Carbohydrate Antigens Tn Research Articles

Monophosphoryl Lipid A-Rhamnose Conjugates as a New Class of Vaccine Adjuvants.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and β-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-β-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.

Abstract B28: Targeting the tumor-associated carbohydrate antigen STn with humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibits ovarian cancer tumor growth in vitro and in vivo

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies in the U.S. The current standard of care is tumor debulking followed by chemotherapy. This treatment results in approximately 70% of patients achieving an initial complete clinical response. However, many of these patients will unfortunately relapse with chemoresistant disease developing in part to the presence of cancer stem cells (CSCs) within the tumor. Indeed, ovarian CSCs have been identified and shown to be resistant to chemo- and radiotherapy. Cancer cell surface glycans, called tumor-associated carbohydrate antigens (TACAs), are a class of attractive cancer-specific targets found on the cell surface of many solid tumors. Siamab is targeting a glycan structure (Sialyl-Tn, STn) that is cancer-specific and a major reported constituent of two well-known CSC biomarkers, CD44 and MUC1, residing on both CSCs and mature malignant cells in some cancer types. Although CD44 and MUC1 are also present on normal tissue and normal stem cell surfaces, tumor and CSC specificity is conferred the presence of STn-glycosylated variants of these CSC markers and other cell surface proteins that are essentially absent from normal human tissues. Importantly, STn can be a component of CA-125 (MUC16), conferring additional specificity to this diagnostic marker for ovarian carcinoma. The elevated presence of STn in tumors is associated with metastatic disease, poor prognosis, and reduced overall survival. In addition, elevations in STn expression are linked to chemotherapy resistance and enable tumors to evade the host immune system. We have observed that STn levels are elevated in cancer cells following chemotherapy treatment in both in vitro and in vivo model systems and STn is present on specific subtype of tumor-infiltrating myeloid-derived suppressor cells. Both observations support the role of STn in tumor chemoresistance and immunomodulation. While attempts have been made to target STn clinically with a cancer vaccine, efficacy has been limited. Siamab has developed a unique immunotherapeutic solution aimed at eradicating human neoplasias by targeting both CSCs and bulk tumor to enable whole tumor killing. We have identified and humanized a panel of mouse monoclonal novel antibodies (Abs) that specifically target with high affinity the STn glycan independent of its carrier protein. These Abs are highly selective in binding assays demonstrating robust nanomolar EC50s, possess specificity on tissues as determined with neuraminidase treatment, and exhibit STn-specific glycan binding on Siamab’s proprietary glycan array. Antitumor efficacy was evaluated in vitro and in vivo utilizing humanized anti-STn antibody drug conjugated (ADC) material. Cell cytotoxicity was demonstrated in a panel of STn-expressing cell lines with low nanomolar IC50s. We confirmed that the decrease in proliferation in vitro can be translated to a reduction in tumor size in vivo, through a series of cell line and patient-derived ovarian cancer xenograft models. Inhibition of tumor progression were observed in all models, with complete regressions observed in some treatment arms. No significant weight loss was observed for any treatment groups, indicating the therapy was well tolerated by all the groups. A decrease in STn expression was noted in the tumors following treatment, indicating that we were hitting the target against which these Abs were generated. In addition, we have begun the development of both tissue- and serum-based biomarker assays utilizing these selective anti-STn Abs. Our data demonstrate that high-affinity, STn-selective humanized mAbs show promise as therapies for ovarian tumors, as well as tools for patient stratification and pharmacodynamic biomarker assessments. Citation Format: Daniel T. Dransfield, Jillian M. Prendergast, David A. Eavarone, Rawan Nazer, Linah Al-Alem, Jenna Stein, Jeff Behrens, Bo Rueda. Targeting the tumor-associated carbohydrate antigen STn with humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibits ovarian cancer tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B28.

Abstract P3-05-14: Expression of the tumor associated carbohydrate antigen Tn and immune effectors in invasive breast cancer

Abstract Background: Glycosylation is a post-translational modification generating complex carbohydrate O-glycans. Tn antigen, a N-acetyl-galactosamine-α-O-Ser/Thr residue that is the core glycosylated component of complex mucin-type O-glycans, is expressed in breast cancers. Tn has been associated with invasion, survival and decreased T-cell mediated immune response in several types of cancer including breast cancer. There is therefore a potential rationale to combine Tn vaccine with checkpoint blockers such as PD-1/PDL-1 inhibitors. Little is known regarding the link between the respective expression of Tn antigen, PDL-1, and TILs, in breast cancer. Our study aimed at characterizing Tn expression in a series of invasive breast cancers, and its correlation with immune response. Methods Tn expression was analyzed by immunohistochemistry performed on a tissue microarray of 1864 invasive breast cancer samples (1850 patients for statistical analyses), consisting of 189 (10%) TNBC, 1601 (86%) ER and/or PR positive à checker tumors and 191 (10%) HER-2 positive tumors treated at our institution between 2005 and 2013. Among these cases, 50 luminal B tumors and 50 TNBC were selected for full section and immune analysis, i.e. to study the pattern of Tn expression across whole tumor section and the link between Tn expression and the level of CD3+ and CD8+ infiltrates, and PDL-1 expression. The level of Tn expression was assessed using H-score, combining the percentage of stained cells with staining intensity. Results: In the 100 initial samples, Tn expression was observed in 95%, with a H-Score>10 in 85% of the cases, and with homogeneous staining across the whole tumor section in 86% of the cases. Tn expression was increased in Luminal B subtype as compared to TNBC, with a mean H-Score of 100.9 vs 55, respectively (p <0,0001). The CD3+ infiltrate was more important in TNBC as compared to luminal B tumors (mean stromal CD3+: 34.3% vs 21.2%, p=0,0052). No significant difference was found for the CD8+ infiltrate. PDL-1 expression in stromal cells (≥1%) was observed in 43% of the cases, and increased in TBNC as compared to Luminal B (mean of 7.3% vs 2.2% of cells respectively, p=0.0252). In both tumor subtypes, we observed a positive correlation between the CD3/CD8 infiltrates and PDL-1 expression in stroma cells (p<0,0001), but not with Tn expression. In the TMA cohort, Tn expression was observed in 1723 (92%) tumors with a mean H-Score of 82.75. Similarly, to the results obtained in full section, Tn expression was less intense in TNBC as compared to other subtypes (mean H-Score of 62 vs 85, p<0.001). Tn expression was lower in histological grade I as compared to grade II/III tumors (64,69 for grade I, 88,84 for grade II and 88.99 for grade III, p<0.0001), and significantly higher in HER2 positive tumors versus others (mean H score 62,06 for HER2-/RE-, 119,05 for HER2+ and 80,25 for HER2-/RE+, p<0.0001). Conclusion: Our study shows a lower level of expression of Tn antigen in TNBC as compared to luminal tumors. We could not identify any correlation between Tn antigen expression and immune status of the tumor as defined by CD3+, CD8+ and PDL-1+ infiltrate in tumor stroma. The role of Tn antigen in the HER2-positive subgroup requires further investigation. Citation Format: Verret B, Rossoni C, Lebeherec D, Michiels S, Castanon-Alvarez E, Leclerc C, Delaloge S, Artaud C, Lacroix-Tikri M. Expression of the tumor associated carbohydrate antigen Tn and immune effectors in invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-14.

Mucoepidermoid carcinoma-associated expression of MUC5AC, MUC5B and mucin-type carbohydrate antigen sialyl-Tn in the parotid gland

ObjectivesThe aberrant expression of mucins and mucin-type carbohydrates has been described in many types of cancer, including mucoepidermoid carcinoma (MEC), a malignant salivary gland tumor. In this study, we examined the aberrant expression patterns of mucins (MUC1, MUC4, MUC5AC and MUC5B), simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) and mature carbohydrate antigens (Lewisa and sulfo-Lewisa antigens) in MEC originating from the parotid gland, which normally does not secrete mucins. DesignWe conducted an immunohistochemical study to investigate the presence of mucins and carbohydrates in 24 MEC samples originating from the parotid gland and in surrounding normal tissue of the same gland in comparison 6 samples of normal salivary glands. The expression levels were compared with respect to the histological grading. Furthermore, 24 MEC samples from non-parotid salivary glands were included. ResultsWe observed loss of topology of membrane-bound MUC1 and MUC4, and de novo expression of MUC5AC, MUC5B and sialyl-Tn in MEC that originated in the parotid gland. Furthermore, mucins MUC1, MUC4 and carbohydrate antigens Tn, sialyl-Tn, T, Lewisa and sulfo-Lewisa were overexpressed in MEC samples compared to surrounding normal salivary gland tissues. MUC1 was expressed in both low- and high grade MECs, whereas MUC4 was not expressed in high grade MECs of the parotid gland. ConclusionDuring the development of MEC in the parotid gland, the genes for gel-forming secretory mucins are switched on. Besides these MEC tissues overexpress short oligosaccharides, suggesting that the glycosylation machinery is altered.

The cytokine-cosmc signaling axis upregulates the tumor-associated carbohydrate antigen Tn.

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.

Challenges in Antibody Development against Tn and Sialyl-Tn Antigens.

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment.

Ultrasensitive photoelectrochemical immunoassay of antibody against tumor-associated carbohydrate antigen amplified by functionalized graphene derivates and enzymatic biocatalytic precipitation

Tumor-associated carbohydrate antigens (TACAs) are often found on the surface of cancer cells. The determination of the carbohydrate components of glycoconjugates is challenging because of the chemical complexity of glycan chains. Through monitoring corresponding antibody, we can get a good solution for clinical diagnosis. Here breast tumor-associated carbohydrate antigens Tn were used as a model and a new photoelectrochemical biosensor for ultrasensitive detection of antibody against Tn was developed. To enhance the sensitivity, both graphene oxide and graphene were used during the construction of biosensor. Through the formation of immunocomplex and the insoluble biocatalytic precipitation (BCP) product, photocurrent intensity was decreased greatly and the antibody could be detected from 0.5 to 500pg/mL with a detection limit of 1.0×10−13g/mL. At the same time, the developed biosensor showed acceptable selectivity and could be used in the complex matrix. Compared with the traditional glycoarray method, this PEC method is more sensitive (5 orders of magnitude), and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.

The promise of glycomics for discovery of new biomarkers

Glycosylation, the attachment of carbohydrates to a protein or lipid, is the most abundant and structurally complex post-translational modification taking place in cells. Approximately two percent of the human genome codes for proteins involved in the glycosylation pathways that modify proteins and lipids. Carbohydrates themselves can be modified in multiple ways, undergoing for example acetylation, sulfation, methylation, phosphorylation, or sialylation. This results in a cellular glycan repertoire composed of vastly diverse and information-rich molecules. Glycans are central to numerous intra-cellular and inter-cellular processes including cell growth, differentiation, adhesion, and cellcell recognition. To date ninety nine distinct human glycosylation disorders, many affecting multiple organ systems, have been identified. The extent of cellular glycosylation is dynamic, changing in response to the phenotype, metabolic state, and developmental stage of the cell. Changes in the expression of one’s cellular glycan profile and the synthetic enzymes involved in their synthesis, as well as the repertoire of one’s serum anti-glycan antibodies, are observed during cellular transformation and metastasis. For example, changes in N-glycan branching, the extent of sialylation or the rate of glycosylation of mucins, the over expression of sialylated gangliosides, and loss of expression of glycosylphosphatidylinositol (GPI), have all been correlated with malignant disease and its progression. This makes glycans promising molecules for the discovery of biomarkers to detect and monitor disease. However, until recently the study of carbohydrates as potential biomarkers has suffered from a lack of tools and technologies capable of carrying out rapid, robust analysis of structural and functional changes in cellular glycans, anti-glycan antibodies, and serum glycoproteins and glycolipids. The development of glycan array technology for determining the binding specificity of glycan binding proteins and serum anti-glycan antibodies, and advances in high-throughput analytical technologies capable of defining glycan structure, are now opening up new avenues for the discovery of glycan biomarkers with specific prognostic and diagnostic value. This special issue of Cancer Biomarkers will review the clinical findings and genetic and glycobiologic basis of glycosylation disorders, and the need for better serum based diagnostic biomarkers, beyond the presently used blood test for N-linked glycan disorders. New glyco-analytical tools for study of glycosylation changes in serum glycoproteins and how such data can improve our understanding of disease pathways and lead to discovery of pre-clinical and clinical glycan based disease biomarkers will be discussed. Likewise, the use of glycan array technology to identify new disease biomarkers, and the power of these arrays to determine carbohydrate binding specificity for use as a diagnostic tool, using flu as a model system will be presented. Finally, we will look at the presence of glycosylphosphatidylinositol anchored proteins in serum and how they may serve as biomarkers for detection of cancer, and how truncation of O-glycans (expression of the tumor-associated carbohydrate antigen Tn and/or Sialyl Tn antigen) through genetic and epigenetic pathways, or metabolic regulation results in malignant disease.

Expression of tumor-associated carbohydrate antigen Tn in cancer cells is stimulated by cytokine secreted from cancer cells

Meeting abstracts Tumour-associated carbohydrate antigen (TACA) Tn is always observed on the cell surface of malignant tumour cells. Tn is presented as mucin-type carbohydrate. It has been reported to be associated with cancer progression, but the regulation on Tn expression in cancer cells is not

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Lea (SLea) and Sialyl-Lex (SLex) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLea/SLex-MUC1 and STn/SLea/SLex-MUC2 glycoforms in $50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLea-MUC2, STn/T/SLea/SLex-MUC5AC and STn/T/SLea/SLex-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.

Recognition profile of Morus nigra agglutinin (Morniga G) expressed by monomeric ligands, simple clusters and mammalian polyvalent glycotopes

The carbohydrate binding properties of a novel member of the subfamily of galactose-specific jacalin-related lectin isolated from the bark of black mulberry ( Morus nigra) (Morniga G) was studied in detail by enzyme-linked lectinosorbent and inhibition assays using panels of monomeric saccharides, mammalian polyvalent glycotopes and polysaccharides. Among the natural glycans tested for lectin binding, Morniga G reacted best with glycoproteins (gps) presenting a high density of tumor-associated carbohydrate antigens Tn (GalNAcα1-Ser/Thr) and T α (Galβ1-3GalNAcα1-). Their reactivities, on a nanogram basis, were up to 72.5, 3.9 × 10 3, 6.0 × 10 3, 8.8 × 10 3 and 2.9 × 10 4 times higher than that of Tn-containing glycopeptides (M.W. < 3000 Da), monomeric T, Tn, GalNAc and Gal, respectively. It also reacted well with many multi-antennary N-glycans with II (Galβ1-4GlcNAc) termini, ABH histo-blood group antigens and their precursors containing high densities of I/ II and T/ Tn glycotopes, and sialylated T/ Tn. Among the mono-, di- and oligosaccharides tested, Thomsen-Friedenreich ( T) disaccharide with aromatic aglycon [Galβ1-3GalNAcα1-benzyl ( T α1-benzyl)] and Tn glycopeptides were the best inhibitors. Molecular modeling and docking studies indicated the occurrence of a primary GalNAcα1- and Galβ1-3GalNAc glycotope-binding site in Morniga G. Using a recently proposed system [Wu, A.M., 2003. Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins. J. Biomed. Sci. 10, 676–688], the binding properties of the combining sites of Morniga G can be defined as follows: (i) the monosaccharide specificity is GalNAc/Gal ≫ Man/Glc, GlcNAc and lFuc; (ii) the mammalian glycotope specificity is T α1-benzyl > T > Tn > GalNAcβ1-3Gal (P), while B/ E (Galα1-3/4Gal), I/ II (Galβ1-3/4GlcNAc), S (GalNAcβ1-4Gal), F/ A (GalNAcα1-3GalNAc/Gal) and L (Galβ1-4Glc) are inactive; (iii) the most active ligand is T/ Tn; (iv) simple clustered Tn or triantennary N-glycans with II termini (Tri- II) have limited impact; (v) high-density polyvalent glycotopes play a prominent role for enhancing Morniga G reactivity. These results provide evidence for the binding of this lectin to dense cell surface T/ Tn glycoconjugates and facilitate future usage of this lectin in biotechnological and medical applications.

Formation of lactones from sialylated MUC1 glycopeptides

The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found that the model compound sialyl T benzyl glycoside , and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-Ala from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen, lactonized during treatment with glacial acetic acid. Compound gave the 1''--> 2' lactone as the major product and the corresponding 1''--> 4' lactone as the minor product. For glycopeptide the 1''--> 4' lactone constitued the major product, whereas the 1''--> 2' lactone was the minor one. When lactonized was dissolved in water the 1''--> 4' lactone underwent slow hydrolysis, whereas the 1''--> 2' remained stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide did not lactonize in glacial acetic acid.

Isolation and Characterization of Thomsen-Friedenreich-Specific Antibodies from Human Serum

The Thomsen-Friedenreich (TF) disaccharide, galactose (Gal)β1–3GalNAcα-, is a blood group-related oncofetal antigen with remarkable tumor specificity. Postpartum, carbohydrate structures on the cell walls of the gastrointestinal flora evoke natural antibodies of presumed TF specificity. These antibodies may provide an early barrier against TF-carrying tumor cells. Their possible role, however, has been difficult to assess, since so far only a multivalent immunosorbent, asialoglycophorin (aGP), has been employed for their preparation, and therefore their fine specificities have been only insufficiently defined. We have used a novel immunosorbent consisting of synthetic TFα disaccharides (Galβ1–3GalNAcα-) coupled to polyacrylamide (PAA), which itself was covalently bound to cross-linked sepharose. For specificity analyses, aGP and a panel of PAA-conjugated mono- and oligosaccharides were employed. Binding to the PAA moiety was excluded. The affinity-purified anti-TFα antibodies were of the IgM (≧0.5 mg/100 ml of serum) and IgG (approximately 0.05 mg/100 ml of serum) classes. They did partially cross-react with TFβ, although we detected a second group of anti-TFβ antibodies (both IgM and IgG) which did not cross-react with TFα. The affinity-purified TFα antibodies showed only marginal cross-reactivity with the related antigens lactose, Gal, Tn or the Forssman antigen. Besides TF-specific antibodies, we found antibodies to the carbohydrate antigens Tn, Forssman and β-D-Gal as well as to noncarbohydrate epitopes of glycophorin in human serum.

Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis.

Biliary papillomatosis is characterized by papillary proliferations of biliary lining cells without invasion or metastasis. The neoplastic character and biological behaviour of this disease remain still speculative. These issues were examined in this study. Mucin core protein MUC1, MUC2, MUC3, MUC5AC and carbohydrate antigens (T, Tn and sialosyl Tn) were immunohistochemically examined, using 11 lesions of biliary papillomatosis from seven patients, and five lesions of biliary papillomatosis with foci of carcinoma from four patients. Five cases of papillary intrahepatic cholangiocarcinoma and 12 histologically normal livers were used as a control. Patients with biliary papillomatosis alone or with carcinoma were middle-aged or elderly (five men and six women). Microscopically, biliary papillomatosis showed a villous, papillo-tubular, papillary, or papillo-villous pattern with a thin fibrovascular core. Cytologically, they were classifiable into biliary epithelial or pyloric gland-like type. The former was frequent in the cases associated with carcinoma. Expression of MUC1, Tn antigen and sialosyl Tn antigen was frequent and marked in biliary papillomatosis alone and with carcinoma and also intrahepatic papillary carcinoma. In addition, marked expression of MUC1 and Tn antigen were rather frequent in biliary papillomatosis with carcinoma and intrahepatic biliary papillary carcinoma compared with biliary papillomatosis. MUC2 was rather frequent and marked in biliary papillomatosis alone compared to other two disease groups. Focal expression of MUC5AC and MUC2 was rather frequent and infrequent irrespective of disease group, respectively. Focal expression of T antigen was frequent in papillary ICC. Biliary papillomatosis could undergo overt malignant transformation along with altered phenotypic expression of MUC proteins and mucin carbohydrate antigens.

The immunoexpression of Tn, sialyl-Tn and T antigens in chronic active gastritis in relation to Helicobacter pylori infection

The simple mucin-type carbohydrate antigens Tn, sialyl-Tn and T represent the mucin core oligosaccharide structures that are produced in the initial steps of mucin biosynthetic pathway. Utilising monoclonal antibodies anti-Tn antigen, anti-sialyl-Tn antigen and anti-T antigen, we have investigated the expression of the simple mucin-type carbohydrate antigens in 47 biopsy specimens of antral mucosa with chronic active gastritis, 25 of which had Helicobacter pylori infection. The Tn immunoreactivity, localised at the supranuclear region of surface and glandular mucous cells, was observed in all samples, independently from H. pylori status. The sialyl-Tn antigen, mainly localised in the cytoplasm of glandular mucous cells and in goblet cells vacuoles, was seen in 56% of the cases with H. pylori infection and in 41% of the cases in the H. pylori-negative group. In addition, the T antigen was found in the cytoplasm of surface and glandular mucous cells in 16% of the H. pylori-positive group, whereas the percentage of positive cases was reduced to 5% in H. pylori-negative patients, with an exclusive localisation in the cytoplasm of glandular mucous cells; after neuraminidase treatment, the percentage of T antigen-positive cases was increased to 28% in H. pylori-positive cases and to 27% in negative cases. No significant relationships between H. pylori infection and Tn, sialyl-Tn or T antigen immunoexpression were encountered in our cases. Therefore, we maintain that the inflammatory infiltrate may itself play an important role in the expression of simple mucin-type carbohydrate antigens in chronic active antral gastritis.

Enzymatic basis for sialyl-Tn expression in human colon cancer cells.

Sialyl-Tn antigen (SAalpha2-6 GalNAc alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc alpha-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal beta1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R beta3-Gal-transferase (core 1 beta3-Gal-transferase) was lacking. Core 1 beta3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc beta1-6 (Gal beta1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 beta3-Gal-transferase activity. The lack of core 1 beta3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 beta3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.

Simple mucin-type carbohydrate antigens in major salivary glands.

Simple mucin-type carbohydrate antigens Tn, sialosyl-Tn and T are often markers of neoplastic transformation and have very limited expression in normal tissues. We performed an immunohistological study of simple mucin-type carbohydrate antigens, including H and A variants, with well-defined monoclonal antibodies (MAb) on frozen and paraffin-embedded normal salivary gland tissue from 22 parotid, 14 submandibular, six sublingual, and 13 labial glands to elucidate the simple mucin-type glycosylation pattern in relation to cyto- and histodifferentiation. The investigated carbohydrate structures were predominantly observed in the cell cytoplasm, most often in the supranuclear area, suggesting localization to the Golgi region, whereas ductal contents were unstained. Mucous acinar cells expressed Tn, sialosyl-Tn, and H and A antigens, regardless of glandular location. Serous acinar cells, on the other hand, expressed A, H, and inconstantly sialosyl-T, Tn, and sialosyl-Tn antigens in major salivary glands, whereas serous cells of minor (labial) salivary glands expressed H exclusively, Tn and sialosyl-T antigens inconstantly, but never sialosyl-Tn and A antigens. The difference may be related to a more simple cytodifferentiation of serous cells of minor (labial) salivary glands as compared with major salivary glands. Duct cells in major salivary glands expressed A, H, and inconstantly T, sialosyl-T, and Tn antigens, whereas minor (labial) salivary glands ducts exclusively expressed H, T and sialosyl-T antigens, differences that may be related to dissimilarities in the duct system. Myoepithelial cells and basal cells exclusively expressed T and sialosyl-T antigens, which may prove useful in studies of salivary gland tumors, since these cells are known to play a key role in the histological characteristics of some salivary gland tumors. The results indicate a similar glycosylation pattern in the different major salivary glands, whereas minor (labial) salivary gland differ slightly in serous and duct cells. The limited and exclusive intracellular expression of the immature Tn, sialosyl-Tn, and T antigens indicates that these structures may be of value as markers of salivary gland tumors.