- Email: [email protected]
Detection of cytokeratin 20-MRNA in peripheral blood: A reliable test for circulating gastric epithelial cells?
A1394 AGA ABSTRACTS
6333 GENETIC ALTERATIONS IN GASTRIC CARCINOMA AND ADJACENT MUCOSA DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION(CGH). Jayoung Koo, Mooin Park, Seonja Park, Sunhoe Koo, Kosin Med Ctr, Pusan, MD, South Korea; Chungnam Univ, Daejeon , MD, South Korea. Twent y-six gastric carcinomas and their adjacent mucosa were screened for chromosomal aberrations using CGH. All carcinomas had chromosomal aberrations, and the mean number of chromosomal aberrations per tumor was 3.2. gain of chromosomal material was more common than loss(gainlloss ratio 4.3:1).10 of total 26 adjancet mucosa had chromosom al aberrations , and the mean number of chromosomal aberrations per adjancet mucosa was 2.3. Loss of chromo somal material per adjacent mucosa was almost the same as that of carcinoma(0.5/0.7), but, gain was less frequently observed than tumor( 1.6/2.6). Gains of chromosomal material per adjacent mucosa was more frequent in advanced stages(II1-IV), The most common gains in tumor were detected on 13q(58.3%, 14cases), 8q(30.8%, 8cases), 6q(27.0%, 7cases), 20p(l9.2%, 5cases). Other recurrent gains of 2q, 7p, 20q were found in more than 10%(3cases) and gain of 3q, Sp, lOp, l lq, 18q was found in 7.2%(2cases) of tumor. The most frequent loss in tumor was detected on I7p(38.5%, lOcases), followed by 16q(7.2%, 2cases). The most common chromosomal aberrations in adjacent mucosa were gain of l3q(l1.5 %, 3cases) and loss of 17q(11.5%, 3cases). Other less common chromosomal aberrations were gain of 5q, lOp, llq, 18q (2 cases). Tumors had more chromosomal gains of 2q, 3q, 13q than adjacent mucosa and especially gain of 13q in tumor was significantly frequent(53.8% versus 11.5%). Tumor had more losses of 17p(38.5% versus 11.5%) and l6q . Gains of 6q, 7p, Sq. 20p, 20q were observed only in tumor, while chromosomal gains of Ip, Zp, 5p, lOp, lip, 18q and loss of 16p, 17q, 21q was same in tumor and adjacent mucosa. Gains of 6q(7case s) and 2Oq(3cases) were observed exclusively in intestinal type carcinoma, but gain of 2q was observed only in diffuse type. Based on the positions of chromosomal gains or losses, more study for several candidate genes less-well defined in gastric carcinoma, for example , DPI , FLTl , c-myb, AIBI , BTAK, ZNF217, IL-I R, Skp2, PIK3A, telomerase RNA gene and CBP may be needed to clarify gastric carcinogenesis and progression. Our comparative genomic hybridization results indicate the presence of multistep genetic alterations that may play some important role in the development and progression of gastric carcinom a, and suggest possible role of CGH in studying carcinogenic process, in predicting prognosis or therapeutic decision of gastric carcinoma.
6334 MUTATION AND EXPRESSION OF THE P511P63 GENE IN HUMAN COLORECTAL CARCINOMA. Toru Koyama, Kunihiro Hamada, Kimihiro Shimizu, Jun Yokota. Susumu Ohwada, Yasuo Morishita, Gunma Univ Sch of Medicine, Maebashi, Japan ; Bioi Div , Natl Cancer Ctr Research Inst, Tokyo, Japan; Gunma Univ Sch of Medicine, MaebashiMaeba shi, Japan . Background : The 1'51/1'63 (1'51) gene on chromo some 3q28 encodes a protein with significant homology to 1'53, and shares several functions with 1'53, including the transcription al activation of 1'21 and the induction of apoptosis . Some 1'51 isotypes are known to have transdominant activity against 1'53. To elucidate the role of the 1'51 gene in colorectal carcinogenesis, we performed a mutation analysis as well as a detailed analysis of gene expression. Material and Method: High molecular weight DNA was prepared from 96 colorectal carcinomas paired with adj acent non-cancerous tissues. All samples were examined for mutations in the entire coding sequence of the 1'51 gene (exons 2-15 and exon 3' ) as well as the core domain of the 1'53 gene (exons 4-8) by PCR-SSCP analysis and direct sequencing. In 24 cases, total RNA was prepared from tumors and adjacent non-cancer ous tissues, and the levels and types of 1'51 expression were examined by RT-PCR analysis. Results: A missense mutation, which changed the amino acid at codon 240 in exon 6 from serine to proline, was detected in I of 96 primary co lorectal carcinomas. ln addition , genetic polymorphi sms were found at codon 248 in exon 6 and in introns 5 and 8. By RT-PCR , pSI transcripts were detected in all 24 tumors examined . In 10 of 24 cases, p5I was expressed less in tumors than in adjacent noncancerous tissues, and these cases showed a higher incidence of 1'53 mutations . Conclusion: These results indicate that 1'51 does not play a major role in colorectal carcinogenesis as a tumor suppressor. However it is possible that 1'51 expression is downregulated in tumors with 1'53 mutations to repress the function of pSI protein in tumor cells. Further analysis of 1'51 expression and function is necessary to fully understand the role of the pSI gene in human colorectal carcinogenesis.
6335 DETECTION OF CYTOKERA TIN 20-MRNA IN PERIPHERAL BLOOD: A RELIABLE TEST FOR CIRCULATING GASTRIC Ep· ITHELIAL CELLS? Udo Kronberg, Juan Eiki Nishizawa, Concepcion Risueno, Sergio Guzman, Manuel Alvarez, CATHOLIC Univ Sch OF MEDICINE, Santiago , Chile. Burchill described in 1995 a method for the detection of mRNA of cytokeratin 20 (CK20) by a highly specific and sensitive RT-PCR in blood samples in order to determin e its prognostic value in gastrointestinal cancer patients. We used a similar nested RT-PCR to evaluate the impact of
GASTROENTEROLOGY Vol. 118, No.4
biopsy-taking during gastroscopy on cell dissemination in patients with or without gastric cancer. We examined blood samples from 38 patients undergoing gastroscopy, 7 with gastric carcinoma, 31 with benign gastrointestinal disorders. Samples were taken from each patient before starting endoscopy, after the third biopsy, and 15 min after the withdrawal of the endoscope. As a control we examined blood samples from 55 healthy donors. Examining the first sample of the patients, CK20·mRNA was detected in 2/7 patients (28.6%) with gastric cancer, and in 18/31 patients (57.3%) with benign gastrointestinal disease. In II patients, all three consecutive samples were positive, and 6 were all negative ; in 9 patients results became positive in the 2nd and/or 3rd sample. In 9 patients with a positive first sample, results were negative in the consecutive samples, and in 3 patients with negative first sample, the 2nd sample was positive, the 3rd not. Samples of healthy donors were positive in 5155 (9%) cases during the first assay: in repetitions of CK20 RT-PCR (2-5 times) on 25 of these samples. 7/25 (28%) remained negative, whereas 18/25 (72%) were positive at least in one of the repetitions. In conclusion, there is no statistically significant difference between samples taken before and after biopsytaking. Due to the fact that the detection of CK20-mRNA by RT-PCR showed positive results in 72% of blood samples of healthy donors and lacked of reproducibilit y, this method does not permit the evaluation of the presence of circulating gastrointestinal epithelial cells in peripheral blood.
6336 IS THERE A GENETIC PREDISPOSITION OF PATIENTS WITH NON·HEREDITARY CHRONIC PANCREATITIS TO DEVELOP PANCREATIC CANCER? Axel Kropp, Stefan B. Hosch, Wolfram T. Knoefel, Asad Kutup, Vacislav N. Kalinin, Jakob R. Izbicki, Dept of Surg, Univ Hamburg, 20246 Hamburg, Germany . Background : Hereditary chronic pancreatiti s (HP) is an autosomal inherited disease based on a gene-mutation in the trypsinogen-gene mapped to the chromosome 7q35.These patients have an increased risk to develop pancreatic cancer. In this study we screened patients with pancreatic cancer, chronic pancreat itis and patiens who developed pancreatic cancer on chronic pancreatitis. Methods: For detection of the mutations of the trypsinogen gene (Trp 4) we extracteted the DNA from frozen tissue specimens from 55 patients with chronic pancreatitis and 36 patients with pancreatic cancer. 20 patients with pancreatic cancer had a preexisting chronic pancreatitis . With polymerase chain reaction (PCR) and subsequent direct sequencing by big dye terminator kit (Perkin Elmer) we examined exon 2 and 3 as described in the literature. For selective amplification of exon 2 we developed a new intronic primer to amplify a 722 bp fragment. Results: None of the patients displayed a mutation in the cationic trypsinogen gene of R 117 H in exon 3 or N21 1- mutation in exon 2. Also in the sequence-analysis no mutations of the cationic trypsinogen gene (A I6V) and no further mutations were found Conclusions: Our results emphasize the specificity of the mutation in the cationic trypsinogen gene for hereditary pancreat itis. In patients where pancreatic cancer had developed in chronic non hereditary pancreatitis the evaluated mutations seem to be irrelevant. Thus the risk to develop cancer in chronic non hereditary pancreatitis cannot be estimated by screening for these mutations
6337 DIAGNOSTIC ACCURACY OF ENDOCAVITAL ULTRASONOG· RAPHY (ENDOSONOGRAPHY) IN STAGING OF GI MALIGNANCIES. Jan Kulig, Wojciech Nowak, Piotr Kolodziejczyk, Bartlomiej Zarebski. Tadeusz Popiela, 1st Dept of Gen and GI Surg, Jagiellonian Univ, Krakow, Poland. Objective.Adequate preoperative and intraoperative staging is crucial for the planning of successful multimodal management of GI malignancies . The aim of the study was to assess diagnostic accuracy of endocavital ultrasonography (ECUS) including endoscopic (EUS), endorectal (ERUS), intraoperative (IOUS) intraductal (IDU), and laparoscopic (LUS) ultrasonography in the staging of patients with GI tumors . Method :2628 ECUS examinations (EUS - 375, ERUS - 1190, 10US - 1004, LUS -35, IDU - 24) were performed in patients undergoing surgery for GI malignant tumors. The results were compared with intraoperati ve findings, and final pathologic reports. The TNM classification was used to evaluate locoregional spread of cancer. Results:Accuracy of ECUS for determin ing tumor size and depth of invasion (T) depended on the site of cancer and was: 84.1% for esophagus, 78.5% for stomach, 99.4 % for liver, 85.3% for pancreas, 87.0 % for rectum, and 91% for other abdominal tumors. The overall accuracy of ECUS for the detection of lymph nodes metastases (N) ranged from 75.8% to 87.5% depend ing on the site and applied techniques ( or their combination). Conclusions: Combination of ECUS methods are useful for staging of GI malignancies and should become routine procedures in planning of the most accurate treatment.
6333 GENETIC ALTERATIONS IN GASTRIC CARCINOMA AND ADJACENT MUCOSA DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION(CGH). Jayoung Koo, Mooin Park, Seonja Park, Sunhoe Koo, Kosin Med Ctr, Pusan, MD, South Korea; Chungnam Univ, Daejeon , MD, South Korea. Twent y-six gastric carcinomas and their adjacent mucosa were screened for chromosomal aberrations using CGH. All carcinomas had chromosomal aberrations, and the mean number of chromosomal aberrations per tumor was 3.2. gain of chromosomal material was more common than loss(gainlloss ratio 4.3:1).10 of total 26 adjancet mucosa had chromosom al aberrations , and the mean number of chromosomal aberrations per adjancet mucosa was 2.3. Loss of chromo somal material per adjacent mucosa was almost the same as that of carcinoma(0.5/0.7), but, gain was less frequently observed than tumor( 1.6/2.6). Gains of chromosomal material per adjacent mucosa was more frequent in advanced stages(II1-IV), The most common gains in tumor were detected on 13q(58.3%, 14cases), 8q(30.8%, 8cases), 6q(27.0%, 7cases), 20p(l9.2%, 5cases). Other recurrent gains of 2q, 7p, 20q were found in more than 10%(3cases) and gain of 3q, Sp, lOp, l lq, 18q was found in 7.2%(2cases) of tumor. The most frequent loss in tumor was detected on I7p(38.5%, lOcases), followed by 16q(7.2%, 2cases). The most common chromosomal aberrations in adjacent mucosa were gain of l3q(l1.5 %, 3cases) and loss of 17q(11.5%, 3cases). Other less common chromosomal aberrations were gain of 5q, lOp, llq, 18q (2 cases). Tumors had more chromosomal gains of 2q, 3q, 13q than adjacent mucosa and especially gain of 13q in tumor was significantly frequent(53.8% versus 11.5%). Tumor had more losses of 17p(38.5% versus 11.5%) and l6q . Gains of 6q, 7p, Sq. 20p, 20q were observed only in tumor, while chromosomal gains of Ip, Zp, 5p, lOp, lip, 18q and loss of 16p, 17q, 21q was same in tumor and adjacent mucosa. Gains of 6q(7case s) and 2Oq(3cases) were observed exclusively in intestinal type carcinoma, but gain of 2q was observed only in diffuse type. Based on the positions of chromosomal gains or losses, more study for several candidate genes less-well defined in gastric carcinoma, for example , DPI , FLTl , c-myb, AIBI , BTAK, ZNF217, IL-I R, Skp2, PIK3A, telomerase RNA gene and CBP may be needed to clarify gastric carcinogenesis and progression. Our comparative genomic hybridization results indicate the presence of multistep genetic alterations that may play some important role in the development and progression of gastric carcinom a, and suggest possible role of CGH in studying carcinogenic process, in predicting prognosis or therapeutic decision of gastric carcinoma.
6334 MUTATION AND EXPRESSION OF THE P511P63 GENE IN HUMAN COLORECTAL CARCINOMA. Toru Koyama, Kunihiro Hamada, Kimihiro Shimizu, Jun Yokota. Susumu Ohwada, Yasuo Morishita, Gunma Univ Sch of Medicine, Maebashi, Japan ; Bioi Div , Natl Cancer Ctr Research Inst, Tokyo, Japan; Gunma Univ Sch of Medicine, MaebashiMaeba shi, Japan . Background : The 1'51/1'63 (1'51) gene on chromo some 3q28 encodes a protein with significant homology to 1'53, and shares several functions with 1'53, including the transcription al activation of 1'21 and the induction of apoptosis . Some 1'51 isotypes are known to have transdominant activity against 1'53. To elucidate the role of the 1'51 gene in colorectal carcinogenesis, we performed a mutation analysis as well as a detailed analysis of gene expression. Material and Method: High molecular weight DNA was prepared from 96 colorectal carcinomas paired with adj acent non-cancerous tissues. All samples were examined for mutations in the entire coding sequence of the 1'51 gene (exons 2-15 and exon 3' ) as well as the core domain of the 1'53 gene (exons 4-8) by PCR-SSCP analysis and direct sequencing. In 24 cases, total RNA was prepared from tumors and adjacent non-cancer ous tissues, and the levels and types of 1'51 expression were examined by RT-PCR analysis. Results: A missense mutation, which changed the amino acid at codon 240 in exon 6 from serine to proline, was detected in I of 96 primary co lorectal carcinomas. ln addition , genetic polymorphi sms were found at codon 248 in exon 6 and in introns 5 and 8. By RT-PCR , pSI transcripts were detected in all 24 tumors examined . In 10 of 24 cases, p5I was expressed less in tumors than in adjacent noncancerous tissues, and these cases showed a higher incidence of 1'53 mutations . Conclusion: These results indicate that 1'51 does not play a major role in colorectal carcinogenesis as a tumor suppressor. However it is possible that 1'51 expression is downregulated in tumors with 1'53 mutations to repress the function of pSI protein in tumor cells. Further analysis of 1'51 expression and function is necessary to fully understand the role of the pSI gene in human colorectal carcinogenesis.
6335 DETECTION OF CYTOKERA TIN 20-MRNA IN PERIPHERAL BLOOD: A RELIABLE TEST FOR CIRCULATING GASTRIC Ep· ITHELIAL CELLS? Udo Kronberg, Juan Eiki Nishizawa, Concepcion Risueno, Sergio Guzman, Manuel Alvarez, CATHOLIC Univ Sch OF MEDICINE, Santiago , Chile. Burchill described in 1995 a method for the detection of mRNA of cytokeratin 20 (CK20) by a highly specific and sensitive RT-PCR in blood samples in order to determin e its prognostic value in gastrointestinal cancer patients. We used a similar nested RT-PCR to evaluate the impact of
GASTROENTEROLOGY Vol. 118, No.4
biopsy-taking during gastroscopy on cell dissemination in patients with or without gastric cancer. We examined blood samples from 38 patients undergoing gastroscopy, 7 with gastric carcinoma, 31 with benign gastrointestinal disorders. Samples were taken from each patient before starting endoscopy, after the third biopsy, and 15 min after the withdrawal of the endoscope. As a control we examined blood samples from 55 healthy donors. Examining the first sample of the patients, CK20·mRNA was detected in 2/7 patients (28.6%) with gastric cancer, and in 18/31 patients (57.3%) with benign gastrointestinal disease. In II patients, all three consecutive samples were positive, and 6 were all negative ; in 9 patients results became positive in the 2nd and/or 3rd sample. In 9 patients with a positive first sample, results were negative in the consecutive samples, and in 3 patients with negative first sample, the 2nd sample was positive, the 3rd not. Samples of healthy donors were positive in 5155 (9%) cases during the first assay: in repetitions of CK20 RT-PCR (2-5 times) on 25 of these samples. 7/25 (28%) remained negative, whereas 18/25 (72%) were positive at least in one of the repetitions. In conclusion, there is no statistically significant difference between samples taken before and after biopsytaking. Due to the fact that the detection of CK20-mRNA by RT-PCR showed positive results in 72% of blood samples of healthy donors and lacked of reproducibilit y, this method does not permit the evaluation of the presence of circulating gastrointestinal epithelial cells in peripheral blood.
6336 IS THERE A GENETIC PREDISPOSITION OF PATIENTS WITH NON·HEREDITARY CHRONIC PANCREATITIS TO DEVELOP PANCREATIC CANCER? Axel Kropp, Stefan B. Hosch, Wolfram T. Knoefel, Asad Kutup, Vacislav N. Kalinin, Jakob R. Izbicki, Dept of Surg, Univ Hamburg, 20246 Hamburg, Germany . Background : Hereditary chronic pancreatiti s (HP) is an autosomal inherited disease based on a gene-mutation in the trypsinogen-gene mapped to the chromosome 7q35.These patients have an increased risk to develop pancreatic cancer. In this study we screened patients with pancreatic cancer, chronic pancreat itis and patiens who developed pancreatic cancer on chronic pancreatitis. Methods: For detection of the mutations of the trypsinogen gene (Trp 4) we extracteted the DNA from frozen tissue specimens from 55 patients with chronic pancreatitis and 36 patients with pancreatic cancer. 20 patients with pancreatic cancer had a preexisting chronic pancreatitis . With polymerase chain reaction (PCR) and subsequent direct sequencing by big dye terminator kit (Perkin Elmer) we examined exon 2 and 3 as described in the literature. For selective amplification of exon 2 we developed a new intronic primer to amplify a 722 bp fragment. Results: None of the patients displayed a mutation in the cationic trypsinogen gene of R 117 H in exon 3 or N21 1- mutation in exon 2. Also in the sequence-analysis no mutations of the cationic trypsinogen gene (A I6V) and no further mutations were found Conclusions: Our results emphasize the specificity of the mutation in the cationic trypsinogen gene for hereditary pancreat itis. In patients where pancreatic cancer had developed in chronic non hereditary pancreatitis the evaluated mutations seem to be irrelevant. Thus the risk to develop cancer in chronic non hereditary pancreatitis cannot be estimated by screening for these mutations
6337 DIAGNOSTIC ACCURACY OF ENDOCAVITAL ULTRASONOG· RAPHY (ENDOSONOGRAPHY) IN STAGING OF GI MALIGNANCIES. Jan Kulig, Wojciech Nowak, Piotr Kolodziejczyk, Bartlomiej Zarebski. Tadeusz Popiela, 1st Dept of Gen and GI Surg, Jagiellonian Univ, Krakow, Poland. Objective.Adequate preoperative and intraoperative staging is crucial for the planning of successful multimodal management of GI malignancies . The aim of the study was to assess diagnostic accuracy of endocavital ultrasonography (ECUS) including endoscopic (EUS), endorectal (ERUS), intraoperative (IOUS) intraductal (IDU), and laparoscopic (LUS) ultrasonography in the staging of patients with GI tumors . Method :2628 ECUS examinations (EUS - 375, ERUS - 1190, 10US - 1004, LUS -35, IDU - 24) were performed in patients undergoing surgery for GI malignant tumors. The results were compared with intraoperati ve findings, and final pathologic reports. The TNM classification was used to evaluate locoregional spread of cancer. Results:Accuracy of ECUS for determin ing tumor size and depth of invasion (T) depended on the site of cancer and was: 84.1% for esophagus, 78.5% for stomach, 99.4 % for liver, 85.3% for pancreas, 87.0 % for rectum, and 91% for other abdominal tumors. The overall accuracy of ECUS for the detection of lymph nodes metastases (N) ranged from 75.8% to 87.5% depend ing on the site and applied techniques ( or their combination). Conclusions: Combination of ECUS methods are useful for staging of GI malignancies and should become routine procedures in planning of the most accurate treatment.