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Recurrence of high-grade gastric MALT lymphoma after long-term regression of low-grade gastroduodenal MALT lymphoma with anti-HP treatment
A622 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 114, No. 4
• G2557 GASTRIN-MEDIATED ALTERATIONS IN GASTRIC EPITHELIAL APOPTOSIS AND PROLIFERATION IN A RODENT MODEL OF GASTRIC NEOPLASIA. M. Kidd, *SF Moss, LH Tang, T. Zhang, *K. Chin, *P. Holt and IM Modlin. Yale University School of Medicine, Surgical Gastric Pathobiology Research Group, and West Haven VA Medical Center, New Haven, CT 06520 & *St. Luke's Roosevelt Hospital Center, Columbia University, NY 10025. The balance between apoptosis and proliferation is believed to maintain tissue mass whilst allowing for rapid changes in size and function in response to physiological stimuli. We have previously established and characterized an experimental model of the African rodent species Mastomys, which is susceptible to gastric carcinoid formation under low acid state induced hypergastrinemia. The aim of this study was to investigate the effect of gastrin on the relationship between apoptosis and proliferation in the gastric epithelium. Hypergastrinemia was generated by the irreversible H2 blockade, loxtidine (lmg/kg/day), and serum gastrin levels determined by RIA. Hyperplastic fundic mucosa (HYP) was obtained from animals treated with Lox for 8 weeks. A hyperplastic withdrawn (HWD) group was generated from animals on Lox for 8 weeks followed by treatment withdrawal for 10 days to normalize gastrin levels prior to harvesting of fundic mucosa. Proliferative cells were identified by Mib-5 immunoreactivity and apoptotic cells by in situ terminal uridine deoxynucleotide end labeling histochemistry. The number of proliferative and apoptotic epithelial ceils were quantitated in a minimum of 3 well-orientated sections and expressed as percentage of the total number of epithelial cells. Expression of the apoptosis-regulatory Bcl-2 family (Bcl-2, Bak, Bax & Bcl-x), was evaluated by immunohistochemisty. The results were as follows: 1) Mib-5 immunoreactivity was confined principally to the middle third of the fundic mucosa; the percentage of the Mib-5 positive cells was increased by ~4-fold in the HYP mucosa (0.27 -+0.07 vs control 0.07 +-0.01, p = 0.01), and decreased by 3-fold in the HWD group (0.1 + 0.01, p < 0.05 vs. HYP). 2) Apoptotic cells were localized in the most superficial epithelium and were increased by 1.8-fold in the hypergastrinemia induced HYP group (0.17 -+0.02 vs control 0.3 --. 0.08, p = 0.05), but returned to the control level in the HWD group. 3) Consistent with expanded tissue mass, the ratio of apoptotic to proliferative cells was significantly lower in the HYP (1.3 ± 0.2) and HWD (2.1 ± 0.3) groups than the controls (3.9 ± 0.8) (p < 0.05). 4) In parallel with the alteration in the number of apoptotic cells, hypergastrinemia invoked an increased expression of Bcl-2 and Bak and decreased expression of Bax confined to the middle third of the gland. These results confirm that gastrin modulates both proliferation and apoptosis of the gastric epithelium in mastomys, and the altered ratio of proliferating to apoptotic ceils may contribute to the pathogenesis of mucosal hyperplasia and probably also neoplasia noted in this model. • G2558 HIGH-RESOLUTION COLONOSCOPY AND CHROMOENDOSCOPY FOR CLASSIFYING COLONIC POLYPS: FINAL RESULTS OF A MULTICENTER STUDY. CY Kim÷+, DE Fleischer*, RA Kozarek**, DL Carr-Locke***, TCM Li*, CJ Gostout+, SJ Heller***, EA Montgomery*, FH A1-Kawas*, MI Avigan*, GM Eisen*, JH Lewis*, SB Benjamin*. *Georgetown University Medical Center, Washington, DC; **Virginia Mason Medical Center, Seattle, WA; ***Brigham & Women's Hospital, Boston, MA; +Mayo Clinic, Rochester, MN; ++Medical University of South Carolina, Charleston, SC. Background: Chromoendoscopy with a conventional high-resolution colonoscope (HRC) adds little time and expense to conventional colonoscopy. We recently reported using an affordable conventional HRC (Fujinon EC200LR) in combination with indigo carmine dye (ICD) at our institution for the diagnosis of small polyps. The sensitivity of our technique in distinguishing adenomatous from non-adenomatous polyps was 96%, and the negative predictive value was 96%. We now report on the final results of our five-center trial (4 academic GI units and 1 primary care practice). Methods: Complete colonoscopy or flexible sigmoidoscopy using the Fujinon 200 series scopes (EC-200LR or ES-200ER) was performed in patients with polyps. All polyps <10mm were sprayed with 0.8% ICD. Histologic diagnosis was predicted based on the polyps's surface architecture ("pits" = hyperplastic (HP), "sulci" - adenomatous (AP)). All polyps were removed. Polyps seen and stained on flex sigmoidoscopy were removed on a followup colonoscopy. Results: 513 polyps with a mean diameter of 4.7 mm were evaluated from 293 patients. 40 polyps were excluded because no prediction could be made.
Actual Histology Predict Histo: AP[Prediet Histo: H A P ( n = 182) 148 [ " ~ HP(n = 210) 37 [ ~ OT* (n = 81) 15 [ ~ * OT (Other) = colonic mucosa or lymphoid aggregate
P Jr I T
~ 81°/° N'~
When attempting to separate AP from non-AP (HP+Other), this technique has a sensitivity of 81%, a specificity of 82%, and a negative predictive value of 88% (i.e. if a polyp is predicted to be hyperplastic, there is a 88% (239/273) likelihood that it is not an adenomatous polyp).
Conclusions: The final results confirm the findings of our preliminary study. With a conventional HRC with ICD, it is possible to separate non-AP from AP without biopsy. Since hyperplastic polyps on flexible sigmoidoscopy do not require further workup, then important reductions in the cost of colon cancer screening may be possible. This research was supported in part by Fujinon, Inc., Wayne, NJ. G2559
CYTOKINE-MEDIATED PROSTAGLANDIN EXPRESSION BY PRIMARY COLONIC FIBROBLAST STRAINS. E.C. Kim. Y. Zhu, P. Lance. Division of Gastroenterology, Department of Medicine, State University of New York and Veterans Affairs of Western New York Health Care System, Buffalo, New York Increased prostaglandin production is characteristic of colorectal adanocarcinomas but the colonic cells from which prostanoids emanate have not been defined. Increasingly, fibroblasts are understood to comprise a heterogeneous population and possess tissue-specific phenotypes. We demonstrated previously that a colonic fibroblast strain (CCD-18Co) is capable of high levels of prostaglandin E2 (PGE2) production. In this report, we investigated PGE 2 synthesis in primary colonic fibroblast strains established from four subjects with normal or diseased large bowels. Methods: Primary fibroblast cultures were initiated from colonoscopic biopsies from patients with normal colons (Patients 1 and 3), a colonic adenoma (Patient 2), and ulcerative colitis (Patient 4). PGE2 levels in medium before and after treatment with interleukin-1 beta (IL-I[~) and tumor necrosis factor-alpha (TNF-ct) were measured by radioimmunoassay. Results: Primary fibroblast cultures from colonoscopic biopsies taken from Patient 1 and 2 were initiated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) 20%. More successful propagation of primary fibroblast cultures was achieved by utilizing MCDB 153 medium enriched with epidermal growth factor (EGF) 10 ng/ml, insulintransferrin-selenium-A, and FBS 20% and this technique was employed with biopsies obtained from Patients 3 and two separate biopsies from Patient 4. Patients 1 and 2 displayed similar PGE2 biosynthetic profiles with a basal level of 3.6 ng/mg protein and an 8-fold and 9-fold increase at 24 h in response to IL-113 (10 ng/ml), respectively. However, the three strains established from Patients 3 and 4 in the enriched MCDB medium demonstrated extremely high levels of PGE2 synthesis (>500 ng/mg protein) both basally and after treatment with IL-l[3 and TNF-~x (50 ng/ml) for 24 h. These strains maintained similarly high levels of PGE2 production even after switching the MCDB medium to DMEM with FBS 10% for four weeks and passaging the fibroblast cultures up to seven times. Conclusions: We have demonstrated that primary human colonic fibroblasts are highly sensitive to both growth factor- and cytokine-mediated induction of PGE2 synthesis indicating that colonic fibroblasts share with pulmonary, synovial, and orbital fibroblasts the property of expressing highly inducible PGE2. This upregulation of PGEz biosynthesis persists in colonic fibroblast cultures for an extended period of time. These findings support the potentially prominent role of fibroblats in prostanoid synthesis in the colon. G2560
RECURRENCE OF HIGH-GRADE GASTRIC MALT LYMPHOMA AFTER LONG-TERM REGRESSION OF LOW-GRADE GASTRODUODENAL MALT LYMPHOMA WITH ANTI-HP TREATMENT. HJ Kim, SG Chi*, YW Kim*, SH Dong, BH Kim, JI Lee, YW Chang, R Chang. Departments of Internal Medicine and Pathology*, Kyung Hee University, College of Medicine, Seoul, Korea Evidence links Helicobacter pylori (Hp) gastritis with the development of mucosa-associated lymphoid tissue (MALT) in the stomach and duodenum, and some studies have suggested that such low-grade MALT lymphoma regress after eradication of Hp infection. However, it is still unknown whether treatment for Hp will avert relapse and, therby actually cure. Using a nestPCR analysis of IgH region with DNA extracted both from freshly frozen and paraffin-embedded tissues, we investigated the clonality of MALT lymphoma tissues serially obtained from a 40-year-old male patient with a low grade MALT lymphoma of the stomach and duodenum associated Hp, which were initially responding to eradication of Hp and showed a subsequent local recurrence of a high-grade gastric MALT lymphoma after 33 months after Hp eradication. With DNA or cDNA which was reverse-transcribed from RNA as template, we performed the two-round PCR amplification of IgH region using previously described primer sets (FR2A/JH or FRIII/JH for 1st PCR and FR2A/VHJH or FRIII/VHJH for nest-PCR). The nest-PCR products were resolved on a 8% polyacrylamide gel and approximately 240 bp (FR2A/VHJH) and 105 bp (FRIII/VHJH) length of discret bands were consistently found in a separate and grossly normal appearing region of stomach and duodenum during the course of disease, which strongly indicates the monoclonal origin of these lymphoma tissues. This study suggest that recurrence of high-grade gastric MALT-lymphoma might be due to a hidden focus of a high grade gastric lymphoma which obscured by the initial low grade gastroduodenal lymphoma and that patients with low-grade MALT lymphoma treated with antibiotics should be under constant surveillance. In addition, we also analysed amplification of c-erbB-2 as a possible molecular event that can cause progression into high-grade lymphoma but no alteration was noted both from low-grade and high-grade MALT lylphoma
GASTROENTEROLOGY Vol. 114, No. 4
• G2557 GASTRIN-MEDIATED ALTERATIONS IN GASTRIC EPITHELIAL APOPTOSIS AND PROLIFERATION IN A RODENT MODEL OF GASTRIC NEOPLASIA. M. Kidd, *SF Moss, LH Tang, T. Zhang, *K. Chin, *P. Holt and IM Modlin. Yale University School of Medicine, Surgical Gastric Pathobiology Research Group, and West Haven VA Medical Center, New Haven, CT 06520 & *St. Luke's Roosevelt Hospital Center, Columbia University, NY 10025. The balance between apoptosis and proliferation is believed to maintain tissue mass whilst allowing for rapid changes in size and function in response to physiological stimuli. We have previously established and characterized an experimental model of the African rodent species Mastomys, which is susceptible to gastric carcinoid formation under low acid state induced hypergastrinemia. The aim of this study was to investigate the effect of gastrin on the relationship between apoptosis and proliferation in the gastric epithelium. Hypergastrinemia was generated by the irreversible H2 blockade, loxtidine (lmg/kg/day), and serum gastrin levels determined by RIA. Hyperplastic fundic mucosa (HYP) was obtained from animals treated with Lox for 8 weeks. A hyperplastic withdrawn (HWD) group was generated from animals on Lox for 8 weeks followed by treatment withdrawal for 10 days to normalize gastrin levels prior to harvesting of fundic mucosa. Proliferative cells were identified by Mib-5 immunoreactivity and apoptotic cells by in situ terminal uridine deoxynucleotide end labeling histochemistry. The number of proliferative and apoptotic epithelial ceils were quantitated in a minimum of 3 well-orientated sections and expressed as percentage of the total number of epithelial cells. Expression of the apoptosis-regulatory Bcl-2 family (Bcl-2, Bak, Bax & Bcl-x), was evaluated by immunohistochemisty. The results were as follows: 1) Mib-5 immunoreactivity was confined principally to the middle third of the fundic mucosa; the percentage of the Mib-5 positive cells was increased by ~4-fold in the HYP mucosa (0.27 -+0.07 vs control 0.07 +-0.01, p = 0.01), and decreased by 3-fold in the HWD group (0.1 + 0.01, p < 0.05 vs. HYP). 2) Apoptotic cells were localized in the most superficial epithelium and were increased by 1.8-fold in the hypergastrinemia induced HYP group (0.17 -+0.02 vs control 0.3 --. 0.08, p = 0.05), but returned to the control level in the HWD group. 3) Consistent with expanded tissue mass, the ratio of apoptotic to proliferative cells was significantly lower in the HYP (1.3 ± 0.2) and HWD (2.1 ± 0.3) groups than the controls (3.9 ± 0.8) (p < 0.05). 4) In parallel with the alteration in the number of apoptotic cells, hypergastrinemia invoked an increased expression of Bcl-2 and Bak and decreased expression of Bax confined to the middle third of the gland. These results confirm that gastrin modulates both proliferation and apoptosis of the gastric epithelium in mastomys, and the altered ratio of proliferating to apoptotic ceils may contribute to the pathogenesis of mucosal hyperplasia and probably also neoplasia noted in this model. • G2558 HIGH-RESOLUTION COLONOSCOPY AND CHROMOENDOSCOPY FOR CLASSIFYING COLONIC POLYPS: FINAL RESULTS OF A MULTICENTER STUDY. CY Kim÷+, DE Fleischer*, RA Kozarek**, DL Carr-Locke***, TCM Li*, CJ Gostout+, SJ Heller***, EA Montgomery*, FH A1-Kawas*, MI Avigan*, GM Eisen*, JH Lewis*, SB Benjamin*. *Georgetown University Medical Center, Washington, DC; **Virginia Mason Medical Center, Seattle, WA; ***Brigham & Women's Hospital, Boston, MA; +Mayo Clinic, Rochester, MN; ++Medical University of South Carolina, Charleston, SC. Background: Chromoendoscopy with a conventional high-resolution colonoscope (HRC) adds little time and expense to conventional colonoscopy. We recently reported using an affordable conventional HRC (Fujinon EC200LR) in combination with indigo carmine dye (ICD) at our institution for the diagnosis of small polyps. The sensitivity of our technique in distinguishing adenomatous from non-adenomatous polyps was 96%, and the negative predictive value was 96%. We now report on the final results of our five-center trial (4 academic GI units and 1 primary care practice). Methods: Complete colonoscopy or flexible sigmoidoscopy using the Fujinon 200 series scopes (EC-200LR or ES-200ER) was performed in patients with polyps. All polyps <10mm were sprayed with 0.8% ICD. Histologic diagnosis was predicted based on the polyps's surface architecture ("pits" = hyperplastic (HP), "sulci" - adenomatous (AP)). All polyps were removed. Polyps seen and stained on flex sigmoidoscopy were removed on a followup colonoscopy. Results: 513 polyps with a mean diameter of 4.7 mm were evaluated from 293 patients. 40 polyps were excluded because no prediction could be made.
Actual Histology Predict Histo: AP[Prediet Histo: H A P ( n = 182) 148 [ " ~ HP(n = 210) 37 [ ~ OT* (n = 81) 15 [ ~ * OT (Other) = colonic mucosa or lymphoid aggregate
P Jr I T
~ 81°/° N'~
When attempting to separate AP from non-AP (HP+Other), this technique has a sensitivity of 81%, a specificity of 82%, and a negative predictive value of 88% (i.e. if a polyp is predicted to be hyperplastic, there is a 88% (239/273) likelihood that it is not an adenomatous polyp).
Conclusions: The final results confirm the findings of our preliminary study. With a conventional HRC with ICD, it is possible to separate non-AP from AP without biopsy. Since hyperplastic polyps on flexible sigmoidoscopy do not require further workup, then important reductions in the cost of colon cancer screening may be possible. This research was supported in part by Fujinon, Inc., Wayne, NJ. G2559
CYTOKINE-MEDIATED PROSTAGLANDIN EXPRESSION BY PRIMARY COLONIC FIBROBLAST STRAINS. E.C. Kim. Y. Zhu, P. Lance. Division of Gastroenterology, Department of Medicine, State University of New York and Veterans Affairs of Western New York Health Care System, Buffalo, New York Increased prostaglandin production is characteristic of colorectal adanocarcinomas but the colonic cells from which prostanoids emanate have not been defined. Increasingly, fibroblasts are understood to comprise a heterogeneous population and possess tissue-specific phenotypes. We demonstrated previously that a colonic fibroblast strain (CCD-18Co) is capable of high levels of prostaglandin E2 (PGE2) production. In this report, we investigated PGE 2 synthesis in primary colonic fibroblast strains established from four subjects with normal or diseased large bowels. Methods: Primary fibroblast cultures were initiated from colonoscopic biopsies from patients with normal colons (Patients 1 and 3), a colonic adenoma (Patient 2), and ulcerative colitis (Patient 4). PGE2 levels in medium before and after treatment with interleukin-1 beta (IL-I[~) and tumor necrosis factor-alpha (TNF-ct) were measured by radioimmunoassay. Results: Primary fibroblast cultures from colonoscopic biopsies taken from Patient 1 and 2 were initiated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) 20%. More successful propagation of primary fibroblast cultures was achieved by utilizing MCDB 153 medium enriched with epidermal growth factor (EGF) 10 ng/ml, insulintransferrin-selenium-A, and FBS 20% and this technique was employed with biopsies obtained from Patients 3 and two separate biopsies from Patient 4. Patients 1 and 2 displayed similar PGE2 biosynthetic profiles with a basal level of 3.6 ng/mg protein and an 8-fold and 9-fold increase at 24 h in response to IL-113 (10 ng/ml), respectively. However, the three strains established from Patients 3 and 4 in the enriched MCDB medium demonstrated extremely high levels of PGE2 synthesis (>500 ng/mg protein) both basally and after treatment with IL-l[3 and TNF-~x (50 ng/ml) for 24 h. These strains maintained similarly high levels of PGE2 production even after switching the MCDB medium to DMEM with FBS 10% for four weeks and passaging the fibroblast cultures up to seven times. Conclusions: We have demonstrated that primary human colonic fibroblasts are highly sensitive to both growth factor- and cytokine-mediated induction of PGE2 synthesis indicating that colonic fibroblasts share with pulmonary, synovial, and orbital fibroblasts the property of expressing highly inducible PGE2. This upregulation of PGEz biosynthesis persists in colonic fibroblast cultures for an extended period of time. These findings support the potentially prominent role of fibroblats in prostanoid synthesis in the colon. G2560
RECURRENCE OF HIGH-GRADE GASTRIC MALT LYMPHOMA AFTER LONG-TERM REGRESSION OF LOW-GRADE GASTRODUODENAL MALT LYMPHOMA WITH ANTI-HP TREATMENT. HJ Kim, SG Chi*, YW Kim*, SH Dong, BH Kim, JI Lee, YW Chang, R Chang. Departments of Internal Medicine and Pathology*, Kyung Hee University, College of Medicine, Seoul, Korea Evidence links Helicobacter pylori (Hp) gastritis with the development of mucosa-associated lymphoid tissue (MALT) in the stomach and duodenum, and some studies have suggested that such low-grade MALT lymphoma regress after eradication of Hp infection. However, it is still unknown whether treatment for Hp will avert relapse and, therby actually cure. Using a nestPCR analysis of IgH region with DNA extracted both from freshly frozen and paraffin-embedded tissues, we investigated the clonality of MALT lymphoma tissues serially obtained from a 40-year-old male patient with a low grade MALT lymphoma of the stomach and duodenum associated Hp, which were initially responding to eradication of Hp and showed a subsequent local recurrence of a high-grade gastric MALT lymphoma after 33 months after Hp eradication. With DNA or cDNA which was reverse-transcribed from RNA as template, we performed the two-round PCR amplification of IgH region using previously described primer sets (FR2A/JH or FRIII/JH for 1st PCR and FR2A/VHJH or FRIII/VHJH for nest-PCR). The nest-PCR products were resolved on a 8% polyacrylamide gel and approximately 240 bp (FR2A/VHJH) and 105 bp (FRIII/VHJH) length of discret bands were consistently found in a separate and grossly normal appearing region of stomach and duodenum during the course of disease, which strongly indicates the monoclonal origin of these lymphoma tissues. This study suggest that recurrence of high-grade gastric MALT-lymphoma might be due to a hidden focus of a high grade gastric lymphoma which obscured by the initial low grade gastroduodenal lymphoma and that patients with low-grade MALT lymphoma treated with antibiotics should be under constant surveillance. In addition, we also analysed amplification of c-erbB-2 as a possible molecular event that can cause progression into high-grade lymphoma but no alteration was noted both from low-grade and high-grade MALT lylphoma