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Apoptosis and cell proliferation in various macroscopic types of intramucosal colorectal carcinoma
April 1998 Conclusion: Dietary intake of fiber and fat does not appear to affect colonic mucosal levels of GST and QR, although these enzymes are more highly expressed in the sigmoid colon. • G2839 THE ROLE OF c-kit MUTATIONS IN HUMAN GASTROINTESTINAL STROMAL TUMORS. M. Taniguchi, T. Nishida, T. Ito, T. Nomura, S. Hirota, Y. Kitamura, H. Matsuda, Osaka, Japan.
The origin and diagnosis of gastrointestinal stromal tumor (GIST) are unknown and still controversial. CD34 was reported to be a good molecular marker of GIST. Recently, we reported gain-of-function mutations in c-kit expressed in GIST (Hirota et al. Science 1997 accepted). However, the role and impact on the morbidity and mortality of mutated c-kit are under investigation. AIM: The aim of the present study is to clarify a role of mutations in c-kit of GIST and its effect on recurrences and the prognosis. METHODS: c-kit in extracted DNA and/or RNA from surgically resected GISTs of 75 patients (detail locus; 1 esophagus, 53 stomach, 6 duodenum, 12 small intestine and 3 colon) were amplified by polymerase chain reaction. The products were analyzed by single strand conformation polymorphysm and sequenced. RESULTS: In immunohistochemistory, c-kit was overexpressed in 64 cases (85%), which usually co-expressed with CD34 and vimentin, but not with ¢t -smooth muscle actin, desmin and S-100. Mutations of c-kit gene were detected in 34 patients (22 deletions, 7 point mutations, 2 deletions and insertion, 2 point mutations and insertions, 1 point mutations and insertions) and were similar to previously reported gain-of-function mutations. Mutations limitedly occurred between Codon 550 and 560 in Exon 11, a juxtamembrane domain. The presence of c-kit mutations did not show any correlation to age, gender, locus, size, cell type, the mitosis number, and cellularity. Recurrences were observed in 21 patients and detail recurrent sites were 5 local recurrences, 13 liver metastasis and 3 peritonitis carcinomatosis. The recurrence rate of GISTs with mutated c-kit (79%) was significantly higher than that of GISTs without mutation (15%) (p=0.034). The 5-year survival rates of GISTs with and without mutated c-kit were 63% and 90%, respectively, and the prognosis of GISTs without mutations was significantly better than that of GISTs with mutations (p=0.0358). CONCLUSIONS: These results suggest that mutations in proto-oncogene c-kit are associated with poor prognosis and may confer malignant transformation on GIST and that c-kit could be a good prognostic factor of GIST. • G2840 APOPTOSIS AND CELL PROLIFERATION IN VARIOUS MACROSCOPIC TYPES OF INTRAMUCOSAL COLORECTAL CARCINOMA. T Tanimoto 1, S Tanaka 1, K Haruma l, Y Kitadai 1, M Yoshihara1, K Sumii 1, G Kajiyama1, F Shimamoto2. 1GI Unit, First Department of Internal Medicine, Hiroshima University School of Medicine, 2Department of Pathology, Hiroshima University Hospital, Hiroshima, Japan
Apoptotic cell death and cell proliferation play important roles in the histogenesis and development of colorectal carcinoma (CRC). The AIM of this study was to examine the relationship between apoptosis and cell proliferation in various macroscopic types of intramucosal CRC in relation to the expression of p53 and bcl-2. METHODS: One hundred forty cases with endoscopically or surgically resected intramucosal CRC were studied. These were 57 cases of polypoid type carcinoma, 55 superficial type carcinoma, and 28 granular type laterally spreading tumor (G-LST). Polypoid type carcinomas were pedunculated, subpedunculated, or sessile polyps. Superficial type carcinomas were fiat lesions with a smooth and even surface. G-LST were superficially spreading lesions with aggregates of nodules and a granular surface. Apoptotic cells were identified by the in situ DNA nick end labeling method. Ki-67, p53, and bcl-2 expression were examined immunohistochemically. RESULTS: The superficial type carcinoma apoptotic index was significantly (p<0.01) lower than that of polypoid type carcinoma and G-LST. The superficial type carcinoma proliferative index was significantly (p<0.01) higher than that of polypoid type carcinoma and G-LST. In superficial type carcinomas the incidence of high proliferative index lesions with p53 expression was significantly (p<0.05) higher than that without p53 expression, and the incidence of high apoptotic index lesions in carcinomas with lower proliferative index was higher than that in carcinomas with high proliferative index (p<0.05). There was no significant difference in apoptotic index, proliferative index, and p53 protein overexpression between de novo carcinomas and those that had arisen in precursor adenomas. CONCLUSIONS: The pattern of cell death and proliferation may vary with different macroscopic types of intramucosai CRC, and this may be useful in understanding their different biologic behavior. • G2841 HIGH SALT DIET INCREASES H E L I C O B A C T E R P Y L O R I COLONIZATION IN C57B1/6 MICE. N.S. Taylor, J.G. Fox, C.A. Dangler, MIT, Cambridge, MA 02139; T. Wang, MGH, Boston, MA 02114.
A high salt diet in humans and experimental animals is known to cause gastritis, and has been associated with a high risk of atrophic gastritis. When administered simultaneously, salt enhances the carcinogenic effects of known gastric carcinogens in experimental animals. While salt is not an initiator of
Gastrointestinal Oncology A687
carcinogenesis, it has been postulated that its role as a co-carcinogen is due to its capacity to break down the mucus barrier and increase cellular exposure to carcinogens. Since long term colonization with Helicobacter pylori (Hp) has been associated with a progression from gastritis to gastric cancer, this study was designed to determine if excessive dietary NaC1 would have an effect on colonization in the mouse model of Hp infection. Seventy-two 8 week old C57B/6 mice were infected with Hp strain Sydney. Thirty-six control animals were dosed with vehicle only. Half of the infected and control animals were fed a high salt diet (7.5% vs 0.25%) for 2 weeks prior to dosing. Twelve infected and 6 control animals from high salt and normal diet groups were sacrificed at 4, 8, and 16 weeks. Quantitative urease assay and culture, and hist01ogic analysis were carried out on gastric tissue from the antrum and body of each animal. At 4,8, and 16 weeks post infection (PI) colony forming units/mg tissue (cfu) were significantly higher (p<0.05) in the body of animals in the high salt diet group compared to normal diet. At 8 weeks PI, cfu were also significantly higher (p<0.05) in the antrum of animals on the high salt diet. Quantitative urease was significantly higher (p<0.05) in all tissues at all time points in animals on the high salt diet when compared to controls, except in the antrum at 16 weeks. WeeksPI Hpinfected
4 I 8 I 16 Body Antrtma Body AntIum ] Body ColonyFormingUnitshngTissue Norm, Diet 1809±8789 1326±192511071±36381 277:1:354 ] 70±934 618±779 5243±6612 2305±6079 5149±5585 3551±4261 766±6413 1747±4945 QuantitativeUrease NormalDiet 10.4±12.9 6.5±4.9 3 . 9 ± 1 1 9.7±9.5 0.7±1.2 ] 1.9-+3.4 HighSaltDiet 31.9±18.7 25.7±31.1 27.3±32.6 18.6±25.4 8.2±16.3 [ 29.5-+54.7 Anlnma [
I
r~s~,tDi~t]
Mice in both the normal and the high salt diet groups developed marked atrophic gastritis of the fundus in response to Hp infection. However, the foveolae of the fundic mucosa in mice on the high salt diet were elongated and colonized by argyrophilic bacteria resembling Hp more frequently, compared to mice on the normal diet. We conclude that excessive NaCI intake enhances Hp colonization. In humans, chronic salt intake may exacerbate gastritis by increasing Hp colonization.. • G2842 K-RAS MUTATION, P-53 AND C-ERBB-2 OVER-EXPRESSION IN THE NEOPLASTIC PROGRESSION OF INTRADUCTAL PAPILLARYMUCINOUS TUMORS OF THE PANCREAS. S Taylor*, A Gown*, LW Traverso++, R Kozarek+, T Brentnall**, MP Bronner*. Departments of *Pathology and **Gastroenterology, University of Washington, Seatde; and sections of +Gastroenterology and ++General, Thoracic, and Vascular Surgery, Virginia Mason Medical Center, Seattle, WA.
Background: Intraductal papillary-mutinous tumors (IPMT) of the pancreas comprise neoplasms formerly classified as two distinct types of tumors: mucinous ductal ectasia and intraductal papillary neoplasms. Although slowgrowing, decisions regarding clinical management of these lesions can be difficult, as they often extensively involve the ductal system and contain a spectrum of histopathologic abnormalities, ranging from hyperplasia to invasive adenocarcinoma. Aim: The aim of this study is to determine if one or more molecular markers, including K-ras mutation and p53 and c-erbB-2 over-expression, may improve clinical decision making by predicting the stage of neoplastic progression in IPMT. Methods: 41 sites from 30 patients with IPMT were microdissected to include a spectrum of ductai histopathology, including hyperplasia, low-grade dysplasia ("adenoma" or "atypical hyperplasia"), high-grade dysplasia ("carcinoma in-situ"), and invasive adenocarcinoma. DNA was extracted and the presence of K-ras mutation at codon 12 determined by a PCR with BstN-1 restriction enzyme assay. Over-expression of p53 and c-erbB-2 was determined by standard immunohistochemistry. Results: Highest Histologie Grade Per Hyperplasia Low-grade dysplasia K-ras mutation 0/3 (0%) 10/13 (77%) p53 overexpression 0/3 (0%) 0/11 (0%) c-erbB-2over1/3 (33%) 4/11 (36%) expression
Patient High-grade Invasion dysplasia 6/6 (100%) 6/8 (75%) 0/4 (0%) 7/11 (64%) 1/4 (25%) 5/11 (45%)
Con~lusion~; K-ras mutations were present in the majority of IPMT cases with neoplastic progression, while cases of hyperplasia did not have K-ras mutations (p < 0.0138, Fisher's exact test). The over-expression of c-erbB-2 was found in all stages of progression and was not significantly associated with malignancy. Over-expression of p53 is significantly associated with invasive malignancy (p <_ 0.0002, Fisher's exact test), p53 over-expression was not seen in hyperplastic, low-grade or high-grade dysplastic lesions. These data taken together suggest that K-ras mutation is a very early event in IPMT neoplastic progression, while p53 mutation occurs later, at the highgrade dysplasia ---> invasive carcinoma transition. The detection of p53 mutations may prove useful in predicting malignant behavior in these tumors.
The origin and diagnosis of gastrointestinal stromal tumor (GIST) are unknown and still controversial. CD34 was reported to be a good molecular marker of GIST. Recently, we reported gain-of-function mutations in c-kit expressed in GIST (Hirota et al. Science 1997 accepted). However, the role and impact on the morbidity and mortality of mutated c-kit are under investigation. AIM: The aim of the present study is to clarify a role of mutations in c-kit of GIST and its effect on recurrences and the prognosis. METHODS: c-kit in extracted DNA and/or RNA from surgically resected GISTs of 75 patients (detail locus; 1 esophagus, 53 stomach, 6 duodenum, 12 small intestine and 3 colon) were amplified by polymerase chain reaction. The products were analyzed by single strand conformation polymorphysm and sequenced. RESULTS: In immunohistochemistory, c-kit was overexpressed in 64 cases (85%), which usually co-expressed with CD34 and vimentin, but not with ¢t -smooth muscle actin, desmin and S-100. Mutations of c-kit gene were detected in 34 patients (22 deletions, 7 point mutations, 2 deletions and insertion, 2 point mutations and insertions, 1 point mutations and insertions) and were similar to previously reported gain-of-function mutations. Mutations limitedly occurred between Codon 550 and 560 in Exon 11, a juxtamembrane domain. The presence of c-kit mutations did not show any correlation to age, gender, locus, size, cell type, the mitosis number, and cellularity. Recurrences were observed in 21 patients and detail recurrent sites were 5 local recurrences, 13 liver metastasis and 3 peritonitis carcinomatosis. The recurrence rate of GISTs with mutated c-kit (79%) was significantly higher than that of GISTs without mutation (15%) (p=0.034). The 5-year survival rates of GISTs with and without mutated c-kit were 63% and 90%, respectively, and the prognosis of GISTs without mutations was significantly better than that of GISTs with mutations (p=0.0358). CONCLUSIONS: These results suggest that mutations in proto-oncogene c-kit are associated with poor prognosis and may confer malignant transformation on GIST and that c-kit could be a good prognostic factor of GIST. • G2840 APOPTOSIS AND CELL PROLIFERATION IN VARIOUS MACROSCOPIC TYPES OF INTRAMUCOSAL COLORECTAL CARCINOMA. T Tanimoto 1, S Tanaka 1, K Haruma l, Y Kitadai 1, M Yoshihara1, K Sumii 1, G Kajiyama1, F Shimamoto2. 1GI Unit, First Department of Internal Medicine, Hiroshima University School of Medicine, 2Department of Pathology, Hiroshima University Hospital, Hiroshima, Japan
Apoptotic cell death and cell proliferation play important roles in the histogenesis and development of colorectal carcinoma (CRC). The AIM of this study was to examine the relationship between apoptosis and cell proliferation in various macroscopic types of intramucosal CRC in relation to the expression of p53 and bcl-2. METHODS: One hundred forty cases with endoscopically or surgically resected intramucosal CRC were studied. These were 57 cases of polypoid type carcinoma, 55 superficial type carcinoma, and 28 granular type laterally spreading tumor (G-LST). Polypoid type carcinomas were pedunculated, subpedunculated, or sessile polyps. Superficial type carcinomas were fiat lesions with a smooth and even surface. G-LST were superficially spreading lesions with aggregates of nodules and a granular surface. Apoptotic cells were identified by the in situ DNA nick end labeling method. Ki-67, p53, and bcl-2 expression were examined immunohistochemically. RESULTS: The superficial type carcinoma apoptotic index was significantly (p<0.01) lower than that of polypoid type carcinoma and G-LST. The superficial type carcinoma proliferative index was significantly (p<0.01) higher than that of polypoid type carcinoma and G-LST. In superficial type carcinomas the incidence of high proliferative index lesions with p53 expression was significantly (p<0.05) higher than that without p53 expression, and the incidence of high apoptotic index lesions in carcinomas with lower proliferative index was higher than that in carcinomas with high proliferative index (p<0.05). There was no significant difference in apoptotic index, proliferative index, and p53 protein overexpression between de novo carcinomas and those that had arisen in precursor adenomas. CONCLUSIONS: The pattern of cell death and proliferation may vary with different macroscopic types of intramucosai CRC, and this may be useful in understanding their different biologic behavior. • G2841 HIGH SALT DIET INCREASES H E L I C O B A C T E R P Y L O R I COLONIZATION IN C57B1/6 MICE. N.S. Taylor, J.G. Fox, C.A. Dangler, MIT, Cambridge, MA 02139; T. Wang, MGH, Boston, MA 02114.
A high salt diet in humans and experimental animals is known to cause gastritis, and has been associated with a high risk of atrophic gastritis. When administered simultaneously, salt enhances the carcinogenic effects of known gastric carcinogens in experimental animals. While salt is not an initiator of
Gastrointestinal Oncology A687
carcinogenesis, it has been postulated that its role as a co-carcinogen is due to its capacity to break down the mucus barrier and increase cellular exposure to carcinogens. Since long term colonization with Helicobacter pylori (Hp) has been associated with a progression from gastritis to gastric cancer, this study was designed to determine if excessive dietary NaC1 would have an effect on colonization in the mouse model of Hp infection. Seventy-two 8 week old C57B/6 mice were infected with Hp strain Sydney. Thirty-six control animals were dosed with vehicle only. Half of the infected and control animals were fed a high salt diet (7.5% vs 0.25%) for 2 weeks prior to dosing. Twelve infected and 6 control animals from high salt and normal diet groups were sacrificed at 4, 8, and 16 weeks. Quantitative urease assay and culture, and hist01ogic analysis were carried out on gastric tissue from the antrum and body of each animal. At 4,8, and 16 weeks post infection (PI) colony forming units/mg tissue (cfu) were significantly higher (p<0.05) in the body of animals in the high salt diet group compared to normal diet. At 8 weeks PI, cfu were also significantly higher (p<0.05) in the antrum of animals on the high salt diet. Quantitative urease was significantly higher (p<0.05) in all tissues at all time points in animals on the high salt diet when compared to controls, except in the antrum at 16 weeks. WeeksPI Hpinfected
4 I 8 I 16 Body Antrtma Body AntIum ] Body ColonyFormingUnitshngTissue Norm, Diet 1809±8789 1326±192511071±36381 277:1:354 ] 70±934 618±779 5243±6612 2305±6079 5149±5585 3551±4261 766±6413 1747±4945 QuantitativeUrease NormalDiet 10.4±12.9 6.5±4.9 3 . 9 ± 1 1 9.7±9.5 0.7±1.2 ] 1.9-+3.4 HighSaltDiet 31.9±18.7 25.7±31.1 27.3±32.6 18.6±25.4 8.2±16.3 [ 29.5-+54.7 Anlnma [
I
r~s~,tDi~t]
Mice in both the normal and the high salt diet groups developed marked atrophic gastritis of the fundus in response to Hp infection. However, the foveolae of the fundic mucosa in mice on the high salt diet were elongated and colonized by argyrophilic bacteria resembling Hp more frequently, compared to mice on the normal diet. We conclude that excessive NaCI intake enhances Hp colonization. In humans, chronic salt intake may exacerbate gastritis by increasing Hp colonization.. • G2842 K-RAS MUTATION, P-53 AND C-ERBB-2 OVER-EXPRESSION IN THE NEOPLASTIC PROGRESSION OF INTRADUCTAL PAPILLARYMUCINOUS TUMORS OF THE PANCREAS. S Taylor*, A Gown*, LW Traverso++, R Kozarek+, T Brentnall**, MP Bronner*. Departments of *Pathology and **Gastroenterology, University of Washington, Seatde; and sections of +Gastroenterology and ++General, Thoracic, and Vascular Surgery, Virginia Mason Medical Center, Seattle, WA.
Background: Intraductal papillary-mutinous tumors (IPMT) of the pancreas comprise neoplasms formerly classified as two distinct types of tumors: mucinous ductal ectasia and intraductal papillary neoplasms. Although slowgrowing, decisions regarding clinical management of these lesions can be difficult, as they often extensively involve the ductal system and contain a spectrum of histopathologic abnormalities, ranging from hyperplasia to invasive adenocarcinoma. Aim: The aim of this study is to determine if one or more molecular markers, including K-ras mutation and p53 and c-erbB-2 over-expression, may improve clinical decision making by predicting the stage of neoplastic progression in IPMT. Methods: 41 sites from 30 patients with IPMT were microdissected to include a spectrum of ductai histopathology, including hyperplasia, low-grade dysplasia ("adenoma" or "atypical hyperplasia"), high-grade dysplasia ("carcinoma in-situ"), and invasive adenocarcinoma. DNA was extracted and the presence of K-ras mutation at codon 12 determined by a PCR with BstN-1 restriction enzyme assay. Over-expression of p53 and c-erbB-2 was determined by standard immunohistochemistry. Results: Highest Histologie Grade Per Hyperplasia Low-grade dysplasia K-ras mutation 0/3 (0%) 10/13 (77%) p53 overexpression 0/3 (0%) 0/11 (0%) c-erbB-2over1/3 (33%) 4/11 (36%) expression
Patient High-grade Invasion dysplasia 6/6 (100%) 6/8 (75%) 0/4 (0%) 7/11 (64%) 1/4 (25%) 5/11 (45%)
Con~lusion~; K-ras mutations were present in the majority of IPMT cases with neoplastic progression, while cases of hyperplasia did not have K-ras mutations (p < 0.0138, Fisher's exact test). The over-expression of c-erbB-2 was found in all stages of progression and was not significantly associated with malignancy. Over-expression of p53 is significantly associated with invasive malignancy (p <_ 0.0002, Fisher's exact test), p53 over-expression was not seen in hyperplastic, low-grade or high-grade dysplastic lesions. These data taken together suggest that K-ras mutation is a very early event in IPMT neoplastic progression, while p53 mutation occurs later, at the highgrade dysplasia ---> invasive carcinoma transition. The detection of p53 mutations may prove useful in predicting malignant behavior in these tumors.