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Optimizing the Response to Thiopurine Therapy: A Search for NovelExplanations for Thiopurine HypermethylationPaul A. Blaker, Annemieke M. Peters van Ton, Monica Arenas, Melissa A. Smith,Catherine H. Smith, Peter M. Irving, Anthony M. Marinaki, Jeremy D. Sanderson

Background. Thiopurines are not effective in up to 1/3 of patients with inflammatory boweldisease (IBD) and 1/5 have to discontinue therapy due to side effects. The most importantcause of these problems is thiopurine hypermethylation. This is a catabolic process leadingto an unfavourable thiopurine metabolite profile (high methyl-mercaptopurine (6-MeMP)to low thioguanine nucleotide (TGN) ratio; >11:1). This is observed in 15% of patients andis not predicted by pre-treatment measurement of thiopurine-S-methyltransferase (TPMT)activity. Importantly thiopurine hypermethylation can be circumvented with the use allopuri-nol in combination with low dose AZA/6-MP. The aim of this research is to establish themechanism of thiopurine hypermethylation and identify predictive genetic markers to allowearly combination therapy. We hypothesized that thiopurine hypermethylation occurs as aresult of genetic factors that affect methylation flux and the cellular transport of methylatedmetabolites. Methods. A well-defined cohort of 168 age and dose-matched patients prescribedAZA/6-MP was identified from the records of the Purine Research Laboratory (PRL). GenomicDNA was extracted from EDTA blood samples of 76 patients demonstrating thiopurinehypermethylation and 92 patients with normal methylation profiles. Polymorphic sequencevariants in genes predicted to affect thiopurine methylation flux and cellular metabolitetransport were identified from single nucleotide polymorphism (SNP) databases and geno-typed by Taqman assay. Associations were tested using Fisher's Exact test. Results. We founda significant association between the haplotype of rs9332377 T and rs4646316 C, whichencodes a low-activity synonymous Catechol-O-methyltransferase (COMT) variant, and pro-tection from thiopurine hypermethylation (rs9332377 T, p=0.0178, rs4646316 C, p=0.03).A polymorphism in the nucleo-base transporter, ABCB5, was significantly associated withthiopurine hypermethylation (rs2031641 G/G, p=0.0098). The association was strengthenedwhen patients with 6-MeMP levels >5000 pmol/L versus 6-MeMP <5000 pmol/L werecompared (p=0.0065). In keeping with this we have previously reported that polymorphismin the ABCB5 gene is associated with a lack of clinical response to thiopurines. Conclusions.Changes in methylation flux due to the activity of methyltransferases other than TPMT affectthe formation of thiopurine methylated metabolites, likely through direct competition forthe essential co-factor S-adenosylmethionine. Furthermore, polymorphism in the ABCB5gene, which affects the nucleotide-binding domain of this transporter, is associated withthiopurine hypermethylation, suggesting reduced cellular efflux of methylated metabolites.Further studies are now indicated to establish the role of these genetic markers in clinical prac-tice.

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Therapeutic Diagnostic: Identification of Molecular Mechanisms UnderlyingUlcerative Colitis in Infliximab Non-Responders at BaselineMichael Macoritto, Ty Thomson, Jennifer S. Park, David Drubin, Renee Deehan-Kenney,David De Graaf, Daphna Laifenfeld

One fundamental challenge in the treatment of patients is heterogeneity of the molecularmechanisms of disease resulting in a varied response to treatment among individuals. Inpatients with ulcerative colitis (UC), anti-TNF therapy is successful, with 61-69% of patientsresponding in the first two months of treatment with infliximab, presumably because theirdisease is driven by TNF. There remain 31-39% of patients who do not respond, andmay havealternative disease mechanisms. Another challenge is the co-development of a therapeuticdiagnostic (TDx) to select patients most likely to respond prior to availability of clinicaloutcome data. We applied a systems biology methodology to identify: 1. Alternate molecularmechanisms of UC in infliximab non-responders 2. Candidate TDx to identify patients witha greater prospect of responding to therapies targeting these alternate mechanisms in theabsence of clinical outcomes data Over 2000 “molecular footprints” (MF) (molecular, contextdependent activation patterns (e.g., TNF)) were used to infer measures of activity for allentities in each patient of a public UC transcriptomic data set. Grouping patients with similaractivity patterns identified two subpopulations of patients that differ on their levels of threemain mechanisms, Il6, Vegf, and Il1b, and patients with high activity levels are non-responders to infliximab. The patients were then stratified by the activities of each of thesemolecular mechanisms and the stratification was used to train classifiers for high and lowactivity for each mechanism. The classifiers were applied to UC patients in an independenttest data set, using infliximab response as a basis of successful prediction in lieu of actualresponse data for targeted therapies to these mechanisms. Infliximab non-responders hadrelatively high levels of signaling for these disease mechanisms with a high degree ofconsistency in the test set. Using patient stratification by target activity as a substrate,candidate biomarkers for predicting therapeutic response can be discovered without a prioriknowledge of outcome.We verified this approach retrospectively using data for InflammatoryBowel Disease (IBD) patients treated with infliximab. Using the TNF MF, we stratified atraining set of IBD patients by inferred TNF activity, then generated gene expression andmechanism-based classifiers from patients with high and low TNF signaling. When appliedto a test set, the mechanism and gene expression classifiers predicted responders with positiveand negative predictive values of 79/88% and 71/100% respectively. This method can beapplied to other diseases and may facilitate target pathway identification and TDx develop-ment and enabling the selection of appropriate patient populations prior to treatment andclinical trial participation.

S-115 AGA Abstracts

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Effect of Flexible Sigmoidoscopy Screening on Incidence and Mortality FromColorectal Cancer in the PLCO Screening TrialRobert E. Schoen, Paul Pinsky, Joel Weissfeld, Lance A. Yokochi, Timothy R. Church,Adeyinka O. Laiyemo, Robert S. Bresalier, Thomas L. Riley, Philip C. Prorok, ChristineBerg

Studies in the UK and Italy demonstrate a reduction in distal CRC incidence and mortalitywith FSG screening. No significant effect in the proximal colon was observed, but only 5-8% of subjects underwent colonoscopy. In the U.S., the multi-center, randomized Prostate,Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial compared FSG with usualcare. Most subjects with abnormal FSG underwent colonoscopy for diagnostic follow up.Aim: To evaluate the effect of flexible sigmoidoscopy on CRC incidence and mortality.Methods: From 1993 to 2001, men and women age 55-74 were randomized to FSG screeningwith a repeat exam at 3 or 5 years, versus usual care. Colorectal cancers and deaths wereascertained and cause of death was evaluated in a formal adjudication process. Results: Atotal of 77,445 participants were randomized to intervention and 77,455 to usual care.Mean follow-up time (11.2 years) in the two arms was similar, and extended maximally to13 years. Over 86% of subjects (67,071/77,445) underwent ≥1 screen, 50.9% (39,440/77,445) had two, and 28.5% (N=22083) had ≥1 exam positive for a polyp or mass. Ofsubjects with an abnormal screen, 80.5% (17,772/22,083) underwent diagnostic interventionwithin 1 year, 95.6% (N=16,990/17,772) of whomunderwent colonoscopy, for a colonoscopyrate of 21.9% (16,990/77,445) as a direct effect of FSG screening. The incidence of CRCwas 11.9/10,000 person years (PY) in the intervention arm compared to 15.2/10,000 inusual care, a 21% reduction (RR=0.79; 95% CI .72-.85, P<0.0001). Mortality due to CRCwas observed in 3.9/10,000 PY in the usual care arm (341 deaths) compared with 2.9/10,000 PY in the intervention arm (252 deaths), a 26% reduction (RR=0.74; 95% CI .63-.87, P=0.0003). Conclusions: Screening with flexible sigmoidoscopy resulted in a statisticallysignificant and clinically important decrease in overall CRC incidence and mortality. Informa-tion on the effect of the intervention in the distal and the proximal colon will be forthcoming.

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Modulation of the Brain-Gut Axis After 4-Week Intervention With a ProbioticFermented Dairy ProductKirsten Tillisch, Jennifer S. Labus, Bahar Ebrat, Jean Stains, Bruce D. Naliboff, DenisGuyonnet, Sophie Legrain-Raspaud, Beatrice Trotin, Emeran A. Mayer

BACKGROUND: Preclinical studies in rodents show that changes in gut microbiota altercentral signaling mechanisms and emotional behavior (1, 2). However, such changes havenever been demonstrated in humans by modulating the gut microbiota with probiotics orantibiotics. AIM: To determine whether consumption of a probiotic fermented dairy productalters response in emotional arousal and visceral afferent brain regions during an EmotionalReactivity Task (ERT) in healthy women. METHODS: In this double-blind, controlled,parallel study, 45 healthy women, age 18-50, were screened to rule out medical or psychiatricillness. They were randomly assigned to three intervention groups, no product (NoP),control milk-based non-fermented dairy product (CTRL), or probiotic dairy product (TEST)consisting in a fermented dairy product containing B. lactis CNCM I-2494, a yoghurtsymbiosis L. bulgaricus and S. thermophilus, as well as L. lactis. CTRL and TEST groupsconsumed twice daily 125 gram products for 4 weeks. All groups performed the ERT duringfunctional magnetic resonance imaging (fMRI) at randomization and at the end of the 4-week intervention period. The ERT consisted of viewing negative emotional faces and viewingshapes as a control. fMRI blood oxygen level-dependent (BOLD) response was measuredusing a 3 Tesla scanner. Pre-post intervention BOLD subtraction images were compared usingthe general linear model implemented in SPM. Small volume corrections were performed inpre-hypothesized regions (amygdala, anterior cingulate, insula, prefrontal cortex-PFC). Partialleast squares analysis was performed to assess intervention related connectivity betweeninsula based and amygdala based networks. RESULTS: The TEST group exhibited less BOLDresponse in the mid/posterior insula during the ERT compared to CTRL (pFWE=.002; MNI46, 2, 6) and NoP (pFWE=.016; MNI 36, -18, 20) groups. No BOLD differences were notedin the cingulate, PFC, or amygdala. The TEST group showed decreased connectivity of anamygdala-centered networkwith the insula, dorsal striatum and lateral PFC. CONCLUSIONS:To our knowledge, this is the first study in humans demonstrating that consumption of aprobiotic fermented dairy product can be associated with modulations of brain activity. Thefindings suggest that changes in the gut microbiota by regular intake of a probiotic can (i)affect brain regions concerned with the central processing of afferent signals from the gut,and (ii) reduce the impact of the brain regions involved in emotional arousal on the centralprocessing of gut afferent signals. One may speculate that the observed changes are mediatedby effects on sensory encoding mechanisms in the gut. References: 1) Bravo JA, et al. PNASUSA 2011 2) Diaz Heijtz, R, et al. PNAS USA 2011

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One-Year Effectiveness and Costs of Six Alternative H. pylori Test/Treat andRetest/Retreat Strategies Using Triple, Concomitant or Sequential DrugRegimens in Seven Latin American Sites (SWOG Trial S0701)Javier Torres, Douglas R. Morgan, E. R. Greenberg, Eduardo Salazar-Martinez, RicardoDominguez, Catterina Ferreccio, Luis Eduardo Bravo, Rodolfo Peña, Rolando Herrero,Mercedes Meza-Montenegro, Maria E. Martinez, Pelayo Correa, Manuel Valdivieso, GarnetL. Anderson, William D. Chey, John Crowley, Laurence H. Baker

BACKGROUND: The potential feasibility of H. pylori (Hp) eradication strategies to preventgastric cancer depends on the prevalence of infection in the community, the longer-termprobability of remaining uninfected following treatment, and the program cost per personwho is freed of infection. We assessed the effectiveness and costs of different strategies usingdata from a large clinical trial in Latin America where gastric cancer risks are high. METHODS:We recruited 1852 adults (ages 21-65) from the general populations of seven communitiesand screened them with a urea breath test (UBT). 1463 individuals (79.4%) tested positive

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sand were randomized to three treatment regimens: 14 days of lansoprazole, amoxicillin andclarithromycin (PAC-14); 5 days of lansoprazole and amoxicillin followed by 5 days oflansoprazole, clarithromycin and metronidazole (SEQ-10); or 5 days of lansoprazole, amoxic-illin, clarithromycin, and metronidazole (PACM-5). Participants had UBTs 6-8 weeks afterrandomization, and retreatment with 14-day lansoprazole, bismuth, metronidazole andtetracycline was offered to those remaining positive. Program effectiveness was determinedfrom the 1340 (91%) participants with a definitive UBT 48-52 weeks after randomization,and effectiveness absent retesting and retreatment was estimated assuming all UBT-positivesat 6-8 weeks remained so. Estimated costs were $12 for PAC-14, $7 for SEQ-10, $6 forPACM-5, $10 for retreatment, and $10 for each UBT. RESULTS: Of the 281 UBT-positiveparticipants at 6-8 weeks, 213 (76%) chose to be retreated, and 143 (51%) completedretreatment; 244 had a definitive 1-year UBT result, of whom 93 (38%) tested negative.The observed 1-year eradication probabilities and estimated costs per treatment success forthe three treatment arms were 80.5% and $44 for PAC-14, 79.8% and $39 for SEQ-10,and 77.8% and $39 for PACM-5 (Table). Under the scenario of no retesting and retreatment,the estimated eradication probabilities were 5-8% lower, but the estimated costs per treatmentsuccess declined substantially more, to $32 for PAC-14 and $27 for SEQ-10 and PACM-5.The principal contributor to the cost differential was the cost of the follow-up UBTs ratherthan the low probability of retreatment success. CONCLUSIONS: Data from this LatinAmerican study suggest that repeat UBT testing and retreatment after initial therapy addsubstantially to program costs but only modestly increase the probability of Hp eradicationafter one year. In settings where resources are scarce, retesting and retreating do not appearto be indicated for programs to prevent Hp-associated gastric cancer. Increasing the effect-iveness of eradication therapies and lowering the risk of recurrence will be better approachesfor improving program performance. SUPPORT: Bill & Melinda Gates Foundation grant43930 and NIH grant CA037429.Estimated one-year outcome and costs by treatment arm with and without retesting andretreatment.

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Obese Pediatric Patients Have Increased Cardiovascular Disease RiskComparable to Pediatric Patients With Familial DyslipidemiaAngela Shannon, Suraj Thangada, Christine Carter-Kent, Ariel E. Feldstein, MichaelRocco, Leslie Cho, Stanley L. Hazen, Naim Alkhouri

Pediatric patients with familial dyslipidemia (FD) are known to be at increased risk ofdeveloping cardiovascular disease (CVD) and are often treated aggressively in order to preventmanifestations of CVD. Obese pediatric patients are also at increased cardiovascular riskbut this risk is less well defined. Myeloperoxidase (MPO) and high-sensitive C-reactiveprotein (hsCRP), markers associated with cardiovascular disease(CVD) in adults, may alsobe predictive of future cardiovascular disease in predisposed children. Our aim was toinvestigate the association between MPO and hsCRP with pediatric obesity and then comparethese markers between pediatric patients with FD and those with obesity in order to betterdescribe the CVD risk in obese pediatric patients. Methods: Data was collected prospectivelyon consecutive patients attending the Pediatric Preventive Cardiology and Metabolic Clinic(PPCMC) and theGenetics Clinic at Cleveland Clinic. Data included demographics, anthropo-metric and laboratory data. The PPCMC cohort was further divided into groups based onBMI (overweight, obese, severely obese) to investigate the association between MPO andhsCRP and obesity. Then, the overall group of patients was divided based on having adiagnosis of FD or overweight/obesity to compare CVD markers between the two groups.Results: 323 patients were included for analysis- 151 in the overweight/obese group and172 in the FD group. In the overweight/obese group, mean age was 12.8±3.5 yr, there were56% males, and 63% of the patients were Caucasian. In the FD group, mean age was12.2±4.4 yr, there were 52% males, and 80% of the patients were Caucasian. The meanLDL in the overweight/obese group was 107.7±4.3 mg/dL, and that of patients with familialhypercholesterolemia was 205.1±63.6 mg/dL (p<0.0001). Mean MPO in the overweight/obese group was 417±162 pmol/L and in the FD group was 424±162 pmol/L [CL (55.1,-69.7), p=0.41]. Mean hsCRP was 4.9±7.2 mg/L in the obese group and 5.6±26 mg/L in theFD group [CL (3.8, -5.2), p=0.38]. MPO and hsCRP levels both had a positive correlationwith BMI%ile (rho 0.42 and 0.47, respectively, p<0.001). Conclusions: This is the firstpediatric study evaluating the association between obesity and MPO and comparing CVDrisk between overweight/obese pediatric patients and patients with FD. MPO and hsCRPboth correlate with increasing BMI in pediatric patients. In addition, though overweight/obese pediatric patients may have a lower LDL than pediatric patients with familial dyslipide-mia, there is no significant difference when comparing nontraditional markers of CVD suchas MPO and hsCRP. We conclude that MPO and hsCRP are useful markers to identify obesepediatric patients at increased CVD risk, and obesity may in fact confer a CVD risk burdencomparable to patients with FD and earlier medical intervention may be warranted.

S-116AGA Abstracts

598

Eosinophil Derived TGF-β1 Activates Human Esophageal Mesenchymal Cellsand Alters Esophageal Motility - Implications for Dysphagia in EosinophilicEsophagitis (EoE)Florian Rieder, Ilche T. Nonevski, Jie Ma, Zhufeng Ouyang, Gail West, Cheryl A.Protheroe, Anja Schirbel, John R. Goldblum, Tracey L. Bonfield, Karen M. Harnett, JamesJ. Lee, Ikuo Hirano, Gary W. Falk, Piero Biancani, Claudio Fiocchi

Background: Dysphagia is a frequent symptom of EoE and may result from a combinationof esophageal fibrosis and motility abnormalities. We investigated mechanisms of dysphagiain EoE by assessing the response of primary human esophageal fibroblasts (HEF), musclecells (HEMC) and esophageal muscle strips to TGF-β1 and eosinophil-derived products.Spontaneous production of TGF-β1 in esophageal biopsies from EoE and control patientswas also investigated. Methods: The presence of eosinophils in the esophageal wall of EoEwas investigated by H&E and eosinophil peroxidase (EPX) stain of full thickness sections.Production of fibronectin (FN) and collagen I (Col I) by HEF and HEMC in response toTGF-β1 and eosinophil products was measured. αSMA, ICAM-1 and VCAM-1 expressionwas assessed by flow cytometry and immunocytochemistry. Eosinophil adhesion assays wereperformed using the human eosinophil cell line AML14.3D10. TGF-β1 and eosinophilsonicates were tested in esophageal muscle contraction assays assessing neurogenic andacetylcholine (ACh)-induced contraction. TGF-β1 was measured in undernatants of eso-phageal biopsy organ cultures by ELISA. Results: Activated eosinophils were present in alllayers of the esophageal wall. TGF-β1 induced a dose-dependent increase in FN and Col Isecretion as well as αSMA expression by HEF and HEMC, indicating transformation to apro-fibrogenic phenotype. Sonicates of pre-activated eosinophils also increased FN and ColI production by HEF and HEMC, an effect that was inhibited by blocking TGF-β1 andp38MAKP signaling. Eosinophil binding increased by pre-treating HEF and HEMC withTGF-β1 or eosinophil sonicates. Combined TGF-β1 and p38MAKP blockade reducedsonicate-induced eosinophil adhesion and completely abrogated TGF-β1-induced adhesion.TGF-β1 did not modulate VCAM-1 or ICAM-1 expression. Eosinophil sonicates and TGF-β1reduced electrical field-induced contraction of esophageal muscle strips. Neither eosinophilsonicates nor TGF-β1 influenced ACh-induced contraction, indicating a primary effect onneurotransmitter release without affecting the muscle itself. Significantly higher concentra-tions of TGF-β1 were present in EoE compared to control esophageal mucosal biopsycultures. Conclusion: Eosinophils infiltrate all esophageal layers in EoE. Eosinophil-derivedTGF-β1 increases matrix production by and eosinophil binding to esophageal mesenchymalcells and reduces neurogenic esophageal smooth muscle contraction. TGF-β1 is present inelevated amounts in EoE compared to controls. These results suggest a direct functionallink of TGF-β1 expression with altered fibrogenesis and motility in EoE, both of which maycontribute to development of dysphagia.

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in Esophageal Squamous Epithelial Cells From Patients With EoE,Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by ReducingBinding of STAT6 to the Eotaxin-3 Promoter.Xi Zhang, Edaire Cheng, Xiaofang Huo, David H. Wang, Qiuyang Zhang, Chunhua Yu,Stuart J. Spechler, Rhonda F. Souza

Introduction: EoE and GERD can have similar clinical and histological features. It has beenproposed that patients with these disorders can be distinguished by their response to protonpump inhibitors (PPIs). This is based on the assumption that reduced gastric acid secretionis the only important effect of PPIs and, therefore, only an acid-peptic disorder like GERDcan respond to PPIs. In earlier studies, we showed that omeprazole in high concentration(100 μM) blocked cytokine-stimulated secretion of eotaxin-3 protein (a potent eosinophilchemoattractant) in esophageal squamous cell lines from patients with EoE. This suggeststhat PPIs might have anti-inflammatory actions independent of their effects on gastric acidsecretion. Now, we have studied the effects of omeprazole in lower concentrations, includingthose that can be achieved in blood with conventional dosage, on IL-4-stimulated eotaxin-3 production in primary cultures of esophageal squamous cells from EoE patients. To explorethe anti-inflammatory mechanism further, we also studied the effects of omeprazole on IL-4-stimulated transcriptional activation of eotaxin-3.Methods: Primary cultures of esophagealsquamous cells (EoE1 and EoE2) and telomerase-immortalized esophageal squamous celllines (EoE1-T and EoE2-T) derived from two patients with EoE were treated with IL-4 (10ng/ml) in the presence or absence of 1-50 μM doses of acid-activated omeprazole for 48hours. To address transcriptional activation, EoE1-T and EoE2-T cells were pretreated for24 hours with 50 μM acid-activated omeprazole before the addition of IL-4. We measuredeotaxin-3 protein secretion by ELISA, mRNA by real-time PCR, STAT6 phosphorylation andits nuclear translocation by Western blot, and eotaxin-3 promoter binding by ChIP assay.Results: In primary EoE cells, IL-4-stimulated eotaxin-3 secretionwas blocked by omeprazolein a dose-dependent fashion (Table). Similar results were obtained in the EoE cell lines,although does-dependency was not as clear as in the primary cells. Omeprazole had noeffect on STAT6 phosphorylation or its translocation to the nucleus. However, omeprazolesignificantly reduced IL-4 stimulated STAT6 binding to the eotaxin-3 promoter (to 61.4±6.6%in EoE1-T and to 56 ±9.8% in EoE2-T cells). In addition, omeprazole had no effect oneotaxin-3 mRNA stability. Conclusions: In esophageal squamous epithelial cells from EoEpatients, omeprazole blocks IL-4-stimulated eotaxin-3 protein secretion by reducing bindingof STAT6 to the eotaxin-3 promoter. In primary cells, we observed significant effects on IL-4-stimulated eotaxin-3 secretion with omeprazole in concentrations that are achieved inblood with conventional dosage. These findings elucidate an acid-independent mechanismwhereby PPIs might heal EoE, and cast doubt on the assumption that a response to PPItherapy distinguishes EoE from GERD.Eotaxin-3 Protein Secretion (pg/ml/250Kcells±SEM)