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Absence of somatic alterations of the EB1 gene apcassociated protein in human sporadic colorectal cancer
Gastrointestinal Oncology A615
April 1998 patients a partial remission was observed, a complete tumor response was achieved in 6 patients. One of these six patients died perioperatively, all other are still alive (20 months, range 12-31). Of 17 patients with partial remission 7 are dead now. Average survival of theses 17 patients was 23.5 months (range 7-38). We cannot exclude a raised perioperative morbidity and mortality rate after preoperative radio-chemotherapy. However neoadjuvant therapy seems to be of clear benefit for patients with tumor response to radio-chemotherapy. • G2528 IMMUNOItISTOCItEMICAL ASSESSMENT OF 17-1A AND COMPLEMENT RESISTANCE FACTORS CD55 AND CD59 IN LIVER METASTASES OF COLON CANCER. S. Inndorf, S.B. Hosch, M. Lueth, P. Scheunemann, K. Pantel, W.T. Knoefel, J.R. Izbicki. Departments of Surgery University of Hamburg, and Immunology University of Munich, Germany. Background: Adjuvant therapy with the monoclonal antibody 17-1A has shown to be effective in preventing distant metastases in a certain amount of patients with Dukes C colon cancer. Those patients who develop liver metastases after intentionally curative resection of their primary tumor have a 50% chance to remain without metastatic tumor relapse after resection of their metastases. So far nothing is known about the benefit of an adjuvant therapy with mAB 17-1A in patients with liver metastases. However, there is evidence that the presence of complement resistance factors inhibit complement dependent cytotoxicity in patients treated with monoclonal antibodies. Methods: We therefore examined the expression of 17-1A antigen and the membrane bound complement resistance factors CD55 and CD59 on liver metastases of 42 patients with colon carcinoma who had undergone intentionally curative resection of their metastases using immunohistochemical assays with monoclonal antibodies directed against 17-1A, CD55, and CD59. Results: All 42 patients showed a homogeneous expression of 17-1A antigen. 34 of these patients (80%) were negative for CD55 expression whereas 5 patients (14%) displayed CD55 heterogeneously and 3 patients (6%) displayed CD55 homogeneously. Regarding CD59 expression, 33 patients (78%) had no CD59 expression, compared to 8 patients (20%) with heterogeneous CD59 expression, and 1 patient (2%) with homogeneous CD59 expression. Conclusions: Our data indicate that 17-1A antigen is widely expressed on liver metastases of patients with colon carcinoma. Therefore these patients might be suitable candidates for an adjuvant 17-1A antibody therapy even more since only a minority of these patients display complement resistance factors, which might inhibit the efficacy of the antibody treatment. Consequently immunohistochemical assessment of these factors on liver metastases could be helpful to identify those patients, who might benefit from such therapeutic strategies. • G2529 DEVELOPMENT AND PROGRESSION IN COLORECTAL TUMOR. T, Iwase. H. Konishi, T. Kodarna, K. Kashima and T. Hattori. Third Dep. of Internal Medicine, Kyoto Prefectual University of Medicine, Kyoto and Dep. of Pathology, Shiga University of Medical Science, Shiga, JAPAN. To clarify the mechanism of colorectal tumor development and progression, we focused on early stage of colorectal cancers and studied their proliferation status, cell fates, p53 expression status, and DNA ploidy pattern. MATERIALS AND METHODS: The material studied consisted of 19 cases of early colorectal cancer (9 mucosal cancers and 10 submucosal cancers) which we classified into two groups; adenocarcinoma asociated with adenoma(Group A) and without adenoma(Group B). Immunohistochemistry was performed to study an expression status of p53 (DO7) in which we considered the case as an overexpression if over 60% of nuclears were positively stained at diffuse area. Ki-67(MIB-1) immunostaining and in situ end-labeling were performed to analyze proliferation status and cell fates, respectively, and then proliferation index (PI), apoptotic index (AI) and AI/PI indices were calculated. These parameters were compared among carcinoma, non-neoplastic adjacent mucosa and/or adenoma within the same case and their spatial distributions were also evaluated. Nuclear DNA contents were analyzed b y a static cytofluorometry using isolated nuclei from various lesions, and the specimen which showed an additional peak in DNA contents was considered as an aneuploidy. RESULTS: Overexpression of p53 was seen in all cases of adenocarcinoma with adenoma, whereas some cases without adenoma (30%) did not show overexpression. Group A (%) normal adenoma carcinoma
PI 14.3 28.8 41.8
AI 0.83 3.99 3.30
AI/PI 5.80 13.8 7.90
Group B (%) normal carcinoma
PI 12.6 41.3
AI AUPI 0.63 4.97 3 . 7 1 8.97
AI and PI increased with the advance of disease from normal, adenoma through carcinoma, spatial distributions of both proliferating and apoptotic cells were, however, disorganized only at carcinomas. Furthermore, AI/PI in carcinomas were constantly lower than those in adenoma. Cytofluorometrical analyses showed that all normal and adenoma tissues were diploidy pattern. The majority of carcinoma (78.9%), on the other hand, showed aneuploidy pattern, no matter how adenoma components were accompanied.
CONCLUSION: It is important to analyze these early cancer cases to clarify the very beginings of colorectal cartinogenesis. The classification which divided colorectal cancers according to their accompanied adenoma status may not be sufficient, and further subclassification which considered p53 overexpression, proliferation status, cell fates, and nuclear contents would be considered. • G2530 THE BASIS FOR TIlE DECREASED AUTOFLUORESCENCE OF ADENOMA AND CANCER OF THE COLON BY LASER-INDUCED FLUORESCENCE ENDOSCOPY. K. Izuishi, H. Tajiri, T. Fujii, S. Abe, M. Ryu, S. Yoshida. National Cancer Center Hospital East, Chiba, Japan. Laser-induced fluorescence (LIF) endoscopy for tissue diagnosis has been drawing attention as a new approach in clinical diagnosis. This technique has also been applied to the diagnosis of lesions in the gastrointestinal tract with considerable success. However, the basis for the difference in green autofluorescence between normal and diseased tissues (cancer and adenoma) in the colon observed by laser-induced autofluorescence endoscopy is unclear, although flavins, NAD(P)H and collagen have been regarded as major sources of fluorescence. The purpose of the present study was to clarify the cause of the differences in the green fluorescence of normal and diseased tissues. MATERIALS and METHODS: Human colonic tissues (adenoma: n=6, cancer: n=ll, normal: n=ll) were obtained from resected specimens. The turin content of human colonic tissue was measured by high performance liquid chromatography. Fluorescence microscopy examination of frozen sections was carried out to visually evaluate the fluorescence localization and its properties in normal colon, adenoma, and cancer tissue under blue light excitation (400-440 nm). To clarify the relationship between the mucosal thickness and fluorescence intensity, colonic tissue was cut obliquely as a means of varying mucosal thickness, and fluorescent imaging was performed (Ex: 437 nm, Em: 520 nm) by the autofluorescence endoscopy system (Xillix Technology Co., Vancouver, Canada and Olympus Optical Co., Tokyo, Japan), and fluorescence intensity was analyzed. RESULTS: The flavin content of normal and diseased tissue was not significantly different. Fluorescence microscopy of normal colonic tissue revealed strong fluorescence in the submucosal layer that corresponded to collagen. Tissue fluorescence did not decrease in reducing or acid solution. No difference in fluorescence was detected in normal mucosa, adenoma, and cancer by fluorescence microscopy. These findings indicate that flavins and NAD(P)H do not affect tissue fluorescence, and that submucosal collagen is the main source of tissue fluorescence in the colon. We also found that tissue autofluorescence was dependent on the thickness of mucosa. CONCLUSION: The cause of the decreased fluorescence in diseased tissues appears to be due to a decrease in collagen fluorescence, because of a screening effect by the mucnsal thickness or replacement of the submucosa by cancer cells. • G2531 ABSENCE OF SOMATIC ALTERATIONS OF THE EB1 GENE APCASSOCIATED PROTEIN IN HUMAN SPORADIC COLORECTAL CANCER. P. Ja'fs (1,5), J.C. Sabourin (2), J. Bombled (1), P. Rougier (3), P. Lasser (4), P. Duvillard (2), J. B~nard (1), B. Bressac-de Paillerets (I). (1) Dpt. of Molecular Genetics, (2) Dpt. of Pathology, (3) Dpt. of Surgery, (4) Dpt. Gastroenterology, lnstitut Gustave Roussy, Villejuif, (5) Dpt. Gastroenterology, Hrpital Bichat, Paris -- France. Background: The human EB1 gene product was recently found by a yeast two-hybrid screening to be associated with the carboxyl-terminus of the APC (Adenomatous Polyposis Coli) protein, the product of a tumor suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis. Aims: Because virtually all the APC mutations result in the synthesis of carboxyl-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product. Thus, the interaction between APC and EB1 proteins may be important for the tumor suppressor activity of APC protein, and raises the hypothesis that EB1 gene is also involved in sporadic colorectal tumorigenesis. Methods: To investigate this hypothesis, somatic mutations were searched in the entire coding sequence of EB1 eDNA by reverse transcriptase-single strand conformational polymorphism (SSCP) analysis. To also investigate whether EB1 locus was comprised within a region subjected to losses of heterozygosity, 4 polymorphism markers surrounding EB1 locus were surveyed. Results: None of the 29 colorectal tumors analyzed contained somatic mutation, whereas a silent eDNA variant was identified in 14% of cases. Only one out of 29 colorectal tumors had a loss of heterozygosity for the D20S 107 marker. Conclusions: The present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis.
April 1998 patients a partial remission was observed, a complete tumor response was achieved in 6 patients. One of these six patients died perioperatively, all other are still alive (20 months, range 12-31). Of 17 patients with partial remission 7 are dead now. Average survival of theses 17 patients was 23.5 months (range 7-38). We cannot exclude a raised perioperative morbidity and mortality rate after preoperative radio-chemotherapy. However neoadjuvant therapy seems to be of clear benefit for patients with tumor response to radio-chemotherapy. • G2528 IMMUNOItISTOCItEMICAL ASSESSMENT OF 17-1A AND COMPLEMENT RESISTANCE FACTORS CD55 AND CD59 IN LIVER METASTASES OF COLON CANCER. S. Inndorf, S.B. Hosch, M. Lueth, P. Scheunemann, K. Pantel, W.T. Knoefel, J.R. Izbicki. Departments of Surgery University of Hamburg, and Immunology University of Munich, Germany. Background: Adjuvant therapy with the monoclonal antibody 17-1A has shown to be effective in preventing distant metastases in a certain amount of patients with Dukes C colon cancer. Those patients who develop liver metastases after intentionally curative resection of their primary tumor have a 50% chance to remain without metastatic tumor relapse after resection of their metastases. So far nothing is known about the benefit of an adjuvant therapy with mAB 17-1A in patients with liver metastases. However, there is evidence that the presence of complement resistance factors inhibit complement dependent cytotoxicity in patients treated with monoclonal antibodies. Methods: We therefore examined the expression of 17-1A antigen and the membrane bound complement resistance factors CD55 and CD59 on liver metastases of 42 patients with colon carcinoma who had undergone intentionally curative resection of their metastases using immunohistochemical assays with monoclonal antibodies directed against 17-1A, CD55, and CD59. Results: All 42 patients showed a homogeneous expression of 17-1A antigen. 34 of these patients (80%) were negative for CD55 expression whereas 5 patients (14%) displayed CD55 heterogeneously and 3 patients (6%) displayed CD55 homogeneously. Regarding CD59 expression, 33 patients (78%) had no CD59 expression, compared to 8 patients (20%) with heterogeneous CD59 expression, and 1 patient (2%) with homogeneous CD59 expression. Conclusions: Our data indicate that 17-1A antigen is widely expressed on liver metastases of patients with colon carcinoma. Therefore these patients might be suitable candidates for an adjuvant 17-1A antibody therapy even more since only a minority of these patients display complement resistance factors, which might inhibit the efficacy of the antibody treatment. Consequently immunohistochemical assessment of these factors on liver metastases could be helpful to identify those patients, who might benefit from such therapeutic strategies. • G2529 DEVELOPMENT AND PROGRESSION IN COLORECTAL TUMOR. T, Iwase. H. Konishi, T. Kodarna, K. Kashima and T. Hattori. Third Dep. of Internal Medicine, Kyoto Prefectual University of Medicine, Kyoto and Dep. of Pathology, Shiga University of Medical Science, Shiga, JAPAN. To clarify the mechanism of colorectal tumor development and progression, we focused on early stage of colorectal cancers and studied their proliferation status, cell fates, p53 expression status, and DNA ploidy pattern. MATERIALS AND METHODS: The material studied consisted of 19 cases of early colorectal cancer (9 mucosal cancers and 10 submucosal cancers) which we classified into two groups; adenocarcinoma asociated with adenoma(Group A) and without adenoma(Group B). Immunohistochemistry was performed to study an expression status of p53 (DO7) in which we considered the case as an overexpression if over 60% of nuclears were positively stained at diffuse area. Ki-67(MIB-1) immunostaining and in situ end-labeling were performed to analyze proliferation status and cell fates, respectively, and then proliferation index (PI), apoptotic index (AI) and AI/PI indices were calculated. These parameters were compared among carcinoma, non-neoplastic adjacent mucosa and/or adenoma within the same case and their spatial distributions were also evaluated. Nuclear DNA contents were analyzed b y a static cytofluorometry using isolated nuclei from various lesions, and the specimen which showed an additional peak in DNA contents was considered as an aneuploidy. RESULTS: Overexpression of p53 was seen in all cases of adenocarcinoma with adenoma, whereas some cases without adenoma (30%) did not show overexpression. Group A (%) normal adenoma carcinoma
PI 14.3 28.8 41.8
AI 0.83 3.99 3.30
AI/PI 5.80 13.8 7.90
Group B (%) normal carcinoma
PI 12.6 41.3
AI AUPI 0.63 4.97 3 . 7 1 8.97
AI and PI increased with the advance of disease from normal, adenoma through carcinoma, spatial distributions of both proliferating and apoptotic cells were, however, disorganized only at carcinomas. Furthermore, AI/PI in carcinomas were constantly lower than those in adenoma. Cytofluorometrical analyses showed that all normal and adenoma tissues were diploidy pattern. The majority of carcinoma (78.9%), on the other hand, showed aneuploidy pattern, no matter how adenoma components were accompanied.
CONCLUSION: It is important to analyze these early cancer cases to clarify the very beginings of colorectal cartinogenesis. The classification which divided colorectal cancers according to their accompanied adenoma status may not be sufficient, and further subclassification which considered p53 overexpression, proliferation status, cell fates, and nuclear contents would be considered. • G2530 THE BASIS FOR TIlE DECREASED AUTOFLUORESCENCE OF ADENOMA AND CANCER OF THE COLON BY LASER-INDUCED FLUORESCENCE ENDOSCOPY. K. Izuishi, H. Tajiri, T. Fujii, S. Abe, M. Ryu, S. Yoshida. National Cancer Center Hospital East, Chiba, Japan. Laser-induced fluorescence (LIF) endoscopy for tissue diagnosis has been drawing attention as a new approach in clinical diagnosis. This technique has also been applied to the diagnosis of lesions in the gastrointestinal tract with considerable success. However, the basis for the difference in green autofluorescence between normal and diseased tissues (cancer and adenoma) in the colon observed by laser-induced autofluorescence endoscopy is unclear, although flavins, NAD(P)H and collagen have been regarded as major sources of fluorescence. The purpose of the present study was to clarify the cause of the differences in the green fluorescence of normal and diseased tissues. MATERIALS and METHODS: Human colonic tissues (adenoma: n=6, cancer: n=ll, normal: n=ll) were obtained from resected specimens. The turin content of human colonic tissue was measured by high performance liquid chromatography. Fluorescence microscopy examination of frozen sections was carried out to visually evaluate the fluorescence localization and its properties in normal colon, adenoma, and cancer tissue under blue light excitation (400-440 nm). To clarify the relationship between the mucosal thickness and fluorescence intensity, colonic tissue was cut obliquely as a means of varying mucosal thickness, and fluorescent imaging was performed (Ex: 437 nm, Em: 520 nm) by the autofluorescence endoscopy system (Xillix Technology Co., Vancouver, Canada and Olympus Optical Co., Tokyo, Japan), and fluorescence intensity was analyzed. RESULTS: The flavin content of normal and diseased tissue was not significantly different. Fluorescence microscopy of normal colonic tissue revealed strong fluorescence in the submucosal layer that corresponded to collagen. Tissue fluorescence did not decrease in reducing or acid solution. No difference in fluorescence was detected in normal mucosa, adenoma, and cancer by fluorescence microscopy. These findings indicate that flavins and NAD(P)H do not affect tissue fluorescence, and that submucosal collagen is the main source of tissue fluorescence in the colon. We also found that tissue autofluorescence was dependent on the thickness of mucosa. CONCLUSION: The cause of the decreased fluorescence in diseased tissues appears to be due to a decrease in collagen fluorescence, because of a screening effect by the mucnsal thickness or replacement of the submucosa by cancer cells. • G2531 ABSENCE OF SOMATIC ALTERATIONS OF THE EB1 GENE APCASSOCIATED PROTEIN IN HUMAN SPORADIC COLORECTAL CANCER. P. Ja'fs (1,5), J.C. Sabourin (2), J. Bombled (1), P. Rougier (3), P. Lasser (4), P. Duvillard (2), J. B~nard (1), B. Bressac-de Paillerets (I). (1) Dpt. of Molecular Genetics, (2) Dpt. of Pathology, (3) Dpt. of Surgery, (4) Dpt. Gastroenterology, lnstitut Gustave Roussy, Villejuif, (5) Dpt. Gastroenterology, Hrpital Bichat, Paris -- France. Background: The human EB1 gene product was recently found by a yeast two-hybrid screening to be associated with the carboxyl-terminus of the APC (Adenomatous Polyposis Coli) protein, the product of a tumor suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis. Aims: Because virtually all the APC mutations result in the synthesis of carboxyl-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product. Thus, the interaction between APC and EB1 proteins may be important for the tumor suppressor activity of APC protein, and raises the hypothesis that EB1 gene is also involved in sporadic colorectal tumorigenesis. Methods: To investigate this hypothesis, somatic mutations were searched in the entire coding sequence of EB1 eDNA by reverse transcriptase-single strand conformational polymorphism (SSCP) analysis. To also investigate whether EB1 locus was comprised within a region subjected to losses of heterozygosity, 4 polymorphism markers surrounding EB1 locus were surveyed. Results: None of the 29 colorectal tumors analyzed contained somatic mutation, whereas a silent eDNA variant was identified in 14% of cases. Only one out of 29 colorectal tumors had a loss of heterozygosity for the D20S 107 marker. Conclusions: The present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis.