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Increased cell proliferation in chronic Helicobacter pylori positive gastritis and gastric carcinoma
GASTROENTEROLOGY Vol. 114, No. 4
A664 AGA ABSTRACTS • G2738
EFFICACY OF ADJUVANT CHEMOTHERAPY WITH 5-FU/ LEUCOVORIN AND 5-FU/LEVAMISOLE IN COLON CANCER. R. Porschen, A. Bermann, T. L6ffler, G. Haack, K. Rettig, Y. Anger, G. Strohmeyer for the Study Group Gastrointestinal Oncology. Dept. Internal Medicine I, University Clinic Ttibingen, Germany Previous studies have demonstrated the positive impact of adjuvant chemotherapy with 5-FU/levamisole on survival of patients with stage I11 colon cancer. In metastatic cancer, biochemical modulation of 5-FU leads to increased remission rates. Therefore, the effects of 5-FU/leucovorin (450 and 100 mg/m2 i,v. x 5 d; 12 cycles, arm A) were compared with 5-FU/levamisole (Moertel scheme; arm B) after curative resection of stage III colon cancers in a prospective multicentric study. 703 pts. were entered in this trial. In general, the characteristics of 674 eligible patients (A: 343 pts., B: 331 pts.) were well balanced between the study arms. Multiple tumors (10 vs. 5), tumor obstruction (37 vs. 31) and tumor perforation (9 vs. 4) were more often observed in arm A. The number of lymph nodes analysed amounted to 14.2±10.8 and 13.2±8.6, respectively, The interval between surgery and start of therapy was 29 and 31 days. In arm A, 1055 and in arm B 737 toxic effects were observed during all chemotherapy cycles. Treatment-associated side effects were generally mild in both treatment arms. Grade III/IV toxic effects were documentd in 8.1% of all cycles in arm A and in 3.5% of all cycles in arm B. 95 recurrences in arm A (27.7%) and 107 in arm B (32.3%) have been reported (locoregional: 10 and 15; metastases: 68 and 73; combination: 17 and 19). 112 pts. have died due to relapses (arm A: 49; arm B: 63). Disease-free survival was significantly better in arm A (p < 0.05), whereas results concerning overall survival are still not significant (p=0.09). DFS was significantly influenced by the interval between operation and start of chemotherapy (p=0.0001). In conclusion, adjuvant treatment with 5-FU/leucovorin leads to an improved disease-free survival compared to 5-FU/levamisole after curative resection of a stage III colon cancer. Supported by the AG flir Krebsbek~lmpfung and the Grimmke Stiftung • G2739 INDIA INK AS A COLONIC TATTOOING AGENT. N. Price*, M. Gottfried-, E.Clary*, D.C. Lawson*, J. Baillie ^, K. Mergener ^, C. Westcott*, S. Eubanks*, T. Pappas*. From the Departments of Surgery*, and Pathology-, Division of Gastroenterology ^, Duke University Medical Center and the VA Medical Center, Durham, NC. Background: The safety and efficacy of colonic tattooing for the intraoperative identification of polypectomy sites has not been documented. Our previous work has demonstrated that India ink is superior to Indocyanine green. The purpose of this study is to document the durability of India ink (rabbit) and to show it's visibility by colonoscopy, larparoscopy and laparotomy (dog). Methods: Twenty-two New Zealand white rabbits (2 kg) were anesthetized with Isoflurane at 2.5% and injected with India ink (undiluted, 1:10, 1:50, 1:100, 1:1000, 1:10,000). Tuberculin syringes were used to create a 0.1 ml serosal bleb at two injection sites two centimeters apart. Repeat laparotomy was performed at one and five months. Twelve mongrel dogs (20 kg) were endoscopically injected with India ink (1:100, 1:1,000, 1:10,000) using 0.5 ml and 1.0 ml injection volumes. Re-exploration by repeat colonoscopy, laparoscopy, and laparntomy was done at seven days and one month. Tattoo visibility at re-exploration of both species was graded on a scale (0-agent not seen, 1-seen with difficulty, 2-easily seen with no gross evidence of inflammation, 3-easily seen with gross inflammation). Histology in the rabbit was judged by degrees of inflammation (0-no inflammation, 2-mild, 4-moderate, 6-severe). Results: India ink was studied at one month and five months after injection in the rabbit model and was visible at all concentrations. The undiluted and 1:10 concentrations were easily seen and showed evidence of mucosal ulceration. All other India ink concentrations were visible without gross inflammation. India ink was also studied at seven days and one month in dogs. The 1:100 concentration at 0.5 ml was seen consistently at colonoscopy, laparoscopy, and laparotomy with only a mild submucosal reaction at seven days. The 1:100 and 1:1,000 concentrations at 0.5 ml and 1.0 ml injection volumes were easily seen in all mediums at one month with similar histology. Conclusion: 1)India ink was an effective colonic tattooing agent. 2) Dilute concentrations that caused little to no inflammation, could be visualized at one month in rabbits and dogs and at five months in rabbits. India ink, at appropriate concentrations, appears to be a safe short and long-term colonic tattooing agent in both models. G2740
CORRELATION BETWEEN PCNA IMMUNOCYTOCHEMISTRY AND TV IMAGE CYTOMETRY PARAMETERS IN THE DETECTION OF GASTRIC PROLIFERATION ACITVITY. L, Pr6nai, E. Szaleczky, B. Molnfir, L. Berczi*, Z. Tulassay, Clin. Gastroent., 2nd Dept. Med., *1st Dept. Pathol., Semmelweis Univ., Budapest, Hungary Backgrounds: The correlation between quantitative DNA and immuncytochemistry in the determination of proliferation acitivity of gastric biopsies is not yet known.
Aims: To compare PCNA immuncytochemistry and TV image cytometry in the determination of proliferation analysis of gastric brash smears and biopsies. Methods: In seventy four patients, the proliferation activity labelling index (LI) of gastric epithelial biopsies (10 healthy, chronic gastritis = 39, gastritis with intestinal metaplasia=24, gastric cancer=l 1 patients) was determined by PCNA immunohistochemistry (anti-PCNA Clone PC10, LSAB kit Peroxidase, DAKO Denmark). The count was performed only in well oriented crypts using a grid placed in an ocular. The mean number of nuclei counted per section specimen was 1000. For cytometry, the DNASK TV image analysis system was used (ASK Ltd. Budapest, Hungary). In each smear, at least 200 nuclei was measured. Internal reference control (epithelial cells, lymphocytes) was used. The statistical analysis was performed with the SPSS programpackage using one-way variance analysis and correlation analysis. Res~alts: PCNA-LI was lower when intestinal metaplasia was concomitantlypresent. Histology
normal (n = 10) chronic gastritis (n = 19) gastritis with intestinal metaplasia (n = 19) carcinoma (n = 11)
LI (%) 45.80 + 63.30 ± 52.01 ± 76.18 ±
11.07 *,+ 18.25 *,^ 21.65 # 12.35 +,A,#
Results are expressed as mean ± SD. Statistical significance between groups are marked: *,+,#,^; p < 0.05. By cytometry, significant differences were found between the malignant and non-malignant cases in the DNA Index, 2c Deviation index, G1, S+g2 ratio, surface and diameter parameters. The perimeter and the maximum density was significantly different between the normal group and the gastritis with intestinal metaplasia group (p=<0.05). When immunocytochemistry was compared with TV cytometry, PCNA-LI correlated with G1 phase (r--- -0.415), S Phase (r=0.385), DNA Index (r=0.598), density (r=0.608), surface (r=0.670), layers (r=0.638) and diameter (r=0.73~5), respectively (p<0.05). Conclusions: A significant correlation was found between PCNA LI and Tv image cytometry parameters of G1 and S phase. The PCNA labeling index well correlates with morphometric parameters, • G2741 INCREASED CELL PROLIFERATION IN CHRONIC HELICOBACTER PYLORI POSITIVE GASTRITIS AND GASTRIC CARCINOMA. L Pr6nal. E. Szaleczky, B. Molnfir, Berczi L*, Zs. Tulassay, 2na Dept Internal Medicine, * 1st Dept. Pathology, Semmelweis Medical University, Hungarian Academy of Science, Joint Research Unit, Hungary, Budapest Background: Epithelial cell proliferation rate has been reported both to be decreased and unaltered following H.pylori eradication, but results are controversial if whether the method is reliable when paraffin emberred material is used to measure proliferation cell nuclear antigen labelling index (PCNA-LI, %). Aims: To compare cell proliferation activity of H.pylori positive and negative gastric biopsies in chronic gastritis with and without intestinal metaplasia (IM) and gastric cancer by using both PCNA and Tv image cytometry. To measure the change in cell proliferation activity prior to and after H.pylori eradication. Patients and methods: Brush smears and antral biopsies were taken from 70 patients (42 men, 28 women, mean age 58 ± 15 y.o). Patients were divided into four groups according to the routine histology; normal epithelia (n=10), chronic gastritis without IM (n=25) and with IM (n=24), and gastric carcinoma (n=ll). H.pylori eradication was controlled in 24 cases. Cell proliferation was measured from non-paraffin fixed samples both by immunocytochemistry (anti-PCNA Clone PC10, LSAB kit Peroxidase DAKO, Denmark) and Tv image cytometry (DNASK, ASK Ltd,, Hungary) by evaluating 12 morphometric parameters including DNA index, S-phase and G1-S-G2 ratio. Results; PCNA-LI, DNA index and GI-S-G2 ratio were all significantly higher in chronic gastritis (63.3 ± 18.3) than in the normal epithelium (45.8 ± 11.1), and were further increased in carcinoma (76.2± 12.4, p<0.05). For Tv cytomerty, H.pylori positive cases (n=33) had a higher DNA index than negative ones (1.10 ± 0.07 vs. 1.05 - 0.07, p<0.05), whereas PCNA-LI was not different between the two groups (53 ± 14 vs. 52 ± 11). Both DNA index and PCNA-LI decreased after successful H.pylori eradication. PCNA-LI was lower in chronic gastritis with IM when compared with that without IM (63 -+ 18 vs. 52 ± 22), which corresponds to the lower S phase ratio in IM as determined by Tv cytometry. Conclusion: Epithelial cell turnover is increased when H.pylori is present in chronic gastritis with or without IM, and in gastric carcinoma. Cell proliferation decreases after successful H.pylori eradication. The lower PCNA-LI observed in chronic gastritis with IM is a result of the lower S-phase ratio in metaplastic ceils. When PCNA alone is used to measure cell proliferation, the decreased labeling index observed in IM may overlap the increase induced by H.pylori infection. This work was supported in part by OTKA grant No: 023788.
A664 AGA ABSTRACTS • G2738
EFFICACY OF ADJUVANT CHEMOTHERAPY WITH 5-FU/ LEUCOVORIN AND 5-FU/LEVAMISOLE IN COLON CANCER. R. Porschen, A. Bermann, T. L6ffler, G. Haack, K. Rettig, Y. Anger, G. Strohmeyer for the Study Group Gastrointestinal Oncology. Dept. Internal Medicine I, University Clinic Ttibingen, Germany Previous studies have demonstrated the positive impact of adjuvant chemotherapy with 5-FU/levamisole on survival of patients with stage I11 colon cancer. In metastatic cancer, biochemical modulation of 5-FU leads to increased remission rates. Therefore, the effects of 5-FU/leucovorin (450 and 100 mg/m2 i,v. x 5 d; 12 cycles, arm A) were compared with 5-FU/levamisole (Moertel scheme; arm B) after curative resection of stage III colon cancers in a prospective multicentric study. 703 pts. were entered in this trial. In general, the characteristics of 674 eligible patients (A: 343 pts., B: 331 pts.) were well balanced between the study arms. Multiple tumors (10 vs. 5), tumor obstruction (37 vs. 31) and tumor perforation (9 vs. 4) were more often observed in arm A. The number of lymph nodes analysed amounted to 14.2±10.8 and 13.2±8.6, respectively, The interval between surgery and start of therapy was 29 and 31 days. In arm A, 1055 and in arm B 737 toxic effects were observed during all chemotherapy cycles. Treatment-associated side effects were generally mild in both treatment arms. Grade III/IV toxic effects were documentd in 8.1% of all cycles in arm A and in 3.5% of all cycles in arm B. 95 recurrences in arm A (27.7%) and 107 in arm B (32.3%) have been reported (locoregional: 10 and 15; metastases: 68 and 73; combination: 17 and 19). 112 pts. have died due to relapses (arm A: 49; arm B: 63). Disease-free survival was significantly better in arm A (p < 0.05), whereas results concerning overall survival are still not significant (p=0.09). DFS was significantly influenced by the interval between operation and start of chemotherapy (p=0.0001). In conclusion, adjuvant treatment with 5-FU/leucovorin leads to an improved disease-free survival compared to 5-FU/levamisole after curative resection of a stage III colon cancer. Supported by the AG flir Krebsbek~lmpfung and the Grimmke Stiftung • G2739 INDIA INK AS A COLONIC TATTOOING AGENT. N. Price*, M. Gottfried-, E.Clary*, D.C. Lawson*, J. Baillie ^, K. Mergener ^, C. Westcott*, S. Eubanks*, T. Pappas*. From the Departments of Surgery*, and Pathology-, Division of Gastroenterology ^, Duke University Medical Center and the VA Medical Center, Durham, NC. Background: The safety and efficacy of colonic tattooing for the intraoperative identification of polypectomy sites has not been documented. Our previous work has demonstrated that India ink is superior to Indocyanine green. The purpose of this study is to document the durability of India ink (rabbit) and to show it's visibility by colonoscopy, larparoscopy and laparotomy (dog). Methods: Twenty-two New Zealand white rabbits (2 kg) were anesthetized with Isoflurane at 2.5% and injected with India ink (undiluted, 1:10, 1:50, 1:100, 1:1000, 1:10,000). Tuberculin syringes were used to create a 0.1 ml serosal bleb at two injection sites two centimeters apart. Repeat laparotomy was performed at one and five months. Twelve mongrel dogs (20 kg) were endoscopically injected with India ink (1:100, 1:1,000, 1:10,000) using 0.5 ml and 1.0 ml injection volumes. Re-exploration by repeat colonoscopy, laparoscopy, and laparntomy was done at seven days and one month. Tattoo visibility at re-exploration of both species was graded on a scale (0-agent not seen, 1-seen with difficulty, 2-easily seen with no gross evidence of inflammation, 3-easily seen with gross inflammation). Histology in the rabbit was judged by degrees of inflammation (0-no inflammation, 2-mild, 4-moderate, 6-severe). Results: India ink was studied at one month and five months after injection in the rabbit model and was visible at all concentrations. The undiluted and 1:10 concentrations were easily seen and showed evidence of mucosal ulceration. All other India ink concentrations were visible without gross inflammation. India ink was also studied at seven days and one month in dogs. The 1:100 concentration at 0.5 ml was seen consistently at colonoscopy, laparoscopy, and laparotomy with only a mild submucosal reaction at seven days. The 1:100 and 1:1,000 concentrations at 0.5 ml and 1.0 ml injection volumes were easily seen in all mediums at one month with similar histology. Conclusion: 1)India ink was an effective colonic tattooing agent. 2) Dilute concentrations that caused little to no inflammation, could be visualized at one month in rabbits and dogs and at five months in rabbits. India ink, at appropriate concentrations, appears to be a safe short and long-term colonic tattooing agent in both models. G2740
CORRELATION BETWEEN PCNA IMMUNOCYTOCHEMISTRY AND TV IMAGE CYTOMETRY PARAMETERS IN THE DETECTION OF GASTRIC PROLIFERATION ACITVITY. L, Pr6nai, E. Szaleczky, B. Molnfir, L. Berczi*, Z. Tulassay, Clin. Gastroent., 2nd Dept. Med., *1st Dept. Pathol., Semmelweis Univ., Budapest, Hungary Backgrounds: The correlation between quantitative DNA and immuncytochemistry in the determination of proliferation acitivity of gastric biopsies is not yet known.
Aims: To compare PCNA immuncytochemistry and TV image cytometry in the determination of proliferation analysis of gastric brash smears and biopsies. Methods: In seventy four patients, the proliferation activity labelling index (LI) of gastric epithelial biopsies (10 healthy, chronic gastritis = 39, gastritis with intestinal metaplasia=24, gastric cancer=l 1 patients) was determined by PCNA immunohistochemistry (anti-PCNA Clone PC10, LSAB kit Peroxidase, DAKO Denmark). The count was performed only in well oriented crypts using a grid placed in an ocular. The mean number of nuclei counted per section specimen was 1000. For cytometry, the DNASK TV image analysis system was used (ASK Ltd. Budapest, Hungary). In each smear, at least 200 nuclei was measured. Internal reference control (epithelial cells, lymphocytes) was used. The statistical analysis was performed with the SPSS programpackage using one-way variance analysis and correlation analysis. Res~alts: PCNA-LI was lower when intestinal metaplasia was concomitantlypresent. Histology
normal (n = 10) chronic gastritis (n = 19) gastritis with intestinal metaplasia (n = 19) carcinoma (n = 11)
LI (%) 45.80 + 63.30 ± 52.01 ± 76.18 ±
11.07 *,+ 18.25 *,^ 21.65 # 12.35 +,A,#
Results are expressed as mean ± SD. Statistical significance between groups are marked: *,+,#,^; p < 0.05. By cytometry, significant differences were found between the malignant and non-malignant cases in the DNA Index, 2c Deviation index, G1, S+g2 ratio, surface and diameter parameters. The perimeter and the maximum density was significantly different between the normal group and the gastritis with intestinal metaplasia group (p=<0.05). When immunocytochemistry was compared with TV cytometry, PCNA-LI correlated with G1 phase (r--- -0.415), S Phase (r=0.385), DNA Index (r=0.598), density (r=0.608), surface (r=0.670), layers (r=0.638) and diameter (r=0.73~5), respectively (p<0.05). Conclusions: A significant correlation was found between PCNA LI and Tv image cytometry parameters of G1 and S phase. The PCNA labeling index well correlates with morphometric parameters, • G2741 INCREASED CELL PROLIFERATION IN CHRONIC HELICOBACTER PYLORI POSITIVE GASTRITIS AND GASTRIC CARCINOMA. L Pr6nal. E. Szaleczky, B. Molnfir, Berczi L*, Zs. Tulassay, 2na Dept Internal Medicine, * 1st Dept. Pathology, Semmelweis Medical University, Hungarian Academy of Science, Joint Research Unit, Hungary, Budapest Background: Epithelial cell proliferation rate has been reported both to be decreased and unaltered following H.pylori eradication, but results are controversial if whether the method is reliable when paraffin emberred material is used to measure proliferation cell nuclear antigen labelling index (PCNA-LI, %). Aims: To compare cell proliferation activity of H.pylori positive and negative gastric biopsies in chronic gastritis with and without intestinal metaplasia (IM) and gastric cancer by using both PCNA and Tv image cytometry. To measure the change in cell proliferation activity prior to and after H.pylori eradication. Patients and methods: Brush smears and antral biopsies were taken from 70 patients (42 men, 28 women, mean age 58 ± 15 y.o). Patients were divided into four groups according to the routine histology; normal epithelia (n=10), chronic gastritis without IM (n=25) and with IM (n=24), and gastric carcinoma (n=ll). H.pylori eradication was controlled in 24 cases. Cell proliferation was measured from non-paraffin fixed samples both by immunocytochemistry (anti-PCNA Clone PC10, LSAB kit Peroxidase DAKO, Denmark) and Tv image cytometry (DNASK, ASK Ltd,, Hungary) by evaluating 12 morphometric parameters including DNA index, S-phase and G1-S-G2 ratio. Results; PCNA-LI, DNA index and GI-S-G2 ratio were all significantly higher in chronic gastritis (63.3 ± 18.3) than in the normal epithelium (45.8 ± 11.1), and were further increased in carcinoma (76.2± 12.4, p<0.05). For Tv cytomerty, H.pylori positive cases (n=33) had a higher DNA index than negative ones (1.10 ± 0.07 vs. 1.05 - 0.07, p<0.05), whereas PCNA-LI was not different between the two groups (53 ± 14 vs. 52 ± 11). Both DNA index and PCNA-LI decreased after successful H.pylori eradication. PCNA-LI was lower in chronic gastritis with IM when compared with that without IM (63 -+ 18 vs. 52 ± 22), which corresponds to the lower S phase ratio in IM as determined by Tv cytometry. Conclusion: Epithelial cell turnover is increased when H.pylori is present in chronic gastritis with or without IM, and in gastric carcinoma. Cell proliferation decreases after successful H.pylori eradication. The lower PCNA-LI observed in chronic gastritis with IM is a result of the lower S-phase ratio in metaplastic ceils. When PCNA alone is used to measure cell proliferation, the decreased labeling index observed in IM may overlap the increase induced by H.pylori infection. This work was supported in part by OTKA grant No: 023788.