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Mucin expression in gastrointestinal neoplasms: molecular background and correlations with clinico-pathological characteristics
Posters: Esophagus,Stomach and GIST / Pathology - Research and Practice 200 (2004) 311-317 Regarding the cyclinD1 polymorphism, genotype distribution in gastric cancer patients (21% G/G, 60% A/G, 19% A/A) and esophageal cancer patients (29% G/G, 46% A/G, 25% AA) was not significantly different to the control group (25% G/G, 54% A/G, 21% A/A). Conclusions: Our results suggest, that the frequencies o f p ] 6 ~NK4A or cyclin D1 polymorphisms in gastric and esophageal adenocarcinoma do not differ significantly from the control group and therefore seem not to be relevant for the predisposition to these malignancies.
226
Mucin expression in gastrointestinal neoplasms: Molecular background and correlations with dinicopathological characteristics S.E. BALDUS Institut ftir Pathologie, Universit~it zu K61n
Aims: Numerous normal and neoplastic epithelial tissues express various mucins which are characterized by peptide cores exhibiting variable number of tandem repeat (VNTR) sequences as well as a strong glycosylation. Our studies focussed on the characterization of genetic polymorphisms of MUC1 and the effects of cytokine stimulation on the MUC1 protein expression by gastric carcinoma cells. Additionally, correlations with clinico-pathological variables in gastrointestinal neoplasia were investigated Methods and Results: Genetic polymorphisms of MUC1 VNTR domains were characterized by molecular methods including minisatellite variant-repeat-PCR. Four sequence variants and their topology could be demonstrated. One of them affects a potential glycosylation site. Gastric carcinoma cell lines AGS and KATOIII were stimulated by various cytokines in vitro and alterations of MUC1 protein expression were measured by FACS analysis. Only TNF-c~ caused a significant stimulation of MUCI and sialyl-Lewis A in AGS cells, whereas other cytokines and cell line KATOIII did not show such an effect. Mucin-associated peptide as well as carbohydrate antigens in gastrointestinal tumor tissues were visualized by immunohistochemistry. A strong MUC 1 and Thomsen-Friedenreich (TF) expression correlated with a worse prognosis in gastric and colorectal carcinoma, whereas MUC5AC showed an inverse effect in the stomach. Mucin-associated carbohydrates like sialylLewis A and sialyl-Lewis X antigens did not show good correlations with tumor progression or survival probability. Conclusions: Mucin peptides exhibit genetic polymorphisms. Like certain associated carbohydrates, especially TF antigen, they represent clinically relevant markers in gastrointestinal neoplasia.
2 2 7 A34 - Expression of a Novel Differentiation Antigen of the Stomach Mucosa on a Protein Level A.A. JUNGBLUTH, C. WILLIAMS, D. KOLB, K. COPLAN, E. SATO, G. RITTER, L.J. OLD, M.J. SCANLAN Ludwig Institute for Cancer Research New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Aims: A34 is a novel gastric differentiation antigen, which was recently identified in silico by database analysis based on sequence homologies to A34, an intestinal differentiation antigen, which is present in normal intestinal mucosa and in >90% of intestinal carcinomas. On an mRNA level, A34 is solely expressed in stomach,
3t 5
testis and at low levels in pancreas. The present analysis was done to analyze the expression of A34 on a protein level in order to assess its potential value as a diagnostic marker and/or as a target for immunotherapy of cancer. Methods: Mice were immunized with bacterially expressed A34 antigen and monoclonal antibodies (mAbs) were generated by standard hybridoma techniques. Newly generated hybridomas were screened by ELISA with A34 antigen and then tested by immunohistochemistry (IHC) using frozen and paraffin-embedded tissues employing antigen "retrieval techniques. Panels of normal and tumor tissues were then analyzed by IHC. Results: Three A34-specific clones were isolated and proved to stain frozen and paraffin-embedded tissues. In normal tissues, A34 was intensely expressed in the epithelia throughout the gastric mucosa, in single pancreatic duct epithelia and in the cytoplasm of testicular germ cells. In tumors, A34 was detected in 1/3 of gastric carcinomas, while no reactivity was found in carcinomas of the breast, kidney, urinary bladder and ovary while A34 was focally detected in colon and lung adenocarcinomas. Conclusions: A34 is mainly expressed in gastric mucosa and in tumors thereof, suggesting a novel differentiation antigen. A34 may be useful as a target for the immunotherapy of gastric carcinomas or as a diagnostic tool for the differential diagnosis of gastric adenocarcinomas.
228 Stimulation of Cathepsin X by Helicobacter pylori in human gastric epithelial cells: potential role in progression of gastric cancer S. KRUEGER 1, T. HUNDERTMARK 1, T. KALINSKI 1, U. PEITZ 2, A. ROESSNER l 11nstitut ftir Pathologie, 2Klinik fur Gastroenterologie, Otto-von-Guericke Universit~it Magdeburg
Aims: There is cumulative evidence that cysteine proteases are involved in the progression of different types of cancer. Epithelial cell responses to bacterial infection include induction of matrixdegrading enzymes. Here, we identified increased cathepsin X expression in the gastric epithelium in response to the oncogenic bacterium H. pylori, and report on the mechanisms of gastric epithelial cell migration and consequences for cancer progression. Methods: Paraffin-embedded biopsies and gastric cancer samples were processed for cathepsin X immunohistochemistry. Frozen samples were homogenized for Western Blots and for quantitative real-time RT-PCR of cathepsin X. Induction of cathepsin X expression in epithelial cells and inflammatory cells caused by H. pylori infection was tested in 3D-cocultures of infected AGS- or primary epithelial cells and primary macrophages/fibroblasts embedded in collagen I. Results: Cathepsin X was significantly higher in chronic atrophic gastritis than in controls. The same was observed for cancer. In parallel, cathepsin X moves toward the basal region of the epithelial cells during progression from normal tissue to carcinoma. In vitro experiments gave evidence that these alterations may be mediated by Rac/Rho signal-transduction pathways activated by H. pylori, resulting in increased migration. Intercellular signalling between H. pylori-infected epitelial cells and macrophages via soluble factors in the culture medium are responsible for increased cathepsin X. Conclusions: Our study demonstrates that cathepsin X may have a role not only in invasion, but also in the progression of gastric cancer.
226
Mucin expression in gastrointestinal neoplasms: Molecular background and correlations with dinicopathological characteristics S.E. BALDUS Institut ftir Pathologie, Universit~it zu K61n
Aims: Numerous normal and neoplastic epithelial tissues express various mucins which are characterized by peptide cores exhibiting variable number of tandem repeat (VNTR) sequences as well as a strong glycosylation. Our studies focussed on the characterization of genetic polymorphisms of MUC1 and the effects of cytokine stimulation on the MUC1 protein expression by gastric carcinoma cells. Additionally, correlations with clinico-pathological variables in gastrointestinal neoplasia were investigated Methods and Results: Genetic polymorphisms of MUC1 VNTR domains were characterized by molecular methods including minisatellite variant-repeat-PCR. Four sequence variants and their topology could be demonstrated. One of them affects a potential glycosylation site. Gastric carcinoma cell lines AGS and KATOIII were stimulated by various cytokines in vitro and alterations of MUC1 protein expression were measured by FACS analysis. Only TNF-c~ caused a significant stimulation of MUCI and sialyl-Lewis A in AGS cells, whereas other cytokines and cell line KATOIII did not show such an effect. Mucin-associated peptide as well as carbohydrate antigens in gastrointestinal tumor tissues were visualized by immunohistochemistry. A strong MUC 1 and Thomsen-Friedenreich (TF) expression correlated with a worse prognosis in gastric and colorectal carcinoma, whereas MUC5AC showed an inverse effect in the stomach. Mucin-associated carbohydrates like sialylLewis A and sialyl-Lewis X antigens did not show good correlations with tumor progression or survival probability. Conclusions: Mucin peptides exhibit genetic polymorphisms. Like certain associated carbohydrates, especially TF antigen, they represent clinically relevant markers in gastrointestinal neoplasia.
2 2 7 A34 - Expression of a Novel Differentiation Antigen of the Stomach Mucosa on a Protein Level A.A. JUNGBLUTH, C. WILLIAMS, D. KOLB, K. COPLAN, E. SATO, G. RITTER, L.J. OLD, M.J. SCANLAN Ludwig Institute for Cancer Research New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Aims: A34 is a novel gastric differentiation antigen, which was recently identified in silico by database analysis based on sequence homologies to A34, an intestinal differentiation antigen, which is present in normal intestinal mucosa and in >90% of intestinal carcinomas. On an mRNA level, A34 is solely expressed in stomach,
3t 5
testis and at low levels in pancreas. The present analysis was done to analyze the expression of A34 on a protein level in order to assess its potential value as a diagnostic marker and/or as a target for immunotherapy of cancer. Methods: Mice were immunized with bacterially expressed A34 antigen and monoclonal antibodies (mAbs) were generated by standard hybridoma techniques. Newly generated hybridomas were screened by ELISA with A34 antigen and then tested by immunohistochemistry (IHC) using frozen and paraffin-embedded tissues employing antigen "retrieval techniques. Panels of normal and tumor tissues were then analyzed by IHC. Results: Three A34-specific clones were isolated and proved to stain frozen and paraffin-embedded tissues. In normal tissues, A34 was intensely expressed in the epithelia throughout the gastric mucosa, in single pancreatic duct epithelia and in the cytoplasm of testicular germ cells. In tumors, A34 was detected in 1/3 of gastric carcinomas, while no reactivity was found in carcinomas of the breast, kidney, urinary bladder and ovary while A34 was focally detected in colon and lung adenocarcinomas. Conclusions: A34 is mainly expressed in gastric mucosa and in tumors thereof, suggesting a novel differentiation antigen. A34 may be useful as a target for the immunotherapy of gastric carcinomas or as a diagnostic tool for the differential diagnosis of gastric adenocarcinomas.
228 Stimulation of Cathepsin X by Helicobacter pylori in human gastric epithelial cells: potential role in progression of gastric cancer S. KRUEGER 1, T. HUNDERTMARK 1, T. KALINSKI 1, U. PEITZ 2, A. ROESSNER l 11nstitut ftir Pathologie, 2Klinik fur Gastroenterologie, Otto-von-Guericke Universit~it Magdeburg
Aims: There is cumulative evidence that cysteine proteases are involved in the progression of different types of cancer. Epithelial cell responses to bacterial infection include induction of matrixdegrading enzymes. Here, we identified increased cathepsin X expression in the gastric epithelium in response to the oncogenic bacterium H. pylori, and report on the mechanisms of gastric epithelial cell migration and consequences for cancer progression. Methods: Paraffin-embedded biopsies and gastric cancer samples were processed for cathepsin X immunohistochemistry. Frozen samples were homogenized for Western Blots and for quantitative real-time RT-PCR of cathepsin X. Induction of cathepsin X expression in epithelial cells and inflammatory cells caused by H. pylori infection was tested in 3D-cocultures of infected AGS- or primary epithelial cells and primary macrophages/fibroblasts embedded in collagen I. Results: Cathepsin X was significantly higher in chronic atrophic gastritis than in controls. The same was observed for cancer. In parallel, cathepsin X moves toward the basal region of the epithelial cells during progression from normal tissue to carcinoma. In vitro experiments gave evidence that these alterations may be mediated by Rac/Rho signal-transduction pathways activated by H. pylori, resulting in increased migration. Intercellular signalling between H. pylori-infected epitelial cells and macrophages via soluble factors in the culture medium are responsible for increased cathepsin X. Conclusions: Our study demonstrates that cathepsin X may have a role not only in invasion, but also in the progression of gastric cancer.