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Expression of chimeric IgG-ξ and IgG-λ receptors with specificity for TAG72
GASTROENTEROLOGY VoL 114, No. 4
A612 AGA ABSTRACTS
• G2515 MALIGNANT ESOPHAGEAL STENOSES PREOPERATIVE STAGING BY BOUGINAGE ENDOSONOGRAPHY. N. Hoepffner, J. Pohle, J. Menzel, W. Domschke, E.C. Foerster. Department of Medicine B, University of Muenster, Muenster, Germany Endoscopic ultrasound (EUS) is regarded to be the most precise method in pre-operative assessment of the TNM stage of esophageal carcinoma today. However, on establishing the diagnosis 25-62% of patients reveal to have such high-grade stenoses due to advanced tumor stage that complete passage of a conventional EUS-instrument is impossible. In these cases the examination either has to remain incomplete which due to reduced accuracy makes the explicitness of the result questionable or the stenoses have to he bouginaged prior to the examination which bears high perforation and complication risks. For this reason a EUS-instrument with a certain bouginage effect has been developed which due to its small external diameter of 7.9 mm allows for complete and less invasive examinations even in patients with high-grade esophageal stenoses. From May 1996 until February 1997 62 patients (7 women, 55 men, age 58 [41-82] years) with histologically confirmed malignant esophageal stenosis (17x adenocarcinoma, 39x squamous cell carcinoma) were examined endosonographically as part of a preoperative staging. As standard instruments Olympus UM3 (7.5 and 12 MHz) and the bougie instrument Olympus MH 908 (7.5 MHz, 7 mm diameter, no optics) were used. Beforehand, an esophagogastroduodenoscopy was tried in all patients, and for the bougie instrument a guide wire was positioned under endoscopic view. In 47 patients the EUS result could be compared with the postoperative histology. In 55.8% a stenosis which could not be passed by the standard instrument was present so that an accuracy of only 41% in the T- and 56% in the N-stage revealed whereas the bougie-EUS-instrument reached an accuracy of 75% in the T- and 63% in the N-stage (p<0.001). Bouginage endosonography has proven a secure, uncomplicated, and precise method in preoperative assessment of stenosing esophageal carcinoma. These results appear encouraging and justify further prospective investigations, especially in comparison with endosonographic miniprobes. • G2516 CHEMOPREVENTION OF COLON CARCINOGENESIS BY DIETARY SELENOMETHIONINE. Holubec, H., Baines, A.T., Bayse J.L., Waite S., Bhattacharyya, A.K., Clark, L.C., Payne, C.M., Earnest, D.L., and Nelson, M.A.. Arizona Cancer Center, University of Arizona, Tucson, AZ, 85724. Selenium supplementation has recently been reported in humans to cause a significant reduction in colon cancer incidence. Although the intervention agent, high Se Brewer's yeast, contained predominately selenomethionine, there is concern as to what form of Se may be active in the yeast formulation. Furthermore, the mechanism of action of selenomethionine as a cancer preventive agent is not known. Therefore, we evaluated the ability of dietary selenomethionine supplementation to modulate the carcinogenic activity of azoxymethane (AOM) in the rat colon. Four week old male F344 rats were treated with 15 mg/kg body weight of AOM once a week, for two weeks. Selenomethionine (0, 1, 2 ppm) in AIN-76 diet was fed for 16 weeks. Thereafter, the colons were removed, formalin fixed and stained with methylene blue. The number of aberrant crypt loci (ACF) and their size (crypt multiplicity) were quantitated by light microscopy. Microtumors were excised from the colonic muscosa and processed for histopathologic assessment. Results; Virtually all control diet rats (90%) treated with AOM had colonic microtumors, whereas only 40% and 20% of the animals on dietary selenomethionine (1 ppm and 2 ppm respectively) had neoplasms. In addition, the adenomas of animals fed selenomethionine exhibited increased apoptosis as compared to those fed the control diet. Neither 1 ppm or 2 ppm dietary selenomethionine had an effect on the incidence of aberrant crypt foci (ACF). However, both dietary levels of selenomethionine supplementation significantly reduced the incidence of large AOM-induced ACF (>7 aberrant crypts/focus). Conclusions: These results demonstrate that dietary selenomethionine supplementation was effective in suppressing ACF growth and formation of microtumors in the AOM-model of colon cancer. The chemopreventive activity of selenomethionine appeared to be mediated through cell loss by apoptosis. Supported in Part by CA 70145-01, ES 06694, and ES 07091. • G2517 EXPRESSION OF CHIMERIC IgG-~ AND I g G q RECEPTORS WITH SPECIFICITY FOR TAG72. A. Hombachl, C. Pohl 2, C. Heuserl, R. Sircar l, T. Tillmann l, V. Diehl l, W. Kruis 2, and H. Abken I. 1Klinik I fiir Innere Medizin, Universitat KNn & 2Inhere Abteilung, Evangelisches Krankenhaus Kalk, 50133 Cologne, Germany Back~,round: Chimeric T cell recptors with specificity for defined tumor associated antigens are valuable tools to target T cells to tumor ceils. We have recently described the generation and expression of a chimeric T-ceU receptor with specificity for the tumor antigen TAG72 consisting of the TAG72 specific single chain antibody (scfv) B72.3-scfv and the "/-chain of the Fc~l-receptor (Hombach et al. Gastroenterology 113, 1163-1170, 1997) Methods: Here we describe a new set of chimeric anti-TAG72 receptors that
consist of a scfv derived from a high affinitiy TAG72 specific second generation antibody (CC49-scfv) fused to the CH2/3 domains of the human lgGl and the CD3-~-chain and the "/-chain of the Fcl;1-receptor respectively. Results: The recombinant CC49-scfv-CH2/3-~ and CC49-CH2/3-y-cDNA respectively under control of the RSV LTR was transfected into MD45 T-cells. Both receptors were expressed as lgG-like homodimers by the transfected MD 45 cells as was demonstrated by FACS and Western blot analysis with an anti-human-lgG-Fc antibody directed against the lgG CH2/3 domains. Specific crosslinking of the chimeric ~-receptor as well as the chimeric "/-receptor resulted in the TAG72+ specific cellular activation of the transfected cell. Conclusion: Our results demonstrate, that expression of the chimeric lgG-like CC49-scfv-CH2/3-~-receptor as well as the CC49-CH2/3-"/ -receptor converts T cells to specificity for TAG72. These new chimeric receptors with higher affinity for the tumor antigen will facilitate the development of efficient gene transfer protocols into peripheral T cells and may be valuable tools for the cellular immunotherapy of CA72--4+ carcinomas. G2518 PERIODATE MEDIATED [I-ELIMINATION: A NOVEL METHOD OF CHEMICAL DEGLYCOSYLATION OF MUCIN GENE PRODUCTS ON PARAFFIN EMBEDDED SECTIONS OF COLORECTAL CANCER. LC. Hon~,. J.R. Gum, J.P. Terdiman, K.R. McQuaid, M.H. Sleisenger, Y.S. Kim. Department of Medicine, University of California San Francisco and GI Research Lab, VA Medical Center, San Francisco, CA.
Altered expression of mucin genes has been reported in many epithelial cancers including colorectal cancer. However, mucins are heavily glycosylated making the study of apomucin expression difficult. To investigate apomucin expression, various deglycosylation techniques have been tried with limited success. Aim: We sought to establish a method to remove carbohydrates and unmask the mucin peptide epitopes, enabling us to identify and ascertain the expression of mucin gene products. Methods: The following chemical deglycosylation methods were tested on 8 formalin fixed colon cancer specimens with adjacent normal mucosa: alkali (A), alkali+periodate oxidation (A+PO), and aikali+periodate mediated 13-elimination (A+PO+BE). PAS staining and immunohistochemical methods with antigen retrieval using antibodies against sialyl-Le x (SHN4), sialyl-Le 2 (CA19-9), tandem repeat regions of MUC1 (139H2), MUC2 (MRP), and MUC3 (M3P) were used. The samples were scored on a scale based on the intensity (0-3) times the area (0-100%). Results: In normal and colon cancer, under mild conditions (A+PO), expression of both carbohydrate antigens (sialyl-Le x & sialyl-Le 2) were markedly reduced while no change was observed with PAS staining indicating modification but no release of carbohydrates. Interestingly, with this treatment MUC1 expression increased but not MUC2 and MUC3. By contrast, under harsh conditions (A+PO+BE), PAS results show marked decrease in staining concomitant with marked increase in MUC2 & MUC3 apomucin expression indicating substantial removal of carbohydrate side chains. Control A A+PO A+PO+BE
PAS 1.79 ± .22 2.43 ± .28 2.50+-.23 1.28+-.17"*
Sialvl-Le x 0.27± .14 0.81 ± .21 0.09_+.04* 0.11_+.06"
MUC1 0.00 -¢-.00 0.00 ± .00 1.45_+.12" 1.79+-.13"
MUC2 0.00 __..00 0.00 _+.00 0.06+-.03 1.34+-.27"
MUC3 0.00 ± .00 0.00 ± .00 0.01_+.01 0.90±.12"
(Normal goblet vacuole staining. Sialyl-Le 2 results not shown, but expression was similar to Sialyl-Le x. Similar pattern of expression was obtained in colorectal cancer. *P<0.01 **/<0.05) Conclusion: This study describes a novel method of chemical deglycosylation of mucin gene products on tissue sections. Unlike previous methods which results only in the modification of carbohydrate structures, this method cause release of carbohydrate side chains resulting in effective exposure of mucin polypeptides. Thus, this method is extremely useful in investigating the expression of diverse mucin gene products in various cell types of normal and neoplastic tissues. This research was supported by VA Medical Research Service, the Betz Foundation, and USPHS Grant CA24321. • G2519 REDUCED EXPRESSION OF PI.AKOGLOBIN INDICATES AN UNFAVORABLE PROGNOSIS IN PATIENTS WITH ESOPHAGEAL CANCER, S.B. Hosch, N.H. Stoecklein, J.R. Izbicki, U. Pichlmeier, K. Pantel. Departments of Surgery and Statistics University of Hamburg, Immunology University of Munich, Germany Background: Esophageal cancer is a very aggressive turnout with poor prognosis, because turnout cell dissemination occurs early in the course of the disease. A prerequisite for this dissemination is loss of homotypic cell adhesion, which may be mediated by plakoglobin (PKG), the common constituent of adhering junctions and desmosomes in epithelial cells. Methods: We therefore assessed the prognostic significance of PKG expression on primary esophageal carcinomas, lymph node metastases and on single tumour ceils in lymph nodes from 53 patients who had undergone radical en-bloc esophagectomy using immunohistochemical assays with the monoclonai antibody (mAb) PG5.1 against PKG.
A612 AGA ABSTRACTS
• G2515 MALIGNANT ESOPHAGEAL STENOSES PREOPERATIVE STAGING BY BOUGINAGE ENDOSONOGRAPHY. N. Hoepffner, J. Pohle, J. Menzel, W. Domschke, E.C. Foerster. Department of Medicine B, University of Muenster, Muenster, Germany Endoscopic ultrasound (EUS) is regarded to be the most precise method in pre-operative assessment of the TNM stage of esophageal carcinoma today. However, on establishing the diagnosis 25-62% of patients reveal to have such high-grade stenoses due to advanced tumor stage that complete passage of a conventional EUS-instrument is impossible. In these cases the examination either has to remain incomplete which due to reduced accuracy makes the explicitness of the result questionable or the stenoses have to he bouginaged prior to the examination which bears high perforation and complication risks. For this reason a EUS-instrument with a certain bouginage effect has been developed which due to its small external diameter of 7.9 mm allows for complete and less invasive examinations even in patients with high-grade esophageal stenoses. From May 1996 until February 1997 62 patients (7 women, 55 men, age 58 [41-82] years) with histologically confirmed malignant esophageal stenosis (17x adenocarcinoma, 39x squamous cell carcinoma) were examined endosonographically as part of a preoperative staging. As standard instruments Olympus UM3 (7.5 and 12 MHz) and the bougie instrument Olympus MH 908 (7.5 MHz, 7 mm diameter, no optics) were used. Beforehand, an esophagogastroduodenoscopy was tried in all patients, and for the bougie instrument a guide wire was positioned under endoscopic view. In 47 patients the EUS result could be compared with the postoperative histology. In 55.8% a stenosis which could not be passed by the standard instrument was present so that an accuracy of only 41% in the T- and 56% in the N-stage revealed whereas the bougie-EUS-instrument reached an accuracy of 75% in the T- and 63% in the N-stage (p<0.001). Bouginage endosonography has proven a secure, uncomplicated, and precise method in preoperative assessment of stenosing esophageal carcinoma. These results appear encouraging and justify further prospective investigations, especially in comparison with endosonographic miniprobes. • G2516 CHEMOPREVENTION OF COLON CARCINOGENESIS BY DIETARY SELENOMETHIONINE. Holubec, H., Baines, A.T., Bayse J.L., Waite S., Bhattacharyya, A.K., Clark, L.C., Payne, C.M., Earnest, D.L., and Nelson, M.A.. Arizona Cancer Center, University of Arizona, Tucson, AZ, 85724. Selenium supplementation has recently been reported in humans to cause a significant reduction in colon cancer incidence. Although the intervention agent, high Se Brewer's yeast, contained predominately selenomethionine, there is concern as to what form of Se may be active in the yeast formulation. Furthermore, the mechanism of action of selenomethionine as a cancer preventive agent is not known. Therefore, we evaluated the ability of dietary selenomethionine supplementation to modulate the carcinogenic activity of azoxymethane (AOM) in the rat colon. Four week old male F344 rats were treated with 15 mg/kg body weight of AOM once a week, for two weeks. Selenomethionine (0, 1, 2 ppm) in AIN-76 diet was fed for 16 weeks. Thereafter, the colons were removed, formalin fixed and stained with methylene blue. The number of aberrant crypt loci (ACF) and their size (crypt multiplicity) were quantitated by light microscopy. Microtumors were excised from the colonic muscosa and processed for histopathologic assessment. Results; Virtually all control diet rats (90%) treated with AOM had colonic microtumors, whereas only 40% and 20% of the animals on dietary selenomethionine (1 ppm and 2 ppm respectively) had neoplasms. In addition, the adenomas of animals fed selenomethionine exhibited increased apoptosis as compared to those fed the control diet. Neither 1 ppm or 2 ppm dietary selenomethionine had an effect on the incidence of aberrant crypt foci (ACF). However, both dietary levels of selenomethionine supplementation significantly reduced the incidence of large AOM-induced ACF (>7 aberrant crypts/focus). Conclusions: These results demonstrate that dietary selenomethionine supplementation was effective in suppressing ACF growth and formation of microtumors in the AOM-model of colon cancer. The chemopreventive activity of selenomethionine appeared to be mediated through cell loss by apoptosis. Supported in Part by CA 70145-01, ES 06694, and ES 07091. • G2517 EXPRESSION OF CHIMERIC IgG-~ AND I g G q RECEPTORS WITH SPECIFICITY FOR TAG72. A. Hombachl, C. Pohl 2, C. Heuserl, R. Sircar l, T. Tillmann l, V. Diehl l, W. Kruis 2, and H. Abken I. 1Klinik I fiir Innere Medizin, Universitat KNn & 2Inhere Abteilung, Evangelisches Krankenhaus Kalk, 50133 Cologne, Germany Back~,round: Chimeric T cell recptors with specificity for defined tumor associated antigens are valuable tools to target T cells to tumor ceils. We have recently described the generation and expression of a chimeric T-ceU receptor with specificity for the tumor antigen TAG72 consisting of the TAG72 specific single chain antibody (scfv) B72.3-scfv and the "/-chain of the Fc~l-receptor (Hombach et al. Gastroenterology 113, 1163-1170, 1997) Methods: Here we describe a new set of chimeric anti-TAG72 receptors that
consist of a scfv derived from a high affinitiy TAG72 specific second generation antibody (CC49-scfv) fused to the CH2/3 domains of the human lgGl and the CD3-~-chain and the "/-chain of the Fcl;1-receptor respectively. Results: The recombinant CC49-scfv-CH2/3-~ and CC49-CH2/3-y-cDNA respectively under control of the RSV LTR was transfected into MD45 T-cells. Both receptors were expressed as lgG-like homodimers by the transfected MD 45 cells as was demonstrated by FACS and Western blot analysis with an anti-human-lgG-Fc antibody directed against the lgG CH2/3 domains. Specific crosslinking of the chimeric ~-receptor as well as the chimeric "/-receptor resulted in the TAG72+ specific cellular activation of the transfected cell. Conclusion: Our results demonstrate, that expression of the chimeric lgG-like CC49-scfv-CH2/3-~-receptor as well as the CC49-CH2/3-"/ -receptor converts T cells to specificity for TAG72. These new chimeric receptors with higher affinity for the tumor antigen will facilitate the development of efficient gene transfer protocols into peripheral T cells and may be valuable tools for the cellular immunotherapy of CA72--4+ carcinomas. G2518 PERIODATE MEDIATED [I-ELIMINATION: A NOVEL METHOD OF CHEMICAL DEGLYCOSYLATION OF MUCIN GENE PRODUCTS ON PARAFFIN EMBEDDED SECTIONS OF COLORECTAL CANCER. LC. Hon~,. J.R. Gum, J.P. Terdiman, K.R. McQuaid, M.H. Sleisenger, Y.S. Kim. Department of Medicine, University of California San Francisco and GI Research Lab, VA Medical Center, San Francisco, CA.
Altered expression of mucin genes has been reported in many epithelial cancers including colorectal cancer. However, mucins are heavily glycosylated making the study of apomucin expression difficult. To investigate apomucin expression, various deglycosylation techniques have been tried with limited success. Aim: We sought to establish a method to remove carbohydrates and unmask the mucin peptide epitopes, enabling us to identify and ascertain the expression of mucin gene products. Methods: The following chemical deglycosylation methods were tested on 8 formalin fixed colon cancer specimens with adjacent normal mucosa: alkali (A), alkali+periodate oxidation (A+PO), and aikali+periodate mediated 13-elimination (A+PO+BE). PAS staining and immunohistochemical methods with antigen retrieval using antibodies against sialyl-Le x (SHN4), sialyl-Le 2 (CA19-9), tandem repeat regions of MUC1 (139H2), MUC2 (MRP), and MUC3 (M3P) were used. The samples were scored on a scale based on the intensity (0-3) times the area (0-100%). Results: In normal and colon cancer, under mild conditions (A+PO), expression of both carbohydrate antigens (sialyl-Le x & sialyl-Le 2) were markedly reduced while no change was observed with PAS staining indicating modification but no release of carbohydrates. Interestingly, with this treatment MUC1 expression increased but not MUC2 and MUC3. By contrast, under harsh conditions (A+PO+BE), PAS results show marked decrease in staining concomitant with marked increase in MUC2 & MUC3 apomucin expression indicating substantial removal of carbohydrate side chains. Control A A+PO A+PO+BE
PAS 1.79 ± .22 2.43 ± .28 2.50+-.23 1.28+-.17"*
Sialvl-Le x 0.27± .14 0.81 ± .21 0.09_+.04* 0.11_+.06"
MUC1 0.00 -¢-.00 0.00 ± .00 1.45_+.12" 1.79+-.13"
MUC2 0.00 __..00 0.00 _+.00 0.06+-.03 1.34+-.27"
MUC3 0.00 ± .00 0.00 ± .00 0.01_+.01 0.90±.12"
(Normal goblet vacuole staining. Sialyl-Le 2 results not shown, but expression was similar to Sialyl-Le x. Similar pattern of expression was obtained in colorectal cancer. *P<0.01 **/<0.05) Conclusion: This study describes a novel method of chemical deglycosylation of mucin gene products on tissue sections. Unlike previous methods which results only in the modification of carbohydrate structures, this method cause release of carbohydrate side chains resulting in effective exposure of mucin polypeptides. Thus, this method is extremely useful in investigating the expression of diverse mucin gene products in various cell types of normal and neoplastic tissues. This research was supported by VA Medical Research Service, the Betz Foundation, and USPHS Grant CA24321. • G2519 REDUCED EXPRESSION OF PI.AKOGLOBIN INDICATES AN UNFAVORABLE PROGNOSIS IN PATIENTS WITH ESOPHAGEAL CANCER, S.B. Hosch, N.H. Stoecklein, J.R. Izbicki, U. Pichlmeier, K. Pantel. Departments of Surgery and Statistics University of Hamburg, Immunology University of Munich, Germany Background: Esophageal cancer is a very aggressive turnout with poor prognosis, because turnout cell dissemination occurs early in the course of the disease. A prerequisite for this dissemination is loss of homotypic cell adhesion, which may be mediated by plakoglobin (PKG), the common constituent of adhering junctions and desmosomes in epithelial cells. Methods: We therefore assessed the prognostic significance of PKG expression on primary esophageal carcinomas, lymph node metastases and on single tumour ceils in lymph nodes from 53 patients who had undergone radical en-bloc esophagectomy using immunohistochemical assays with the monoclonai antibody (mAb) PG5.1 against PKG.