S1061 Risk of Developing Dysplasia/Cancer in Patients With Short Segment Barrett's Oesophagus is...
1
AGA Abstracts BE >2cm, diagnosed 1973-1984. After excluding 2 incident cases of high grade dysplasia (HGD) at BE diagnosis, 19 dying within 2 yrs of BE diagnosis, 1 esophagectomy without HGD/ EAC and 11 lost to follow up, 133 patients (M/F 73/60) were taken into account. We distinguished 3 end points after BE diagnosis: symptomatic HGD/ EAC (sHGD/EAC), death without sHGD/EAC, and survival on 2-28-2008 without sHGD/EAC. Univariate ana- lysis, Kaplan-Meier and Log rank tests were used to analyse the data. Results: (See Table) 13 (10%) cases of sHGD/ EAC (6/7), were observed over 1967.2 persyrs of follow-up; 0.66% p.a (95% CI: 0.63-0.68). Patients who developed sHGD/EAC were at baseline characterized by a higher prevalence of low grade dysplasia (LGD) (54% vs 17%, p=0.005) and significantly larger BE segment (mean length: 8.3 cm vs 6.3, p=0.03). 96 (72%) patients died during follow-up without sHGD/EAC. Survivors without sHGD/ EAC had by far the lowest age at BE diagnosis (mean 42.8 yrs, log rank p<0.001), suggesting onset at early age. This group therefore comprises the best candidates for eventually establishing the maximum incubation period to sHGD/ EAC, which now stands at around 30 years. Conclusion: This is to our knowledge the longest running cohort of Barrett's patients. Our data indicate that the incidence of sHGD/EAC is in accordance with previous data despite the fact that we did not apply surveillance and the long observational period. Furthermore, LGD and a longer BE length at diagnosis were more prevalent in patients that developed sHGD/EAC. Finally, our data show a mean survival of 25,5 years without sHGD/EAC in patients diagnosed with BE at early age (mean 42.8 yrs). Characteristics different groups S1060 Increased Activity of the Leukotriene Pathway in Barrett's Metaplasia and Esophageal Adenocarcinoma James D. Shutt, Philip C. Boger, James Neale, Praful Patel, Anthony P. Sampson Background & Aims 5-lipoxygenase and its constituent pathway proteins metabolise arachi- donic acid to leukotrienes (LT), potent pro-inflammatory mediators. Up-regulation of this pathway is evident in a variety of tumour types and has been shown to play a role in promotion of cancer cell growth. We aimed to evaluate the activity of the 5-lipoxygenase pathway in esophageal adenocarcinoma, and its pre-malignant lesions; Barrett's esophagus, and Barrett's with high grade dysplasia (HGD). Method Esophageal biopsies were taken from patients with Barrett's esophagus (n=16), Barrett's esophagus next to HGD/adenocarcin- oma (4), HGD (2), esophageal adenocarcinoma (13), and squamous (11) controls. The biopsies were equilibrated in DMEM for 5 minutes before culture for a further 60 minutes in the presence of calcium ionophore A23187 (2uM). After methanolic extraction, evaporation and reconstitution in immunoassay buffer, the generated leukotrienes were quantified per mg of wet tissue weight, using highly-sensitive enzyme immunoassays for LTB4 and for total cysteinyl-leukotrienes (LTC4/D4/E4) (Cayman Chem). Adjacent biopsies were assessed by a gastrointestinal pathologist to confirm diagnosis. Results The A23187-stimulated concen- trations of LTB4 and total cysteinyl-leukotrienes generated by esophageal tissues increased significantly with increasing degree of dysplasia (p=0.011 and p=0.018 respectively). Signific- antly more LTB4 was produced by adenocarcinoma (644 +/- 299 pg/mg (mean +/- SEM)) tissue than by Barrett's esophagus (80.8 +/- 22.6 pg/mg, p=0.018) or squamous tissue (36.5 +/- 4.14 pg/mg, p=0.002). Adenocarcinoma also produced significantly more total cysteinyl- leukotrienes (235 +/- 36.9 pg/mg) than Barrett's esophagus (113 +/- 13.6 pg/mg, p=0.003) or squamous tissue (137 +/- 33.6 pg/mg, p=0.019). No difference was seen in LTB4 or cys- LT concentrations between squamous and Barrett's biopsies (p=0.55 and p=0.96). Generation of LTB4 and total cys-LTs by adenocarcinoma tissue achieved nanomolar concentrations within the culture medium, exceeding the published dissociation constants (Kd) of the respective high-affinity LT receptors. Conclusions In this novel study, we have demonstrated generation of biologically-active concentrations of LTB4 and cys-LTs in whole endoscopic esophageal biopsy specimens, with a marked concentration gradient from Barrett's metaplasia through increasing dysplasia to adenocarcinoma. Leukotrienes may play a role in the progres- sion of the metaplasia-dysplasia-adenocarcinoma sequence. S1061 Risk of Developing Dysplasia/Cancer in Patients With Short Segment Barrett's Oesophagus is Similar to Long Segment Barrett's Oesophagus Venkataraman Subramanian, Naaventhan Palaniyappan, Ai May Lee, Jayan Mannath, Chris J. Hawkey, Krish Ragunath Introduction: There is increasing evidence that the incidence of Barrett's oesophagus (BO) is rising and the majority of these are the short segment (SSBO) variety. At the same time the incidence of oesophageal adenocarcinoma is also on the rise. While it is generally accepted that long segment Barrett's oesophagus (LSBO) is associated with an increased risk of developing dysplasia/cancer, the risk associated with SSBO is poorly defined. Aim: to estimate the risk of developing high grade dysplasia/cancer in patients with SSBO and LSBO under- going surveillance endoscopy. Methods: We identified patients, from the endoscopic records of Nottingham University Hospital, with a confirmed endoscopic and histological diagnosis of BO from 2002-2006. Duration of follow up was calculated from the time the first histological diagnosis of BO was made to the time of the last endoscopy. Incidence density (ID) of developing high grade dysplasia/cancer and incidence rate ratios (IRR) were calculated S-170 AGA Abstracts in patients with SSBE and LSBE. A Poisson distribution was assumed for purposes of calculation and analysis for IRR was adjusted for age and sex. Data was analysed using Stata version 9.1. Results: 703 patients with a confirmed histological diagnosis of BO were identified from the database. 470 patients (339 male and 139 female) had LSBO and 233 patients (141 male and 92 female) had SSBO. There were 70 dysplastic lesions (54 HGD/cancer and 26 LGD) in patients with LSBO and 16 dysplastic lesions (10 HGD/cancer and 6 LGD) in patients with SSBO. The crude incidence density of developing HGD/cancer in patients with LSBO was 31.5/1000 pyd (person years duration) (95% CI 23.7-41.2) and in patients with SSBO was 15.1/ 1000 pyd (95% CI 7.3-27.8). The age and sex adjusted incidence rate ratio of developing HGD/cancer among patients with SSBO compared to LSBO was 0.99 (95% CI 0.93-1.06). Conclusions: The risk of developing HGD/cancer in patients with SSBO appear to be similar to those with LSBO. Surveillance guidelines and physicians need to take this into account when planning surveillance strategies for patients with SSBO. S1062 Acid-Induced p16 Hypermethylation Contributes to Development of Esophageal Adenocarcinoma via Activation of NADPH Oxidase NOX5-S Jie Hong, Murray Resnick, Jose Behar, Li Juan Wang, Jack R. Wands, Ronald A. DeLellis, David Lambeth, Rhonda F. Souza, Stuart J. Spechler, Weibiao Cao Background: Inactivation of the tumor suppressor gene p16 may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Hyperme- thylation of the p16 promoter is an important mechanism for inactivating p16. However, the mechanisms involved in p16 hypermethylation in EA are not known. Since acid reflux appears to contribute to the progression from BE to EA by over expressing NADPH oxidase NOX5-S, we examined whether acid increases methylation of the p16 gene promoter by activating NOX5-S in a Barrett's cell line BAR-T and an EA cell line OE33. Methods: P16 mRNA and methylation of p16 promoter were measured by real-time PCR. Transfection of NOX5 siRNA and plasmid was carried out by using Lipofectamine 2000 and Amaxa- Nucleofector-System respectively. Result: BAR-T and OE33 cells expressed NADPH oxidase NOX5-S. Acid-induced increase in H 2 O 2 production was significantly reduced by knockdown of NOX5-S. Exogenous H 2 O 2 and acid treatment significantly increased methylation of p16 promoter. In addition, acid treatment significantly decreased the p16 mRNA level. Knock- down of NOX5-S significantly increased p16 mRNA, inhibited acid-induced down-regulation of p16 mRNA. Knockdown of NOX5-S also blocked the acid-induced increase in p16 methylation. Conversely, overexpression of NOX5-S significantly decreased p16 mRNA and increased p16 methylation. Conclusion: NOX5-S is present in BAR-T cells and OE33 cells, and mediates acid-induced H 2 O 2 production. NOX5-S is also involved in acid-induced hypermethylation of the p16 gene promoter and in downregulation of p16 mRNA. It is possible that acid reflux in BE patients may activate NOX5-S. NOX5-S-derived ROS in turn increases p16 promoter methylation and down-regulates p16 expression, thereby contribut- ing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703. S1063 The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate Bile Acid-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells Jie Hong, Murray Resnick, Jose Behar, Jack R. Wands, Li Juan Wang, Ronald A. DeLellis, David Lambeth, Weibiao Cao Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for the development of esophageal adenocarcinoma (EA). However, the mechanisms of progression from BE to EA are not fully understood. Bile acids may play an important role in this progression. The aim of this study was to determine the expression of a novel bile acid receptor TGR5 in BE and to examine the role of NADPH oxidase NOX5-S and TGR5 in taurodeoxycholic acid (TDCA)-induced increased cell proliferation. Immunohistochemical studies showed that TGR5 membranous staining was identifed in BE and EA, and was almost undetectable in esophageal squamous epithelial cells. TGR5 mRNA levels were significantly higher in EA tissues than in normal esophageal mucosa or Barrett's mucosa. In an EA cell line FLO, NADPH oxidase NOX5-S was present. TDCA significantly increased NOX5-S expression, H 2 O 2 production and thymidine incorporation in these cells. This increase in thymidine incorporation was significantly reduced by knockdown of NOX5-S. Knockdown of TGR5 markedly inhibited TDCA-induced increase in NOX5-S expression, H 2 O 2 produc- tion and thymidine incorporation. Conversely overexpression of TGR5 significantly enhanced TDCA's effects. In FLO cells transfected with NOX5-S luciferase reporter plasmid NOX5LP containing NOX5-S promoter fragment (-2501 to -1 from ATG), TDCA significantly increased the luciferase activity, an increase which was significantly decreased by phosphatidylinositol- specific phospholipase C (PI-PLC) inhibitor U73122. TDCA-induced increase in NOX5-S expression, H 2 O 2 production and cell proliferation was significantly reduced by knockdown of PI-PLC γ2 or ERK-2 MAP kinase using specific siRNAs. TDCA significantly increased ERK-2 phosphorylation, an increase which was decreased by PI-PLCγ2 siRNA. In conclusion, the TDCA-induced increase in cell proliferation depends on upregulation of NOX5-S expres- sion. TDCA-induced NOX5-S expression may be mediated by activation of the TGR5 receptor, PI-PLCγ2 and ERK-2 MAP kinase in FLO EA cells. Our data may provide potential targets to prevent and/or treat Barrett's associated esophageal adenocarcinoma. Supported by NIH NIDDK R01 DK080703.
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Transcript of S1061 Risk of Developing Dysplasia/Cancer in Patients With Short Segment Barrett's Oesophagus is...
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sBE >2cm, diagnosed 1973-1984. After excluding 2 incident cases of high grade dysplasia(HGD) at BE diagnosis, 19 dying within 2 yrs of BE diagnosis, 1 esophagectomy withoutHGD/ EAC and 11 lost to follow up, 133 patients (M/F 73/60) were taken into account.We distinguished 3 end points after BE diagnosis: symptomatic HGD/ EAC (sHGD/EAC),death without sHGD/EAC, and survival on 2-28-2008 without sHGD/EAC. Univariate ana-lysis, Kaplan-Meier and Log rank tests were used to analyse the data. Results: (See Table)13 (10%) cases of sHGD/ EAC (6/7), were observed over 1967.2 persyrs of follow-up; 0.66%p.a (95% CI: 0.63-0.68). Patients who developed sHGD/EAC were at baseline characterizedby a higher prevalence of low grade dysplasia (LGD) (54% vs 17%, p=0.005) and significantlylarger BE segment (mean length: 8.3 cm vs 6.3, p=0.03). 96 (72%) patients died duringfollow-up without sHGD/EAC. Survivors without sHGD/ EAC had by far the lowest age atBE diagnosis (mean 42.8 yrs, log rank p<0.001), suggesting onset at early age. This grouptherefore comprises the best candidates for eventually establishing the maximum incubationperiod to sHGD/ EAC, which now stands at around 30 years. Conclusion: This is to ourknowledge the longest running cohort of Barrett's patients. Our data indicate that theincidence of sHGD/EAC is in accordance with previous data despite the fact that we didnot apply surveillance and the long observational period. Furthermore, LGD and a longerBE length at diagnosis were more prevalent in patients that developed sHGD/EAC. Finally,our data show a mean survival of 25,5 years without sHGD/EAC in patients diagnosed withBE at early age (mean 42.8 yrs).Characteristics different groups
S1060
Increased Activity of the Leukotriene Pathway in Barrett's Metaplasia andEsophageal AdenocarcinomaJames D. Shutt, Philip C. Boger, James Neale, Praful Patel, Anthony P. Sampson
Background & Aims 5-lipoxygenase and its constituent pathway proteins metabolise arachi-donic acid to leukotrienes (LT), potent pro-inflammatory mediators. Up-regulation of thispathway is evident in a variety of tumour types and has been shown to play a role inpromotion of cancer cell growth. We aimed to evaluate the activity of the 5-lipoxygenasepathway in esophageal adenocarcinoma, and its pre-malignant lesions; Barrett's esophagus,and Barrett's with high grade dysplasia (HGD). Method Esophageal biopsies were takenfrom patients with Barrett's esophagus (n=16), Barrett's esophagus next to HGD/adenocarcin-oma (4), HGD (2), esophageal adenocarcinoma (13), and squamous (11) controls. Thebiopsies were equilibrated in DMEM for 5 minutes before culture for a further 60 minutesin the presence of calcium ionophore A23187 (2uM). After methanolic extraction, evaporationand reconstitution in immunoassay buffer, the generated leukotrienes were quantified permg of wet tissue weight, using highly-sensitive enzyme immunoassays for LTB4 and fortotal cysteinyl-leukotrienes (LTC4/D4/E4) (Cayman Chem). Adjacent biopsies were assessedby a gastrointestinal pathologist to confirm diagnosis. Results The A23187-stimulated concen-trations of LTB4 and total cysteinyl-leukotrienes generated by esophageal tissues increasedsignificantly with increasing degree of dysplasia (p=0.011 and p=0.018 respectively). Signific-antly more LTB4 was produced by adenocarcinoma (644 +/- 299 pg/mg (mean +/- SEM))tissue than by Barrett's esophagus (80.8 +/- 22.6 pg/mg, p=0.018) or squamous tissue (36.5+/- 4.14 pg/mg, p=0.002). Adenocarcinoma also produced significantly more total cysteinyl-leukotrienes (235 +/- 36.9 pg/mg) than Barrett's esophagus (113 +/- 13.6 pg/mg, p=0.003)or squamous tissue (137 +/- 33.6 pg/mg, p=0.019). No difference was seen in LTB4 or cys-LT concentrations between squamous and Barrett's biopsies (p=0.55 and p=0.96). Generationof LTB4 and total cys-LTs by adenocarcinoma tissue achieved nanomolar concentrationswithin the culture medium, exceeding the published dissociation constants (Kd) of therespective high-affinity LT receptors. Conclusions In this novel study, we have demonstratedgeneration of biologically-active concentrations of LTB4 and cys-LTs in whole endoscopicesophageal biopsy specimens, with a marked concentration gradient from Barrett's metaplasiathrough increasing dysplasia to adenocarcinoma. Leukotrienes may play a role in the progres-sion of the metaplasia-dysplasia-adenocarcinoma sequence.
S1061
Risk of Developing Dysplasia/Cancer in Patients With Short Segment Barrett'sOesophagus is Similar to Long Segment Barrett's OesophagusVenkataraman Subramanian, Naaventhan Palaniyappan, Ai May Lee, Jayan Mannath,Chris J. Hawkey, Krish Ragunath
Introduction: There is increasing evidence that the incidence of Barrett's oesophagus (BO)is rising and the majority of these are the short segment (SSBO) variety. At the same timethe incidence of oesophageal adenocarcinoma is also on the rise. While it is generally acceptedthat long segment Barrett's oesophagus (LSBO) is associated with an increased risk ofdeveloping dysplasia/cancer, the risk associated with SSBO is poorly defined. Aim: to estimatethe risk of developing high grade dysplasia/cancer in patients with SSBO and LSBO under-going surveillance endoscopy. Methods: We identified patients, from the endoscopic recordsof Nottingham University Hospital, with a confirmed endoscopic and histological diagnosisof BO from 2002-2006. Duration of follow up was calculated from the time the firsthistological diagnosis of BO was made to the time of the last endoscopy. Incidence density(ID) of developing high grade dysplasia/cancer and incidence rate ratios (IRR) were calculated
S-170AGA Abstracts
in patients with SSBE and LSBE. A Poisson distribution was assumed for purposes ofcalculation and analysis for IRR was adjusted for age and sex. Data was analysed using Stataversion 9.1. Results: 703 patients with a confirmed histological diagnosis of BO were identifiedfrom the database. 470 patients (339 male and 139 female) had LSBO and 233 patients(141 male and 92 female) had SSBO. There were 70 dysplastic lesions (54 HGD/cancer and26 LGD) in patients with LSBO and 16 dysplastic lesions (10 HGD/cancer and 6 LGD) inpatients with SSBO. The crude incidence density of developing HGD/cancer in patients withLSBO was 31.5/1000 pyd (person years duration) (95% CI 23.7-41.2) and in patients withSSBO was 15.1/ 1000 pyd (95% CI 7.3-27.8). The age and sex adjusted incidence rate ratioof developing HGD/cancer among patients with SSBO compared to LSBO was 0.99 (95%CI 0.93-1.06). Conclusions: The risk of developing HGD/cancer in patients with SSBOappear to be similar to those with LSBO. Surveillance guidelines and physicians need totake this into account when planning surveillance strategies for patients with SSBO.
S1062
Acid-Induced p16 Hypermethylation Contributes to Development ofEsophageal Adenocarcinoma via Activation of NADPH Oxidase NOX5-SJie Hong, Murray Resnick, Jose Behar, Li Juan Wang, Jack R. Wands, Ronald A. DeLellis,David Lambeth, Rhonda F. Souza, Stuart J. Spechler, Weibiao Cao
Background: Inactivation of the tumor suppressor gene p16 may play an important role inthe progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). Hyperme-thylation of the p16 promoter is an important mechanism for inactivating p16. However,the mechanisms involved in p16 hypermethylation in EA are not known. Since acid refluxappears to contribute to the progression from BE to EA by over expressing NADPH oxidaseNOX5-S, we examined whether acid increases methylation of the p16 gene promoter byactivating NOX5-S in a Barrett's cell line BAR-T and an EA cell line OE33. Methods: P16mRNA and methylation of p16 promoter were measured by real-time PCR. Transfection ofNOX5 siRNA and plasmid was carried out by using Lipofectamine 2000 and Amaxa-Nucleofector-System respectively. Result: BAR-T and OE33 cells expressed NADPH oxidaseNOX5-S. Acid-induced increase in H2O2 production was significantly reduced by knockdownof NOX5-S. Exogenous H2O2 and acid treatment significantly increased methylation of p16promoter. In addition, acid treatment significantly decreased the p16 mRNA level. Knock-down of NOX5-S significantly increased p16 mRNA, inhibited acid-induced down-regulationof p16 mRNA. Knockdown of NOX5-S also blocked the acid-induced increase in p16methylation. Conversely, overexpression of NOX5-S significantly decreased p16 mRNA andincreased p16 methylation. Conclusion: NOX5-S is present in BAR-T cells and OE33 cells,and mediates acid-induced H2O2 production. NOX5-S is also involved in acid-inducedhypermethylation of the p16 gene promoter and in downregulation of p16 mRNA. It ispossible that acid reflux in BE patients may activate NOX5-S. NOX5-S-derived ROS in turnincreases p16 promoter methylation and down-regulates p16 expression, thereby contribut-ing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.
S1063
The Bile Acid Receptor TGR5 and NADPH Oxidase NOX5-S Mediate BileAcid-Induced Increase in Cell Proliferation in Barrett's EsophagealAdenocarcinoma CellsJie Hong, Murray Resnick, Jose Behar, Jack R. Wands, Li Juan Wang, Ronald A. DeLellis,David Lambeth, Weibiao Cao
Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major riskfactor for the development of esophageal adenocarcinoma (EA). However, the mechanismsof progression from BE to EA are not fully understood. Bile acids may play an importantrole in this progression. The aim of this study was to determine the expression of a novelbile acid receptor TGR5 in BE and to examine the role of NADPH oxidase NOX5-S and TGR5in taurodeoxycholic acid (TDCA)-induced increased cell proliferation. Immunohistochemicalstudies showed that TGR5 membranous staining was identifed in BE and EA, and was almostundetectable in esophageal squamous epithelial cells. TGR5 mRNA levels were significantlyhigher in EA tissues than in normal esophageal mucosa or Barrett's mucosa. In an EA cellline FLO, NADPH oxidase NOX5-S was present. TDCA significantly increased NOX5-Sexpression, H2O2 production and thymidine incorporation in these cells. This increase inthymidine incorporation was significantly reduced by knockdown of NOX5-S. Knockdownof TGR5 markedly inhibited TDCA-induced increase in NOX5-S expression, H2O2 produc-tion and thymidine incorporation. Conversely overexpression of TGR5 significantly enhancedTDCA's effects. In FLO cells transfected with NOX5-S luciferase reporter plasmid NOX5LPcontaining NOX5-S promoter fragment (-2501 to -1 from ATG), TDCA significantly increasedthe luciferase activity, an increase which was significantly decreased by phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U73122. TDCA-induced increase in NOX5-Sexpression, H2O2 production and cell proliferation was significantly reduced by knockdownof PI-PLC γ2 or ERK-2 MAP kinase using specific siRNAs. TDCA significantly increasedERK-2 phosphorylation, an increase which was decreased by PI-PLCγ2 siRNA. In conclusion,the TDCA-induced increase in cell proliferation depends on upregulation of NOX5-S expres-sion. TDCA-induced NOX5-S expression may be mediated by activation of the TGR5 receptor,PI-PLCγ2 and ERK-2 MAP kinase in FLO EA cells. Our data may provide potential targetsto prevent and/or treat Barrett's associated esophageal adenocarcinoma. Supported by NIHNIDDK R01 DK080703.
