114 Proton Pump Inhibitor Prevents Clinically Significant Upper Gastrointestinal Evetns in Clopidogrel Users With Ulcer History

113 Inhibition of IDO1 Reduces Human Colon Cancer Cell Proliferation in Cell Culture and Reduces Tumor Burden in Colitis Associated Cancer Model Ameet I. Thaker, Lynne R. Foster, Suprada Rao, William F. Stenson, Matthew A. Ciorba BACKGROUND: Colitis associated cancer is an important complication of the human inflammatory bowel diseases (IBD). Indoleamine 2,3 Dioxygenase-1 (IDO), the rate limiting enzyme in tryptophan catabolism, is over-expressed in the epithelium of active IBD and some cancers. Specific inhibitors of IDO1, including 1-methyl tryptophan (1-mT), are under investigation for their ability to break tumor-induced immune tolerance induced by IDO1 mediated suppression of T-effector cell responses. AIM: Examine the role of IDO1 in a mouse model of colitis associated cancer and human colon cancer cell lines. METHODS: C57B/6 WT and IDO-/- mice were used. 1-mT was administered to WT mice via an implanted time release capsule. Comparator groups were co-caged. Colitis associated cancer induction used a standardized protocol of azoxymethane (AOM: 10mg/kg) IP prior to 3 week-long cycles of 2.5% dextran sodium sulfate (DSS). Clinical Disease Activity Index (DAI), histology and epithelial proliferation was assessed. Proliferation in HCT-116 & HT29 human colon cancer cell lines was assessed by MTT incorporation in the presence and absence of IFNg stimulation. RESULTS: Western blot and qRT-PCR revealed AOM/DSS tumor expression of IDO1 to be elevated over baseline colon levels. Over the 60 day protocol clinical disease activity was similar between IDO-/- vs WT mice. At sacrifice tumor number (4.1 vs 7.5, p<0.01) and total tumor area (13.7 vs 31.2 sq mm, p=0.017) were lower in IDO-/- vs WT mice. Correspondingly, the epithelial proliferation index (BrdU incorporation) was lower in adenomas from IDO-/- vs WT mice (20.2% vs 41%, p=0.04) while cytoplasmic B-catenin staining was stronger in the WT than IDO-/- mice. The IDO1 inhibitor 1-mT was examined both in cell culture and invivo. HCT-116 and HT29 cells express IDO1 at baseline and IFNg increases IDO1 expression. 1-mT added to the media (1-4000 uM) suppressed proliferation (10-71%, p<0.001), an effect that was amplified in the presence of IFNg. In invivo testing WT mice were given the IDO1 inhibitor 1-mT slow release pellet after the first cycle of DSS. 1-mT treated mice demonstrated significantly fewer visible tumors (3.00 vs 5.57, p<.04) and smaller total tumor area (15.3 vs 25 sq mm, p<.05). Importantly colitis severity in 1-mT treated mice was similar to placebo controls both in histology and DAI. CONCLUSIONS/ SIGNIFICANCE: The IDO1 enzyme is expressed both in human cancer cell lines and in a mouse model of colitis associated cancer. IDO1 is an important regulator of colitis associated cancer progression as mice lacking IDO1 produce fewer/smaller tumors. IDO1 inhibition with 1-mT reduced cancer cell proliferation invivo and reduced the tumor burden in an invivo model. Pharmacologic inhibition of IDO1 may be an effective therapeutic strategy for colon cancer, particularly inflammation associated colon cancer.

111 Epigenome-Wide Analysis Identifies Novel Candidate Risk Stratification Markers for Ulcerative Colitis-Associated Dysplasia Andrew Kaz, Yanxin Luo, Chao-Jen Wong, Lisa Lai, William M. Grady, Teresa Brentnall INTRODUCTION: Individuals with long-standing ulcerative colitis (UC) are at increased risk for colorectal cancer (CRC) related to chronic colonic inflammation. To prevent CRC in these individuals, regular surveillance colonoscopy with extensive biopsies to assess for pre-cancerous changes in the colonic mucosa is recommended. Currently, dysplasia in biopsies of normal-appearing colonic mucosa is the best marker for cancer risk, but endoscopic biopsy combined with histology is suboptimal due to its costs, procedural risks, and variability. Thus, there is a need to identify more accurate, less invasive, and less expensive risk markers. Molecular alterations in the normal colon mucosa of UC patients (e.g. DNA methylation) have the potential to be more accurate predictive markers for dysplasia/cancer than histology. HYPOTHESIS: Aberrantly methylated genes occur in the normal-appearing mucosa of UC patients who have colon dysplasia/cancer located remotely in the colon. This phenomenon reflects a field cancerization process and can be used to identify those patients with high-grade dysplasia or cancer from those that are cancer free. METHODS: Epithelial DNA was isolated from: 1) normal mucosa from 11 UC patients without cancer (UC-N), 2) normal mucosa from 10 UC patients with dysplasia/cancer located remotely in the colon (‘remote cancer') (UC-RC), and 3) high-grade dysplasia or cancer from 10 UC patients (UCC). DNA was bisulfite treated and analyzed using the Illumina HumanMethylation 450 BeadChip. The array results were validated by pyrosequencing DNA isolated from several UC-N, UC-RC, and UC-C patients that were used on the array. RESULTS: After filtering the array data using a previously validated approach, we identified 412 CpG sites that were differentially methylated between the UC-N and UC-RC cases (p < 0.001) yet demonstrated a similar methylation pattern between the UC-RC and UC-C cases. The great majority of these CpG sites were located in gene promoters, and all demonstrated increased methylation in the UC-RC and UC-C cases compared to UC-N cases. From this group of CpGs, we generated a list of nine top candidates for further study. We validated the methylation status of one of these genes, ELMO1, in several UC-N, UC-RC, and UC-C cases using pyrosequencing, which confirmed increased methylation in the UC-RC and UC-C cases. DISCUSSION: Using a methylation array-based approach, we identified several novel genes that show low methylation in the normal colonic mucosa of UC patients but become hypermethylated in the normal colonic of mucosa of UC patients who had cancer/dysplasia located elsewhere in the colon. Since these genes appear to remain hypermethylated during the progression to UC-associated dysplasia, they are attractive molecular markers with potential to identify UC patients at risk for developing colonic dysplasia or cancer.

114 Proton Pump Inhibitor Prevents Clinically Significant Upper Gastrointestinal Evetns in Clopidogrel Users With Ulcer History Ping-I Hsu Background and aims: Platelet ADP-receptor antagonists impair the healing of peptic ulcers and potentially convert silent ulcers to bleeding lesions. The aim of this study was to investigate whether proton pump inhibitor (PPI) can prevent clinically significant upper gastrointestinal (GI) events in clopidogrel users with a peptic ulcer history. Methods: From January 2008 to November 2010, long-termed clopidogrel users with peptic ulcer history who did not have peptic ulcers on enrollment were randomly assigned to receive either esomeprazole (20 mg daily, before breakfast) plus clopidogrel (75 mg daily, at bedtime) or clopidogrel alone for 6 months. Eradication therapy was administered if endoscopy on enrollment revealed H. pylori infection. Follow-up endoscopy was carried out at the end of the 6th month and whenever severe epigastric discomfort, hematemesis or melena occurred. Clinically significant upper GI event was defined as the occurrence of upper GI bleeding and/or peptic ulcer with dyspeptic symptoms. Results: The cumulative incidence of peptic ulcer documented by endoscopy during the 6-month period was 1.3% (n = 2) among patients given the combination of esomeprazole and clopidogrel (n = 157) and 11.8% (n = 19) among patients given clopidogrel alone (n = 161) (difference, 10.5%; 95% confidence interval, 5.2%-15.8%, P < 0.001). Significantly clinical upper GI events developed in 0.6% (upper GI bleeding, n = 0; symptomatic ulcer, n = 1) and 5.6% (upper GI bleeding, n = 2; symptomatic ulcer, n = 7) of esomeprazole-plus-clopidogrel group and clopidogrel group, respectively. The incidence of clinically significant upper GI events in the former were less than that in the latter (difference, 5.0%; 95% confidence interval, 1.3-8.8%, P = 0.020). Conclusions: PPI can prevent clinically significant upper GI events in clopidogrel users with a history of peptic ulcer.

112 MTG16 Functions as a Tumor Suppressor in Murine Inflammatory Carcinogenesis Elizabeth M. McDonough, Caitlyn W. Barrett, Amber Bradley, Yash A. Choksi, Wei Ning, Shenika Poindexter, Bobak Parang, Melissa Fischer, Rupesh Chaturvedi, Frank Revetta, Kay Washington, Scott W. Hiebert, Keith T. Wilson, Christopher S. Williams MTGR1, MTG8 and MTG16 are members of the myeloid translocation gene (MTG) family, originally identified in the 8:21 translocation in acute myeloid leukemia. The MTGs function as transcriptional co-repressors, participate in developmental and epithelial wound healing and repair processes. Our group has demonstrated that Mtgr1 and Mtg16 modulate murine colonic injury responses. We also found decreased MTGR1 and MTG16 mRNA levels in moderate-severe ulcerative colitis. Additionally, Mtgr1 has been linked as a tumor modifier in murine colitis associated carcinoma. We postulate that Mtg16 may also contribute to tumor formation after inflammatory injury. To test this hypothesis, age and sex-matched wild-type and Mtg16-/- C57BL6 mice (n=40) were treated with azoxymethane (AOM) followed by two cycles of colonic injury using 2% dextran sodium sulfate (DSS). Weights, stool scores, and mortality were measured during each day of treatment. At the end of the second cycle, colonoscopies were performed to evaluate tumor burden. Mice were sacrificed on day 42 and colons were removed, tumor multiplicity and burden were determined. Half of the colon was swiss-rolled, fixed and embedded, and 5 micron sections were cut and H&E staining was performed. Immunohistochemistry was performed using TUNEL staining for apoptosis, phosphohistone H3 for proliferation, and β-catenin for quantification and nuclear localization. The remainder was saved for RNA and protein analysis. Mtg16-/- mice showed worsening colitis in response to AOM/DSS when compared to wild type mice with more severe weight loss, stool scores and overall mortality. Additionally, Mtg16-/- mice had increased tumor multiplicity (14.5 vs 1.2 P<0.001) and increased tumor burden compared to the wild type mice (4.8 mm2 vs 1.7 mm2 P<0.001). Histologic analysis of colons showed increased

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inflammation scores in the Mtg16-/- mice (10.2 vs 7.2 P<0.05) and more severe dysplasia 89% of WT mice with no or low grade dysplasia and 61.5% of Mtg16-/- with high grade dysplasia or invasive adenocarcinoma (P<0.01). TUNEL staining revealed increased apoptotic cells per crypt in the Mtg16-/- mice (0.49 vs 0.34 P<0.05) and interestingly Mtg16-/- mice had higher intra-tumoral apoptosis (35.5 vs 26.1 p<0.05). Because MTG16 regulated hematopoietic stem cell programs we excluded a hematopoietic cell-autonomous role for MTG16 in this model via transplantation of WT marrow into Mtg16-/- mice - which failed to rescue the Mtg16-/- AOM/DSS phenotype. MTG16 is an important modulator of colitis and tumor development in the AOM/DSS model of inflammatory carcinogenesis. This represents the first observation linking MTG16 to colonic wound healing and carcinogenesis; suggesting that Mtg16 is a tumor suppressor in inflammatory carcinogenesis.

UC and a median IBD duration of 14 (0-45) years. In both groups, 63% were male and median age was 60 years (range 45-72 and 45-77 for cases and controls respectively). Four cases and 5 controls had primary sclerosing cholangitis (PSC). Case neoplasms included 9 cancers [median size 2.3 (0.8-5) cm; 6/9 (66%) were proximal to splenic flexure; median AJCC stage was I (range I-IIIC)], and 10 dysplastic lesions [8 visible adenomas (dysplasia grade: 3 high, 5 low) with median size 2.3 (1.0-6.2) cm and two flat lesions (1 high grade, 1 low grade) detected by random biopsy]. Univariate and multivariate logistic regression tested the association of marker copy numbers, and potential confounders, to IBD-CRN and cancer. Results: All 3 markers individually showed high discrimination for IBD-CRN: areas under the receiver operating characteristics curves (AUC) with VIM, NDRG4 and EYA4 were 0.91, 0.84 and 0.85, respectively. For cancer the AUC with VIM, NDRG4 and EYA4 were 0.97, 0.94 and 0.95, respectively. Stool assay of VIM alone at 95% specificity yielded a sensitivity for IBD-CRN of 68% (95% CI 43-86%) and for cancer of 89% (95% CI 51-99%). Neither IBD disease duration nor comorbid PSC influenced marker levels. Combining markers did not improve discrimination. Conclusions: Stool assay of methylated VIM, NDRG4 or EYA4 highly discriminates IBD-CRN cases from IBD controls. These data corroborate our earlier proof-of-concept report of IBD-CRN detection by stool assay of methylated DNA markers. Further studies are indicated to evaluate this noninvasive approach as a complement to endoscopic strategies in IBD surveillance cohorts.