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Lectin staining patterns in models of gastrointestinal neoplasia
AGAA1387
April 2000
polyposis or cancer. In all subjects basal GH, Insulin Growth Factor-I (IGF- I), maximal suppresion of GH (maxGH) and plasma insulin after a standarized meal were measured and a colonoscopy was performed . All patients had been diagnosed by tipical symptom s, increased GH (greater than 2 nglml after oral glucose overload) and increased levels of IGF-l. Results: 19 patients(50%) had polyps (8 mill 1):15 (39,5 %) adenomatou s and 4 (10,5%) hyperplasic . 19 patients (50 %) had no polyps (9 milO I) . One patient with adenomatous polyps had colonic carcinoma (2.6 %). In a control group (subjects referred to colonoscopy for other reasons with the same age and sex) the prevalence of polyps was 25 %. Conclusion s: The prevalence of polyps and colon cancer in active acromegaly patients is higher than in the general population and in the subjects referred to colonoscopy for other reasons. Except for age, no significant differences were observed in the risk factors studied. We recommend a colonoscop y examination in acromegalic patients, especially those over 50 years of age.
Age (years) Sel (male/female) Disease duration (years) Acrocordons Fasting GH (ng/ml) Fasting IG·1 (ng/ml)
Polyposis+
Polyposis.
p
57.6 8/11 12,3 21,4 9,2±5,2 871+222
49.8
0.01 5
9/10
NS NS NS NS NS
11 .1 15,2 25,4±24,4 825±353
adenomas . MATERIALS AND METHODS: The samples were obtained from 24 colorectal adenomas ( 16 male, mean age 59.3, low grade atypia 12, high grade atypia 12), and its adjacent normal mucosa by surgery or endoscopic mucosal resection . Serial sections (4-JLm) were cut from paraffin-embedded blocks. After activation of antigen with microwave oven, the sections were immunostained using anti-human CQX-2 antibod y and anti-human chromogranin A antibody according to streptavidin biotin method. RESULTS : The immunoreactivity of COX-2 was detected in a few cells of a gland in normal colorectal epitherium (24/24). The immunoreactive products showed granular appearance in the cytoplasm of spindle epitherial cells, and moreover the COX-2 positive cells corresponded in part to chromogranin A positive cells, suggesting that COX-2 positive cells were endocrine cells. In the lamina propria , COX-2 staining was detevtcd in macrophages and fibroblasts. In colorectal adenomas, 14 of 24 tumors showed a characteristic finding: hyperplasia of endocrine-like cells showing positive immunoreactivity of COX-2 ; whereas 10 of 24 tumors were negative. Only a small percent of COX-2 positive endocrine-like cells corresponded to chromogranin A positive cells in colorectal adenomas. No significant correlation was found between the grade of cell atypia and COX-2 expression in adenoma . CONCLUSIONS: we showed that the CQX-2 immunoreactive products showing granular appearance were detected not only in normal endocrine cells but also in endocrine-like cells of colorectal adenomas. Although the role of endocrine-like cells is unknown , the COX-2 produced in these cells may promote the growth of colorectal tumors.
6305
6307
LECTIN STAINING PATTERNS IN MODELS OF GASTROINTES· TINAL NEOPLASIA. Anthony John FitzGerald, Nicholas A. Wright, Robert A. Goodlad , Imperial Coil Sch Of Medicine, London, United Kingdom; ICSM, London, United Kingdom; lerf, London , United Kingdom.
HIGH DETECTION RATE OF ABERRANT METHYLATION OF PI6 GENE IN THE SERUMIPLASMA IN PATIENTS WITH COLORECTAL CANCER. Kaori Fujiya, Toshifumi Ashida, Atsuo Maemoto, Fumika Orii, Takanori Fujiki , Mikihiro Fujiya, Katsuya Einami, Jiroh Watari, Yusuke Saitoh, Yoshimi Shibata, Takeshi Obara, Yutaka Kohgo, Asahikawa Med Coli, Asahikawa, Japan ,
Background : Lectins are highly specific carbohydrate binding proteins . One lectin is peanut agglutinin (PNA) which binds to galactosej3-I,3 -Nacetyl galactosamine-a, the Thomsen Friedenreich (TF ) antigen. This antigen is upregulated in inflammatory bowel diseases, hyperplastic and adenomatous polyps and also colorectal cancer. Lectins are prevalent in our diets. resist digestion, and pass through the gastrointestinal tract where they remain active and can have biological activity in both the small intestine and in the colon. Methods: We investigated the binding of PNA and other lectins, in two experimenta l models of carcinogenesis. The 1,2-dimethylhydrazine (DMH) treated rat, for the formation of colonic tumours and the multiple intestinal neoplasia (MIN) mouse, for the formation of intestinal polyps. Both these models were used in feeding experiments in which a standard chow was supplemented with low doses of various lectins. Results: PNA staining of intestinal polyps and colonic tumours showed unique patterns associated with changes in surface carbohydrate expression, within the tumour but also upon the adjacent yet otherwise normal epithelium . The various lectins showed different staining specificities in respect to surface carbohydrate expression and secreted mucin levels. Specific staining increased as the polyp or tumour progressed . Conclusion: Changes in carbohydrate expression can be useful in the identification of premalignant gastrointestinal epithelium, especially in high-risk subjects; moreover, the biological effects of lectins binding vary as the tissue becomes more neoplastic. 6306 IMMUNOHISTOCHEMICAL EXPRESSION OF CYCLOOXY· GENASE·2 IN COLORECTAL ADENOMAS. Mikio Fujita, Hirokazu Fukui, Toshihiro Kusaka, Tetsuya Nakamura, Yoshihiko Ueda, Takahiro Fujimori, Dokkyo Univ Sch of Medicine, Tochigi, Japan; Kobe National Hosp, Kobe, Japan. BACKGROUNDS : Cycloo xygenase (COX)-2 is recently reported to promote cell growth or inhibit apoptosis. COX-2 is expressed in colorectal cancers, and suggested to associate with its progression. However, immunohistochemical study of COX-2 is not performed on colorectal adenoma s, which are precancerou s lesions . Moreover, the immunohistochemical localization of COX-2 is not clarified in both colorectal normal epitherial cells and adenomas, although COX-2 is prelimina ry reported to express in normal colorectal endocrine cells. In the present study, we showed COX-2 expression immunohistochemicall y in colorectal normal epitherium and
Background!Aim: p16, which is one of G I-specific cell cycle inhibitors, is one of the most commonly detected molecular defects in cancer. The suppressive mechanism of pl6 gene is now considered to be due to an aberrant methylation of CpG islands on promoter region . In colon cancer cell line, pI6 gene methylation is reported to be uniformly detectable. However, using tissue sections, the frequency of p16 gene methylation is reported to be relatively low (55%, RJ Guan, Gastroenterology 1999). Low detection rate of the pl6 methylation may be due to the heterogeneity of sampled tumor tissues . In this study, we purposed to estimate the incidence of pl6 methylation in colon cancer tissue, by examining DNA came from the tumor tissue into circulating blood of host patients with methylation specific PCR (MSP) method. Materials !Methods: Plasma/serum samples were collected from preoperative patients with colorectal cancer. DNAs dissolved in plasma/serum samples were extracted using a QIAamp DNA Kit (Qiagen,Hilden,Germany). Extracted DNAs were treated with CpGenome DNA Modification Kit (Intergen), to convert unmethylated cytosine to uracil (Bisulfite conversion ). pl6 promoter region (exonl 25 to 175) in converted DNA samples were then amplified by MSP, using specific primer pairs corresponding to unmethylated and methylated sequences. To confirm the bisulfite conversion , wild sequence of this region was also amplified. Suppressed expression of p 16 in cancer tissues were examined by immunohistochemical staining of biopsied specimens taken from those patients, using anti-p l 6 monoclonal antibody (Santa Cruz Biotechnology) . Results: We have analyzed blood samples and cancer tissues of 10 patients with colorectal cancer treated in our hospital. Clinical stages of these patients were varied stage 2 to 4 (stage 2; 3 patients, stage 3; 2 patients, stage 4 ; 5 patients). By MSP experiment , we have found that DNA fragment f~om aberrantl y methylated promoter region of pl6 gene was detectable In plasma/serum taken from 80% (8/ 10) of the patients. However, only 4 tissue samples, 50% (4/8) taken from the patients who have methylated p 16 DNA fragment in their blood, showed reduced p16 protein expression. Conclusions Through these results, we can hypothesize that aberrant methylation of p16 gene is a more common genetic abnormalit y of colorectal cancer than expected. This also raises the possibility that MSP of pl6 gene with plasma/ serum DNA will be an alternative screening method for colorectal cancer.
April 2000
polyposis or cancer. In all subjects basal GH, Insulin Growth Factor-I (IGF- I), maximal suppresion of GH (maxGH) and plasma insulin after a standarized meal were measured and a colonoscopy was performed . All patients had been diagnosed by tipical symptom s, increased GH (greater than 2 nglml after oral glucose overload) and increased levels of IGF-l. Results: 19 patients(50%) had polyps (8 mill 1):15 (39,5 %) adenomatou s and 4 (10,5%) hyperplasic . 19 patients (50 %) had no polyps (9 milO I) . One patient with adenomatous polyps had colonic carcinoma (2.6 %). In a control group (subjects referred to colonoscopy for other reasons with the same age and sex) the prevalence of polyps was 25 %. Conclusion s: The prevalence of polyps and colon cancer in active acromegaly patients is higher than in the general population and in the subjects referred to colonoscopy for other reasons. Except for age, no significant differences were observed in the risk factors studied. We recommend a colonoscop y examination in acromegalic patients, especially those over 50 years of age.
Age (years) Sel (male/female) Disease duration (years) Acrocordons Fasting GH (ng/ml) Fasting IG·1 (ng/ml)
Polyposis+
Polyposis.
p
57.6 8/11 12,3 21,4 9,2±5,2 871+222
49.8
0.01 5
9/10
NS NS NS NS NS
11 .1 15,2 25,4±24,4 825±353
adenomas . MATERIALS AND METHODS: The samples were obtained from 24 colorectal adenomas ( 16 male, mean age 59.3, low grade atypia 12, high grade atypia 12), and its adjacent normal mucosa by surgery or endoscopic mucosal resection . Serial sections (4-JLm) were cut from paraffin-embedded blocks. After activation of antigen with microwave oven, the sections were immunostained using anti-human CQX-2 antibod y and anti-human chromogranin A antibody according to streptavidin biotin method. RESULTS : The immunoreactivity of COX-2 was detected in a few cells of a gland in normal colorectal epitherium (24/24). The immunoreactive products showed granular appearance in the cytoplasm of spindle epitherial cells, and moreover the COX-2 positive cells corresponded in part to chromogranin A positive cells, suggesting that COX-2 positive cells were endocrine cells. In the lamina propria , COX-2 staining was detevtcd in macrophages and fibroblasts. In colorectal adenomas, 14 of 24 tumors showed a characteristic finding: hyperplasia of endocrine-like cells showing positive immunoreactivity of COX-2 ; whereas 10 of 24 tumors were negative. Only a small percent of COX-2 positive endocrine-like cells corresponded to chromogranin A positive cells in colorectal adenomas. No significant correlation was found between the grade of cell atypia and COX-2 expression in adenoma . CONCLUSIONS: we showed that the CQX-2 immunoreactive products showing granular appearance were detected not only in normal endocrine cells but also in endocrine-like cells of colorectal adenomas. Although the role of endocrine-like cells is unknown , the COX-2 produced in these cells may promote the growth of colorectal tumors.
6305
6307
LECTIN STAINING PATTERNS IN MODELS OF GASTROINTES· TINAL NEOPLASIA. Anthony John FitzGerald, Nicholas A. Wright, Robert A. Goodlad , Imperial Coil Sch Of Medicine, London, United Kingdom; ICSM, London, United Kingdom; lerf, London , United Kingdom.
HIGH DETECTION RATE OF ABERRANT METHYLATION OF PI6 GENE IN THE SERUMIPLASMA IN PATIENTS WITH COLORECTAL CANCER. Kaori Fujiya, Toshifumi Ashida, Atsuo Maemoto, Fumika Orii, Takanori Fujiki , Mikihiro Fujiya, Katsuya Einami, Jiroh Watari, Yusuke Saitoh, Yoshimi Shibata, Takeshi Obara, Yutaka Kohgo, Asahikawa Med Coli, Asahikawa, Japan ,
Background : Lectins are highly specific carbohydrate binding proteins . One lectin is peanut agglutinin (PNA) which binds to galactosej3-I,3 -Nacetyl galactosamine-a, the Thomsen Friedenreich (TF ) antigen. This antigen is upregulated in inflammatory bowel diseases, hyperplastic and adenomatous polyps and also colorectal cancer. Lectins are prevalent in our diets. resist digestion, and pass through the gastrointestinal tract where they remain active and can have biological activity in both the small intestine and in the colon. Methods: We investigated the binding of PNA and other lectins, in two experimenta l models of carcinogenesis. The 1,2-dimethylhydrazine (DMH) treated rat, for the formation of colonic tumours and the multiple intestinal neoplasia (MIN) mouse, for the formation of intestinal polyps. Both these models were used in feeding experiments in which a standard chow was supplemented with low doses of various lectins. Results: PNA staining of intestinal polyps and colonic tumours showed unique patterns associated with changes in surface carbohydrate expression, within the tumour but also upon the adjacent yet otherwise normal epithelium . The various lectins showed different staining specificities in respect to surface carbohydrate expression and secreted mucin levels. Specific staining increased as the polyp or tumour progressed . Conclusion: Changes in carbohydrate expression can be useful in the identification of premalignant gastrointestinal epithelium, especially in high-risk subjects; moreover, the biological effects of lectins binding vary as the tissue becomes more neoplastic. 6306 IMMUNOHISTOCHEMICAL EXPRESSION OF CYCLOOXY· GENASE·2 IN COLORECTAL ADENOMAS. Mikio Fujita, Hirokazu Fukui, Toshihiro Kusaka, Tetsuya Nakamura, Yoshihiko Ueda, Takahiro Fujimori, Dokkyo Univ Sch of Medicine, Tochigi, Japan; Kobe National Hosp, Kobe, Japan. BACKGROUNDS : Cycloo xygenase (COX)-2 is recently reported to promote cell growth or inhibit apoptosis. COX-2 is expressed in colorectal cancers, and suggested to associate with its progression. However, immunohistochemical study of COX-2 is not performed on colorectal adenoma s, which are precancerou s lesions . Moreover, the immunohistochemical localization of COX-2 is not clarified in both colorectal normal epitherial cells and adenomas, although COX-2 is prelimina ry reported to express in normal colorectal endocrine cells. In the present study, we showed COX-2 expression immunohistochemicall y in colorectal normal epitherium and
Background!Aim: p16, which is one of G I-specific cell cycle inhibitors, is one of the most commonly detected molecular defects in cancer. The suppressive mechanism of pl6 gene is now considered to be due to an aberrant methylation of CpG islands on promoter region . In colon cancer cell line, pI6 gene methylation is reported to be uniformly detectable. However, using tissue sections, the frequency of p16 gene methylation is reported to be relatively low (55%, RJ Guan, Gastroenterology 1999). Low detection rate of the pl6 methylation may be due to the heterogeneity of sampled tumor tissues . In this study, we purposed to estimate the incidence of pl6 methylation in colon cancer tissue, by examining DNA came from the tumor tissue into circulating blood of host patients with methylation specific PCR (MSP) method. Materials !Methods: Plasma/serum samples were collected from preoperative patients with colorectal cancer. DNAs dissolved in plasma/serum samples were extracted using a QIAamp DNA Kit (Qiagen,Hilden,Germany). Extracted DNAs were treated with CpGenome DNA Modification Kit (Intergen), to convert unmethylated cytosine to uracil (Bisulfite conversion ). pl6 promoter region (exonl 25 to 175) in converted DNA samples were then amplified by MSP, using specific primer pairs corresponding to unmethylated and methylated sequences. To confirm the bisulfite conversion , wild sequence of this region was also amplified. Suppressed expression of p 16 in cancer tissues were examined by immunohistochemical staining of biopsied specimens taken from those patients, using anti-p l 6 monoclonal antibody (Santa Cruz Biotechnology) . Results: We have analyzed blood samples and cancer tissues of 10 patients with colorectal cancer treated in our hospital. Clinical stages of these patients were varied stage 2 to 4 (stage 2; 3 patients, stage 3; 2 patients, stage 4 ; 5 patients). By MSP experiment , we have found that DNA fragment f~om aberrantl y methylated promoter region of pl6 gene was detectable In plasma/serum taken from 80% (8/ 10) of the patients. However, only 4 tissue samples, 50% (4/8) taken from the patients who have methylated p 16 DNA fragment in their blood, showed reduced p16 protein expression. Conclusions Through these results, we can hypothesize that aberrant methylation of p16 gene is a more common genetic abnormalit y of colorectal cancer than expected. This also raises the possibility that MSP of pl6 gene with plasma/ serum DNA will be an alternative screening method for colorectal cancer.