t1898 sox2 and cdx2, transcriptional regulators of early differentation, play a key role in the...

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sAdditional application of bile acids results in Barrett like metaplasia and constitutes apromising model for studying Barrett's Esophagus and its progression to adenocarcinoma.

T1896

Stimulatory Effect of Luminal Nitric Oxide On the Development of Barrett'sEsophagus in RatHiroyuki Endo, Kiyotaka Asanuma, Katsunori Iijima, Shuichi Ohara, Tooru Shimosegawa

Aim: Many studies reported that gastric acid and bile acid could affect the development ofBarrett's esophagus (BE), but the pathogenesis of BE has not been clarified. It has beendemonstrated that cytotoxic concentration of nitric oxide (NO) is generated luminally atgastroesophageal junction through the entero-salivary re-circulation of dietary nitrate inhuman. Furthermore, the site of luminal NO generation shifts to the lower esophagus whengastric acid refluxes into the esophagus. In the present study, using a rat model, we investig-ated whether NO generated luminally could affect the development of BE. Method: Wemade a rat model of BE by performing an esophagojejunostomy and gastrojejunostomy aspreviously described (Tao Zhang, et.al. DDS 2007). In this model, it was proved that bothgastric and duodenal contents refluxed into the esophagus with the esophagus maintainedat acidic pH. After the surgery, rats were divided into two groups. One group was adminis-trated 0.05% of sodium-nitrite (NaNO2) in tap water and 1.0% of ascorbic acid in powdereddiet. (Ascorbic acid converts NaNO2 to NO in the acidic condition.) The other group wasadministrated a tap water and conventional diet as controls. Four and eight weeks later,rats were sacrificed and the esophagus was taken. Severity of inflammation was assessedmacroscopically and incidence of BE was assessed microscopically in each group, as well.Furthermore, CDX2, MUC2, MUC5AC and MUC6 expression were investigated by immuno-histochemistry in order to elucidate histogenity of Barrett's esophagus. Results: The severityof inflammation tended to be increased in NaNO2 group compared with controls. At the4 weeks, BE was more frequently emerged significantly in NaNO2-administered groupcompared with controls with the incidence of 40% and 5%, respectively (P<0.05). Sub-sequently, at 8 weeks, BE was observed in substantial parts of both groups (77.8% in NaNO2group v.s. 62.5% in controls). These findings suggest that administration of NaNO2 couldaccelerate the development of BE in the rat model. CDX2, MUC2, MUC6 immunostainingwere positive and MUC5AC immunostaining was negative in the emerged columnar epithe-lium in the rat model, being consistent with immunohistochemical property of BE in human.Conclusion: In this study, we found that administration of nitrite accelerates the developmentof BE in the rat model, suggesting that NO generated luminally in the esophagus may beinvolved in columnar transformation of squamous epithelium of the esophagus.

T1897

Conversion of Goblet to Non-Goblet Columnar Metaplasia of the Esophagus. aClinical/Pathologic and Molecular Study of 10 CasesTanya A. Rege, Carissa A. Sanchez, Xiaohong Li, David Cowan, Brian J. Reid, Patricia L.Blount, Robert D. Odze

Background: In patients with Barrett's esophagus (BE), it is presumed that only esophagealcolumnar epithelium with goblet cells is at risk for neoplastic progression. However, wehave noted, anecdotally, that a small proportion of patients with established BE convert tonon-goblet columnar epithelium over the course of long term endoscopic surveillance. Thisphenomenon has never been investigated, and the aim of this study was to evaluate theclinical, pathologic and flow cytometric abnormalities of BE patients who have convertedfrom goblet to non-goblet columnar epithelium, and to compare the data with a large groupof “non-converters.” The results have implications with regard to the ACG definition of BE,which require goblet cells to be identified in order to establish the diagnosis. Design: Duringa 7 year period (2001-2008) in a prospective BE surveillance cohort in which all patientshad 4 quadrant biopsies every 2 cm of esophagus, 10 BE patients out of 333 (3%) whoregressed from goblet to non-goblet cell columnar epithelium of the esophagus as confirmedin their last 2 surveillance endoscopies, were identified. After exclusion of 137 patients whohad cancer or EMR during surveillance, the final (N=10) cases and 186 controls wereevaluated for a variety of clinical and pathologic features, including flow cytometric character-istics (increased 4N fraction, aneuploidy). Results: BE converters (mean follow-up = 59.6months) showed a similar male/female ratio (8/2), but were significantly older (mean age:67 years) compared to the 186 non-converters (mean follow-up = 48.7 months) (M/F:149/37, p=0.8, mean age: 63.7 years, p=0.07). Converters showed a significantly shorter meanlength of esophageal columnar epithelium at baseline (1.6 cm, range: 1-6) and a significantlyshorter length at last endoscopy (mean 1.3 cm, range 1-4) compared to non-converters (5.4(1-17) and 5.0 (1-16), respectively, p<0.01 for both comparisons). The 10 convertersshowed a similar proportion of cases with tetraploidy (30%), aneuploidy (40%), or any flowabnormality (50%) compared to non-converters (23%, 16% and 31%, respectively, p> 0.05for all comparisons). The prevalence rate of dysplasia/cancer in the two patient groupswere statistically similar. Conclusions: Conversion of goblet (BE) to non-goblet columnarepithelium of the esophagus is rare (prevalence rate = 3%), but is more common in BEpatients with shorter segments of columnar epithelium. Conversion of BE to non-gobletepithelium is not associated with loss of flow cytometric abnormalities, suggesting that thesepatients may still be at risk for cancer and should remain in endoscopic surveillance.

T1898

SOX2 and CDx2, Transcriptional Regulators of Early Differentation, Play aKey Role in the Development of Acid and Bile-Associated ColumnarMetaplasiaYuvnish Bhardwaj, Cathrine J. DeMars, Shalini Achra, Marlys Anderson, Ganapathy A.Prasad, Kenneth K. Wang, Raul A. Urrutia, Navtej Buttar

BACKGROUND: The mechanism of development of Barrett's metaplasia remains to bedefined. Using an endodermal differentiation model, we have previously identified a set ofgenes which show changes in temporal expression during embryonic differentiation of

A-596AGA Abstracts

endoderm to squamous or columnar epithelium. The functional relevance of these differenti-ally expressed genes needs further investigation. The AIM of our study was to examine thefunctional relevance of transcriptional regulators, SOX2 and CDX2, in context of cytokeratinexpression changes that are reminiscent to squamous versus columnar differentiation.METHOD: Primary squamous and Barrett's epithelial cells as well as HaCaT, a humankeratinocyte cell line, were used for the experiments. Treatments included exposure to pulsesof acid for 15 minutes at pH 4.5, four times daily, with bile salts for up to 6 days. Westernblot, immunofluorescence and quantitative PCR were used to quantify the expression ofCK7, CK10/13, CDX2 and SOX2. We used SOX2 siRNA and CDX2 siRNA to examine theeffect of loss of function of these genes on the cytokeratin expression in presence or absenceof acid+bile treatment. RESULTS: We found that exposure of primary squamous cells aswell as HaCaT cells to acid+bile resulted in decreased SOX2 and increased CDX2 expression.As shown in the figure below, we also found that the treatment with acid+bile decreased themarkers of squamous differentiation (CK10/13) and increased the columnar differentiationmarker (CK7). SOX2 knockdown by siRNA in HaCaT cells resulted in decreased squamousand increased columnar differentiation markers. We next treated HaCaT cells with acid+bileto increase CDX2 expression and then treated the cells with control or CDX2 siRNA. Wenoted that in the presence of CDX2 siRNA, acid+bile failed to decrease the markers ofsquamous differentiation as well as failed to increase the columnar differentiation marker.CONCLUSION: We found that SOX2 and CDX2, transcriptional regulators that were pre-dicted by endodermal differentiation model, have functional relevance as shown by thealteration of cytokeratin expression pattern. The mechanisms and the interplay betweenSOX2 and CDX2 are being investigated. Other markers predicted based on this model arealso being examined.

T1899

Insulin Resistance As a Risk Factor for Barrett's EsophagusKatarina B. Greer, Lacie Brenner, Kayode Olowe, Beth Bednarchik, Anokh Kondru, GaryW. Falk, Dawn Dawson, William M. Grady, Joseph Willis, Gregory S. Cooper, Li Li,Amitabh Chak

Background and aims: Obesity has been associated with esophageal adenocarcinoma (EAC)and its precursor Barrett's esophagus (BE), independent of gastroesophageal reflux (GERD).Hyperinsulinemia related to obesity leads to increased cellular proliferation and decreasedapoptosis, which is postulated to be one mechanism for carcinogenesis. The aim of this casecontrol study was to investigate obesity, central adiposity and hyperinsulinemia defined byinsulin resistance (HOMA-IR) as a risk factor for BE. We also explored the role of totaladiponectin and leptin as risk factors for Barrett's esophagus. Methods: BE patients (n=97)were recruited from consecutive patients presenting to a tertiary care institution and comparedwith GERD controls (n=112). Collected data included baseline demographic characteristics,anthropometric measures, fasting glucose, insulin, adiponectin and leptin concentrations.Patients were categorized as overweight or obese based on WHO criteria of calculated bodymass index (BMI). Central adiposity was defined as waist hip ratio (WHR) > 0.85. Insulinresistance was estimated through homeostatic model assessment (HOMA). Proportions ofobese subjects among cases and controls were compared by a chi-square test. Univariateand multivariate regression analysis was performed on all variables of interest. Results:Patients with BE had higher BMI than GERD controls (31.1 vs. 29.4 kg/m2, p=0.04). Afteradjustment for demographic factors, BMI emerged as a consistent risk factor for BE. Riskof BE was increased over 2 fold in obese subjects compared to normal weight controls (OR2.76, 95% CI 1.12, 6.80). Central adiposity did not increase risk of BE in our studypopulation (OR 1.46, 95% CI 0.26, 7.76). Patients with BE had higher baseline insulinresistance as assessed by HOMA (2.42 vs. 1.87, p=0.04). In a multivariate logistic regressionanalysis, insulin resistance was independently associated with the presence of BE afteradjusting for age, sex, and gender (adjusted OR 1.28, 95% CI 1.05, 1.57). There was norelationship between serum adiponectin levels and BE cases status (OR 0.68 95% CI 0.28,1.65). High levels of serum leptin did not increase BE risk in our study sample (1.68, 95%CI 0.61, 4.34). Conclusions: Obesity and insulin resistance are associated with increasedrisk of BE. The role of the proliferative insulin pathway in development of esophageal tissuemetaplasia needs to be explored at the tissue level.

T1900

A Novel 3D Ex Vivo Model of Native Human Barrett's OesophagusNatalia Scobioala-laker, Amy Reynolds, Alyson Parris, Esther M. Mitchell, Michael P.Lewis, Hugh J. Kennedy, William Stebbings, Alison Prior, Martin Phillips, Ian Beales,Mark R. Williams

Barrett's oesophagus is the replacement of the normal oesophageal stratified, squamousepithelial lining of the lower oesophagus with a glandular polarised columnar epithelium.Columnar epithelial cells exhibit an intestinal metaplastic-phenotype characterised by themorphological presence of numerous glands or crypts. An understanding of the molecularand cellular basis for the development and progression of Barrett's metaplasia is requiredto develop effective clinical management strategies. This has been hampered by the lack ofan ex vivo model of human Barrett's oesophagus. Previous work in our laboratory hasdeveloped a novel ex vivo model of the human colonic epithelium (Reynolds et al., J. Physiol2007). AIMS: To develop a 3D ex vivo culture model of isolated Barrett's crypts that isamenable to real-time imaging. METHODS: Tissue samples of Barrett's oesophagus wereobtained at endoscopy (Ethical approval ). Biopsies were incubated in a calcium-free, hepes-buffered saline solution and Barrett's crypts were liberated by vigorous shaking. Barrett'scrypts were placed into a 3D culture system for up to 5 days. Viability of cultured Barrett'scrypts was assessed by calcein-AM uptake and propidium iodide exclusion. The activity ofa number of signalling pathways implicated in the genesis/maintenance of Barrett's crypts