and hence its value as a reliable prognosticator. On the other hand, there is no significant, prognostically useful association between T antigen expression and clinical disease course. Everyone has "preexisting" anticarcinoma anti-Tn and anti-T antibodies, induced predominantly by the intestinal flora, while cellular immune responses to T and Tn epitopes are evoked only by carcinomas and some lymphomas. Carcinoma dedifferentiation leading to predominance of Tn over T epitopes is described, as is the role of Tn and T epitopes in very early, including preclinical, carcinoma detection. The highest sensitivities in carcinoma detection are for preclinical and the earliest clinical stages. Obviously, preclinical carcinoma detection is of practical importance. T/anti-T tests detected preclinical carcinoma in 77% of 48 patients long (mean 6 years) before their biopsy/X-ray results became positive. There were no false predictions of carcinoma in 38 control persons with benign diseases (observation average 4.8 years). These findings open a novel window for both curative approaches and pathophysiological studies. The autoimmunogenicity of carcinoma T/Tn antigen led us more than two decades ago to begin intradermal vaccination of patients with advanced breast carcinoma of stages IV-IIb, predominately after modified radical mastectomy and sometimes lumpectomy plus axillary dissection always followed by adjuvant radio/chemotherapy. The vaccine consists of human group O red blood cell membrane derived, HLA-free T/Tn antigen containing as adjuvant Ca3(PO4)2 plus a trace of phosphoglycolipid A hyperantigen, i.e., S typhi vaccine (USP), which itself has T and Tn specificities. Our efforts have now for up to 20 years remained successful in a large majority of the 32 patients. All 32 patients survived at least 5 years; 10-year survival was statistically highly significantly improved (5-year survival: P1 x 10(-7); 10-year survival: P1 x 10(-5)) compared to statistics of the United States National Cancer Institute. Because these vaccinations are successful, their extension to large populations with major types of carcinomas should be considered, and even immunological carcinoma prevention may be contemplated.

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.

The efficient chemoenzymatic synthesis of the Thomsen-Friedenreich antigen determinant is demonstrated under transglycosylation conditions employing β-galactosidase from bovine testes.

Especially in childhood, the in vivo action of microbial neuraminidase may cause haemolytic anaemia or life-threatening haemolytic uraemic syndrome. The exposure of the Thomsen-Friedenreich (T) crypto-antigen and T-antigen polyagglutinability of erythrocytes has been described as the first sign of toxic cleavage of N-acetylneuraminic acid (Neu5Ac) from sialoglycoproteins of cell membranes. This phenomenon may, however, be too unspecific to initiate treatment for toxin elimination. The present study investigated the diagnostic effectiveness of a panel of three monoclonal antibodies (mcabs) for the estimation of the clinical significance of neuraminidase action in vivo. Depending on the amount of Neu5Ac released, the mcabs I-C4, II-Q9 and III-Y12 recognized different epitopes on erythrocyte asialoglycophorin. In 1345 patients, the mcab II-Q9 detected cleavage of Neu5Ac in 32 children who had T-antigen polyagglutinability and mild to moderate haemolytic anaemia. However, only 10 patients, whose erythrocytes were agglutinated by the mcabs III-Y12 or I-C4, developed severe haemolysis, thrombocytopenia, and finally the life-threatening haemolytic uraemic syndrome (p

Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-density glycosylation, is critical for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.

Aim: Disseminated tumor cells are found in the bone marrow of patients with epithelial carcinoma and are correlated with a poor prognosis of the disease. Their detection is a technical challenge. This report describes a model system for the detection of cancer cells by co-immunostaining of Thomsen–Friedenreich and Her-2 antigens. Methods & results: Small numbers of cancer cells from different cancer cell lines were mixed with blood samples of healthy donors. Cytospins were prepared and double immunostaining against Thomsen–Friedenreich antigen and Her-2 was carried out by fluorochrome-coupled antibodies. Quantification of Thomsen–Friedenreich and/or Her-2-positive cells was performed with an epifluorescence microscope. On average, 83% of cancer cells were recovered by this method. Conclusion: Immunostaining is a useful method for the detection of cancer cells in blood samples. Results of this model system will be transferred to bone marrow patient samples to prove the benefits for detection of disseminated tumor cells.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens, TF (Gal beta 1-3GalNAc alpha 1-R), Tn (TF precursor, GalNAc alpha 1-R), sialosyl-Tn (NeuAc alpha 2-6GalNAc alpha 1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopes, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF > > sialosyl-Tn > Tn > TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase from Vibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-R and Gal beta 1-3 (NeuAc alpha 2-6)GalNAc alpha 1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.

Heat-labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the G(M1) ganglioside receptor. We report here the crystal structure of heat-labile enterotoxin in complex with the disaccharide portion of the Thomsen-Friedenreich (T-antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.13 Angstrom resolution, yielding an R-factor of 18.5%. The T-antigen disaccharide, D-Gal-beta 1,3-GalNAc-Ser/Thr, is present in more than 85% of human carcinomas and monitoring ifs autoimmune response is used for the early detection of tumors. Insight into the molecular recognition of this tumor antigen by sugar binding proteins can benefit the development of a diagnostic tool for human carcinomas as weil as a T-antigen directed anticancer drug delivery system.

Heat-labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the GM1 ganglioside receptor. We report here the crystal structure of heat-labile enterotoxin in complex with the disaccharide portion of the Thomsen-Friedenreich (T-antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.13 A resolution, yielding an R-factor of 18.5%. The T-antigen disaccharide, D-Gal-beta 1,3-GalNAc-Ser/Thr, is present in more than 85% of human carcinomas and monitoring its autoimmune response is used for the early detection of tumors. Insight into the molecular recognition of this tumor antigen by sugar binding proteins can benefit the development of a diagnostic tool for human carcinomas as well as a T-antigen directed anticancer drug delivery system.

Background. Expression of the pancarcinoma Thomsen-Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not generally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence. Methods. Cryostat sections from 52 primary tumors (20 with adjacent transitional mucosa), 22 liver metastases of colorectal carcinomas, and 17 samples of normal mucosae were examined immunohistologically with a panel of at least two monoclonal antibodies (mAbs) each to TF, to its precursor, Tn, and to sialosyl-Tn, among them two newly developed anti-TF mAbs. Results. Of the primary colorectal carcinomas, 60% expressed TF. Staining was more intense with TF-alpha/beta-reactive than with exclusively TF-alpha- or TF-beta-reactive mAbs. Normal and transitional mucosae were negative. Liver metastases were positive for TF in a significantly higher percentage of cases (91%) than primary carcinomas. Patients with TF-positive primary tumors had a significantly higher risk to develop liver metastases compared with patients with TF-negative tumors (57% vs. 14%, respectively). Tn and sialosyl-Tn were expressed concomitantly in most primary (85%) and metastatic (95%) colorectal carcinomas. These antigens also were detected in transitional mucosae (Tn in 25%, sialosyl-Tn in 55% of cases). Normal mucosae were negative. Conclusions. These results prove unequivocally the presence of exposed TF epitopes in a majority of colorectal carcinomas in which both anomers of TF are expressed. These data further suggest that TF favors liver metastasis and that its expression in primary colorectal carcinomas is a significant risk factor for the development of liver metastasis. Cancer 1995; 76:1700–8.

Controversy centres on the role and identification of myoepithelial (MEC) and basal cells in salivary gland tumours, and recent studies suggest that both basal cells and myoepithelial cells participate in the formation of salivary gland tumours. We have correlated the expression of different well-known markers of normal MEC/basal cells (i.e. alpha-smooth muscle actin and cytokeratin 14) with T (Thomsen-Friedenreich) antigen and its sialylated derivative: sialosyl-T antigen,) in 17 normal parotid glands and in two tumour types with MEC participation (i.e pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC)) using immunohistology with well-defined monoclonal antibodies (MAbs). Paraffin-embedded/fresh frozen tissue sections were studied from 33/17 patients with PA and 15/7 patients with ACC. In normal parotid tissue coexpression of alpha-smooth muscle actin, cytokeratin 14, T and sialosyl-T antigens was found in all MEC and in some of the basal cells lining striated ducts. The remaining basal cells exclusively expressed cytokeratin 14, T and sialosyl-T antigens. In the tumours, cells believed to be modified myoepithelial cells showed two different staining patterns: 1) Coexpression of alpha-smooth muscle actin, cytokeratin 14, T and sialosyl-T antigens, and 2) Coexpression of cytokeratin 14, T and sialosyl-T antigens, but no alpha-smooth muscle actin. The epithelial ductular structures in the tumours showed aberrant expression of cytokeratin 14, T and sialosyl-T antigens, and cytokeratin 14 was the only marker of cells in solid undifferentiated areas of adenoid cystic carcinomas. Our study supports the view, that modified "myoepithelial" cells in the tumours consist of a mixture of basal cells and myoepithelial cells. None of the investigated structures was in itself an ideal marker in the identification of MEC/basal cells. The cells can be identified by a combination of markers (i.e. cytokeratin 14, alpha-smooth-muscle actin, T and sialosyl-T antigens).

This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen-Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-β(1-3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an "intestinal loop" mouse model. Quantitative analysis of the data indicated that approximately 2 μg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level.

A novel MUC1-glycopeptide–BSA conjugate vaccine with a specifically fluorinated Thomsen–Friedenreich antigen side chain at Thr6 was prepared. Preliminary immunological experiments reveal specific binding of the tumor-associated glycopeptide antigen analog by anti-MUC1-mouse antibodies.

The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif ((51)AHGDA(55)) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins.

Pemphigus foliaceus (PF) is an autoimmune skin blistering disease mediated by pathogenic autoantibodies against the desmosomal core glycoprotein desmoglein 1 (Dsg1). This study demonstrated that the O-glycan-specific plant lectin jacalin binds Dsg1 and inhibits the interaction of Dsg1/PF IgG. N-glycosylation is not involved in the interaction of Dsg1/jacalin or Dsg1/PF IgG. Subcutaneous injection of jacalin into neonatal mice drastically reduced PF IgG deposition at the epidermal cell surface and blocked PF IgG-induced skin blisters, both clinically and histologically. Interestingly, another plant lectin peanut agglutinin (PNA), which shares the same carbohydrate specificity toward the O-linked carbohydrate structure known as Thomsen-Friedenreich antigen (TF antigen, Galβ1-3GalNAcα-O-Ser/Thr), also bound Dsg1 and blocked the skin blistering. In contrast, the plant lectin vicia villosa-B4 (VVL-B4), which shares the carbohydrate specificity toward the O-linked monosaccharide known as Thomsennouveau antigen (Tn antigen, GalNAc-α1-O-Ser/Thr) did not bind Dsg1 and did not show a protective effect against the disease induced by the autoantibodies. Collectively, these results suggest that the binding of jacalin to O-linked TF carbohydrate motifs on Dsg1 impairs the Dsg1/PF autoantibody interactions and abrogates its pathogenicity in vivo. TF-specific binding ligands may have a potential therapeutic value for PF.

Thomsen-Friedenreich (TF) antigen, which plays an important role in the regulation of cancer cell proliferation, occurs in ∼90% of all human cancers and precancerous conditions. Although TF antigen has been known for almost 80 yr as a pancarcinoma antigen, the recognition mechanism between TF antigen and target protein has not been structurally characterized. A number of studies indicated that TF disaccharide is a potential ligand of the galactoside-binding galectins. In this work, we identified the TF antigen as a potential ligand of the antitumor galectin AAL (Agrocybe aegerita lectin) through glycan array analysis and reported the crystal structure of AAL complexed with the TF antigen. The structure provides a first look at the recognition mode between AAL and TF antigen, which is unique in a conservative (Glu-water-Arg-water) structural motif-based hydrogen bond network. Structure-based mutagenesis analysis further revealed the residues responsible for recognition specificity and binding affinity. Crystal structures of AAL complexed with two other TF-containing glycans showed that the unique TF recognition mode is kept intact, which may be commonly adopted in some cancer-related galectins. The finding provided the new target and approach for the antitumor drug design and relative strategy based on the AAL-TF recognition mode as a prototype model.—Feng, L., Sun, H., Zhang, Y., Li, D.-F., Wang, D.-C. Structural insights into the recognition mechanism between an antitumor galectin AAL and the Thomsen-Friedenreich antigen.

1. 1. Twenty perchloric acid-soluble glycoprotein fractions (PASFs) were separated from carcinomatous and non-carcinomatous regions of the livers of 5 patients with primary hepatocellular carcinoma (HCC) and 5 patients with a complication of HCC and hepatocirrhosis (HC). 2. 2. In autoradiography using 125 ,I-labelled Vicia unijuga lectin (VUA) and 125 I-labelled Arachis hypogaea anti-T lectin as probes, 10 PASFs from carcinomatous regions of the livers of 10 patients gave 1 Vgu glycoprotein with T activity and/or 1–4 Vgu glycoproteins, respectively, and among 10 PASFs from non-carcinomatous regions of the livers of 10 patients, 7 PASFs gave no glycoproteins, respectively. li ]3. These results show that a Vgu glycoprotein with T activity and Vgu glycoproteins occur in HCC and in a complication of HCC and HC as oncofetal antigens.

Michael addition and acrylamide copolymerization of single N-acryloylated carbohydrate precursors of the Thomsen Friedenreich antigen (T-antigen) and an ABH type 2 human blood group trisaccharide determinant afforded both neoglycoprotein and glycopolymer conjugates suitable for immunochemical studies.

Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(beta 1-3)GalNAc(alpha)-O-] and Tn [GalNAc(alpha)-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the alpha and beta forms of different Gal(beta 1-3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.

Methyl 2-acetamido-2-deoxy-5,6-O-isopropylidene-β-D-galactofuranoside was prepared in one step from 2-acetamido-2-deoxy-D-galactose and 2,2-dimeth-oxypropane in good yield. Condensation of this compound with acetobromo-galactose using mercury (II) cyanide as catalyst followed by removal of the protecting groups from the resulting disaccharide derivative yielded the Thomsen-Friedenreich (TF) antigenic disaccharide.

Glycan structures of IgG strongly influence the affinity for Fcgamma receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-alphaGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-alphaGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-alphaGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-alphaGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-alphaGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer.

Background The T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression. Methods Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: ST3Gal.I, ST3Gal.II and ST3Gal.IV mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines. Results In nonmuscle-invasive bladder cancers, ST3Gal.I mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, ST3Gal.I mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed ST3Gal.I mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the ST3Gal.I mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two ST3Gal.I transcript variants were also equally expressed, independently of cell phenotype or malignancy. Conclusion ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of ST3Gal.I seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.

1. 1. Five perchloric acid-soluble fractions (PASFs) obtained from ascitic fluids of three patients with primary hepatocellular carcinoma (HCC), a patient with liver metastatic carcinoma (LUC) from ureteral carcinoma and human normal serum (NS) were subjected to DEAE-cellulose column chromatography to separate seven glycoprotein fractions, respectively. 2. 2. In this chromatography, two HCC-PASFs gave a Thomsen-Friedenreich (T)-active glycoprotein, respectively. 3. 3. Other HCC-PASF gave a T-active glycoprotein, two blood group N antigen precursor glycoproteins and an N antigen precursor glycoprotein with T activity. 4. 4. LUC-PASF gave two T-active glycoproteins and an N antigen precursor glycoprotein with T activity. 5. 5. NS-PASF did not give these serologically active glycoproteins.

1. 1. Four perchloric acid-soluble fractions (PASFs) from human liver metastases of sigmoid colon carcinoma (LSCC), pancreas carcinoma (LPC), breast carcinoma (LBC) and human normal liver (NL) were subjected to DEAE-cellulose column chromatography and separated into three glycoprotein fractions, respectively. 2. 2. In chromatography, LSCC-PASF and LBC-PASF gave Thomsen-Friedenreich (T)-active glyco-protein, blood group N antigen precursor glycoprotein with T activity and N antigen precursor glycoprotein, respectively. 3. 3. LPC-PASF gave N antigen precursor glycoprotein with T activity and two N antigen precursor glycoproteins. 4. 4. The three glycoprotein fractions from NL-PASF did not exhibit both T activity and N antigen precursor activity.

Galectin-3 on vascular endothelium has been shown to facilitate lung specific metastasis. Metastatic variants of B16 melanoma were chosen to identify specific ligands that mediate lung colonization via galectin-3. Flow cytometry showed that, galectin-3 binding to cells correlates with surface expression of poly N-acetyllactosamine (polylacNAc) but not with other reported ligands, e.g. Thomsen-Friedenreich (T/Tn) antigen. Immobilized galectin-3 promoted adhesion of melanoma cells in a metastasis dependent manner. Moreover, adhesion and galectin-3 binding to cells were specifically inhibited with lactose. These properties together with lung metastasis were inhibited with N-glycosylation inhibitor Swainsonine (SW), whereas, O-glycosylation inhibitor Benzyl-alpha-N-acetylgalactosamine (BG) had no effect. BG treatment significantly increased expression of T/Tn antigen on low metastatic cells; however, had no effect on their metastatic potential. The studies very comprehensively demonstrate the importance of polylacNAc substitutions on N-oligosaccharides in galectin-3 mediated lung metastasis.

Semiconductor nanocrystals, also called quantum dots (QDs) are particles that exhibit unique size- and composition-dependent optical properties. Several recent reports have described the synthesis of QDs coated with a variety of biomolecules for cellular imaging applications. We have prepared QDs coated with the tumor-associated carbohydrate antigen (TACA) disaccharide Galβ1-SGalNAcα- (Thomsen Friedenreich antigen) 0-conjugated to various linkers and studied their photoluminescent and binding properties. The de novo synthesis of highly stable, luminescent and functional quantum dots was achieved by the use of a "hybrid" system containing small percentages of different surface passivating agents along with an appropriate neoglycoconjugate. The glycan-coated quantum dots were prepared in a simple one-pot procedure that is amenable to various other saccharides. Initial studies with thiolacetic acid as a co-capping agent produced highly luminescent particles with functional carbohydrate units but the negative charge was undesirable for important applications such as cellular bioimaging. Replacement of this small organic acid with a short modified polyethylene glycol segment yielded quantum dots with very similar properties but lacking charged functionality on the surface. An array of binding and agglutination assays confirmed that the functional characteristics of the sugar were intact on all particles synthesized. These new tools hold great promise for labeling cells that express specific carbohydrate-binding proteins on their surfaces.

Purpose The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galβ1-3GalNAcα-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. Methods BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. Results 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1–10). By enrichment, a median of 23.3 mio cells (0.8–218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1–105, P

Thomsen-Friedenreich antigen (TF-Ag) is expressed in many carcinomas, including those of the breast, colon, bladder, and prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. We hypothesized that passive transfer of JAA-F11, an anti-TF-Ag monoclonal antibody, may create a survival advantage for patients with TF-Ag-expressing tumors by cytotoxicity, blocking of tumor cell adhesion, and inhibition of metastasis. This was tested using in vitro models of tumor cell growth; cytotoxicity assays; in vitro, ex vivo, and in vivo models of cancer metastasis; and, finally, in vivo effects in mice with metastatic breast cancer. Unlike some anti-TF-Ag antibodies, JAA-F11 did not enhance breast carcinoma cell growth. JAA-F11 did not induce the killing of 4T1 tumor cells through complement-dependent cytotoxicity or apoptotic mechanisms. However, JAA-F11 blocked the stages of metastasis that involve the adhesion of human breast carcinoma cells to human endothelial cells (human umbilical vein endothelial cells and human bone marrow endothelial cells 60) in in vitro static adhesion models, in a perfused ex vivo model, and in murine lung vasculature in an in vivo metastatic deposit formation assay. JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a > 50% inhibition of lung metastasis.

The oncofetal Thomsen-Friedenreich carbohydrate antigen (Galbeta1-3GalNAcalpha1-Ser/Thr TF or T antigen) is a pan-carcinoma antigen highly expressed by about 90% of all human carcinomas. Its broad expression and high specificity in cancer have attracted many investigations into its potential use in cancer diagnosis and immunotherapy. Over the past few years increasing evidence suggests that the increased TF occurrence in cancer cells may be functionally important in cancer progression by allowing increased interaction/communication of the cells with endogenous carbohydrate-binding proteins (lectins), particularly the members of the galactoside-binding galectin family. This review focuses on the recent progress in understanding of the regulation and functional significance of increased TF occurrence in cancer progression and metastasis.

The Thomsen-Friedenreich antigen (TF, CD176) is a specific oncofetal carbohydrate epitope (Gal beta1-3GalNAc alpha-O-Ser/Thr) expressed on the surface of various carcinomas. It mediates endothelium adhesion and formation of metastases. As it also causes immune response, its prognostic impact is indeterminate. The presence of disseminated tumor cells in the bone marrow of breast cancer patients (DTC-BM) indicates worse prognosis. We examined the expression of TF in primary breast cancer tissue of 265 patients with known BM status at the time of first diagnosis.BM aspiration, cytospin preparation and immunocytochemical staining with the anti-Cytokeratin antibody A45 B/B3 was done following a standardised protocol. TF expression was examined immunohistochemically on Tissue Micro Arrays (TMA) with the anti-TF antibody A78-G/A7. Evaluation was done using the immunoreactive score (IRS).Median IRS for TF expression was 2 (0-12). 68 of 265 patients (25.7%) showed DTC-BM with a median of 2/2 x 10(6) cells (1-1500). There was no correlation between TF expression and DTC-BM. After a median follow up of 60.1 months (7-119), the detection of DTC-BM showed prognostic significance for overall survival (OS, p = 0.034), whereas TF positivity (IRS2) indicated prolonged disease-free (p = 0.01), distant disease-free (p = 0.005), and overall survival (p = 0.005).Patients with TF-positive tumors had a significantly better prognosis. Dissemination routes, TF-mediated metastasis formation, and the immunogeneity of TF might determine the prognostic impact of TF expression in different tumor entities. Further characterisation of primary tumors and DTC-BM could help to improve the biological understanding of metastases and develop targeted therapies.

In this study, we demonstrate a correlation between T antigen expression on a panel of human carcinoma cell lines and their sensitivity to porcine NK cell lysis. Specifically, the more T antigen is expressed, the more sensitive the cancer cells are to porcine NK cell lysis. Furthermore, this correlation also exists for these cells and their ability to induce tumors in vivo. In this porcine animal model, the less T antigen is expressed, the more prolific the tumor growth in vivo and vice versa. Using the human colorectal adenocarcinoma cell line SW-48, we used limiting dilution to clone 2 populations of cells, one expressing high and the other low levels of T antigen, clones 143 and 111, respectively. In these cloned cells, the clone that expressed more T antigen was more NK-sensitive in vitro and weakly induced tumor growth in vivo. Inversely, the clone that expressed less T antigen clone was more NK-resistant in vitro and grew more prolific tumors in vivo. Using soluble T antigen in a competitive inhibition assay, there was a decrease in porcine NK cell killing of the T antigen+ human cell line Colo 320HSR. Taken together, these findings suggest a novel role for T antigen in the NK cell recognition of cancer cells, specifically as markers for NK sensitivity in carcinoma cell lines. The significance of T antigens as targets for NK cell–mediated lysis is novel and identifies NK cell–T antigen interactions as potentially significant in the immunotherapy of cancer and its associated metastases. © 2004 Wiley-Liss, Inc.

The Thomsen-Friedenreich disaccharide (TF) is a promising target antigen for tumor immunotherapy, since it is almost exclusively expressed in carcinoma tissues. The TF-specific antibodies generated so far are IgMs of mouse origin with limited therapeutic potential. Phage-displayed scFv repertoires are an established source for recombinant antibodies; however, we were unable to identify scFvs binding to TF when applying libraries in the standard monovalent display format of phagemid systems. Here, we report on the successful selection of TF-specific antibody fragments using a multivalent scFv phagemid library format based on shortened linkers (one amino acid residue). The libraries were constructed from mice immunized with asialoglycophorin and selected using TF displayed on two different carrier molecules in combination with the proteolytically cleavable helper phage KM13. All isolated clones encoded the same framework genes and the same complementarity-determining regions. After affinity maturation only scFv with the founder sequence were selected from secondary repertoires. This indicates a very narrow sequence window for TF-specific antibodies. Investigating other linker-length formats revealed a clear inverse correlation between linker length and binding activity both as soluble proteins and displayed on phages. The highest affinity was obtained with the tetrameric format. The selected scFv was specific for TF on various carrier molecules and tumor cells and performed well in ELISA and immunohistochemistry. We postulate that scFv phagemid library formats with short linkers (i.e. multimeric scFvs) may, in general, be advantageous in selections for the generation of scFvs against carbohydrate epitopes or other epitopes associated with low intrinsic affinity per binding site), and expect that they will be superior in applications for diagnosis or therapy.

The differential overexpression of self-antigens on tumor cells is a prime feature of malignant transformation. Thomsen-Friedenreich (TF), a core disaccharide of O-glycosylated complex glycoproteins, is one of many "self" antigens expressed on malignantly transformed cells that has served as a target for immune recognition and attack. Previously, we conducted clinical trials with a series of synthetic glycolipid, peptide and carbohydrate antigens conjugated to the immunological carrier keyhole limpet hemocyanin (KLH) mixed with the immunological saponin adjuvant, QS21. These trials resulted in the generation of high-titer IgM and IgG antibody responses specific for the individual antigens, and, in several cases, the capacity of those antibodies to mediate complement lysis. Four groups of five patients who had evidence of a biochemical relapse defined as rising prostate-specific antigens (PSAs) following primary therapy for prostate cancer with either prostatectomy or radiation were treated with escalating doses of 1, 3, 10 and 30 microg of synthetic TF in a clustered formation (c) which was conjugated to KLH and given with 100 microg of QS21. Patients received a total of five subcutaneous vaccines over 6 months and were monitored expectantly with scans every 3-4 months. Serum samples were obtained at weeks 1, 2, 3, 7, 9, 13, 19, 26, 50 and every 3 months. Antibody titers were monitored by ELISA and antibody binding to the cell surface of prostate cell lines was performed by flow cytometry. Complement-dependent cytotoxicity was performed on selected patients. Twenty evaluable patients were accrued to the study, of whom only one did not receive all six vaccinations. All patients developed maximum IgM and IgG antibody titers by week 9. The median IgM antibody titer by week 7 was 1/1,280 at 10 microg, 1/320 at 30 microg, 1/1,280 at 3 microg and 1/1,280 at 1 microg dose groups. The IgM titers from all groups remained greater than 1/320 by week 32 and beyond through week 50. We report here the results of a dose-escalating trial of a TF(c)-KLH conjugate vaccine in patients in the clinical state of a rising PSA in the absence of radiographic disease. For the first time, a synthetically made TF trimer or cluster (c) was made with three TF disaccharides attached to three sequential threonines on a peptide backbone. TF(c) doses of 1, 3, 10 and 30 microg were conjugated to KLH and administered with QS21. All doses induced high-titer IgM and IgG antibodies against TF. Unlike our findings in previous dose-escalating phase I trials, there did not appear to be increased antibody production with increasing doses of vaccine; higher titers of IgM and IgG antibodies developed at the lowest dose level (1 microg). An anti-tumor effect in the form of a change in post-treatment versus pretreatment logPSA slopes was also observed. The results justify the inclusion of TF(c) at a dose of 1 microg as a relevant antigenic target in a multivalent phase II vaccine trial in patients in the high-risk minimal disease state.

The lectin from the common edible mushroom Agaricus bisporus (ABL) belongs to the group of proteins that have the property of binding the Thomsen-Friedenreich antigen (T-antigen) selectively and with high affinity, but does not show any sequence similarity to the other proteins that share this property. The ABL sequence is instead similar to those of members of the saline-soluble fungal lectins, a protein family with pesticidal properties. The presence of different isoforms has been reported. It has been found that in order to be able to grow diffraction-quality crystals of the lectin, it is essential to separate the isoforms, which was performed by preparative isoelectric focusing. Using standard procedures, it was possible to crystallize the most basic of the forms by either vapour diffusion or equilibrium dialysis, but attempts to grow crystals of the other more acidic forms were unsuccessful. The ABL crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 93.06, b = 98.16, c = 76.38 A, and diffract to a resolution of 2.2 A on a conventional source at room temperature. It is expected that the solution of this structure will yield further valuable information on the differences in the T-antigen-binding folds and will perhaps help to clarify the details of the ligand binding to the protein.

Background The TF (Thomson – Friedenreich) blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen) by Peanut agglutinin (PNA) and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA) and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT) in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple and cost-effective method for the early diagnosis of ESCC. The present study reveals that, during tumorigenesis, an aberrant glycosylation takes place in Golgi apparatus leading to over secretion of TF antigen into the cytoplasm along with mucin granules and later into cell membrane. We suggest that the further characterization of TF antigen may unravel pathogenetic aspects of this silent disease.

Carbohydrate tumor antigens on glycoproteins and glycolipids are targets for active and passive cancer immunotherapy. These highly abundant antigens are de novo expressed or up-regulated due to changes in the complex glycosylation apparatus of tumor cells, involving sets of enzymes like glycosyltransferases, glycosidases, epimerases, and nucleotide sugar transporters. Various lipid or protein bound carbohydrate tumor antigens are described, for example, GM2, GD2, GD3, fucosylated GM1, Globo H, LeY, Lea, Sialyl-Lea and the mucin core structures Tn, Sialyl-Tn, and the Thomsen-Friedenreich Antigen (TF). Carbohydrate tumor antigens are far more abundant than protein tumor antigens rendering them suitable targets especially for antibodies, for example, highly expressed protein tumor markers as Her-2/neu express about 106 and TF about 107 copies per cell. More recent data show that certain carbohydrate structures are not only targets for humoral but also cellular immune responses.

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.