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Loss of Glutathione Peroxidase 7 Promotes TNF-α-Induced NF-kB Activationin Barrett's CarcinogenesisDunFa Peng, Tianling Hu, Mohammed Soutto, Abbes Belkhiri, Wael El-Rifai

BACKGROUND: Esophageal adenocarcinoma (EAC) is a classic example of inflammation-associated cancer, which develops through GERD (gastro-esophageal reflux disease)-Barrett'sesophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence rate for EAC hasincreased 4-10% per year among men since 1976 in the USA, more rapidly than for anyother type of cancer. Patients with Barrett's esophagus can progress to dysplasia and EACat 30-60 times that of the general population. NF-kB plays a key role in inflammationprocess and TNF-α is a pro-inflammatory cytokine and a known NF-kB activator. It hasbeen reported that TNF-α and TNFR1 are significantly increased in BE and EACs. GlutathionePeroxidase 7 (GPX7), a recent member of GPX family which currently has 8 members, isfrequently silenced through location-specific hypermethylation during Barrett's tumorigene-sis. Its functions remain largely uncharacterized. In this study, we investigated the potentialrole of GPX7 in regulating NF-kB activity. METHODS and RESULTS: In vitro experimentsand de-identified human tissue samples were utilized for quantitative real-time PCR, immuno-fluorescence, luciferase reporter, immunoblotting and intracellular ROS (reactive oxygenspecies) assays. Reconstitution of GPX7 in BE and EAC cell models abrogated TNF-α-induced NF-kB reporter activity and nuclear translocation of NF-kB-p65 (P<.01). In addition,we detected marked reduction in phosphorylation levels of components of NF-kB signalingpathway (p-p65, p-IkB-α, and p-IKKα/β) and significant downregulation of NF-kB targetgenes (TNF-α, IL-1β, IL-6, IL-8, CXCL-1, and CXCL-2) in GPX7 expressing cells as comparedto control cells. These effects were validated by knockdown of endogenous GPX7 in HET1Acells. We also found that the GPX7-mediated suppression of TNF-α-induced activation wasindependent of its capacity to regulate ROS. On the other hand, we demonstrated that GPX7promotes protein degradation of TNFR1 and TRAF2 which could explain GPX7-dependentregulation of the IKK - IkB - NF-kB-p65 axis. The relevance of these findings to Barrett'swas further confirmed by showing that GPX7 suppressed bile salts-induced activation ofNF-kB and expression of cytokines in vitro and the presence of inverse relationship betweenGPX7 and cytokines' expression in esophageal tissue samples. CONCLUSIONS: The loss ofGPX7 expression could unleash the TNF-α- and bile salt-induced activation of pro-inflamma-tory NF-kB signaling that contributes to the GERD-associated BE-dysplasia-EAC sequence.

934

Immune Factors Involved in the Promotion of Esophageal Adenocarcinoma AsDefined by siRNA Library ScreeningFiona Behan, Shane P. Duggan, Catherine Garry, Sinead Phipps, Dermot Kelleher

Introduction: There is a pressing need to identify new therapeutic targets for esophagealadenocarcinoma (EAC). Hence, we utilized siRNA-screening libraries to identify genesimpacting on EAC cancer cell growth to identify potential therapeutic targets. Methods: A"druggable genome" library (6022 individual siRNAs) was utilized to examine EAC cellsurvival using the MTT assay. Statistical analysis combined the use of Z-factor, t-test andSSMD in a combined and rigorous set of sequential statistical algorithms. EAC cell linesGohTRT, SKGT4 and OE33 were utilized. Clinical interpretation was defined by Oncomineand transcriptomic studies of EAC carcinogenesis. Functional validation of the resultingsiRNAs utilized RT-PCR, Western Blot, ELISA and TOPFLASH assays. Results: siRNA libraryscreening resulted in positive quality metrics (Z-factor>0.5) confirming its validity. Statisti-cally significant targets were identified and ranked based on Z-score (Z-Score< -1.6), t-test(p<0.01), SSMD (SSMD<-7) and cell viability (%CV< 40%), identifying 118 high confidencegene targets affecting EAC cell growth. Verification of these siRNA targets in multiple celllines indicated a good level of concordance with the primary screening data. Bioinformaticand pathway mapping approaches using these genes emphasized links between EAC cellproliferation and regulators of inflammation (LIF, C1Qa, C1r, C1s, GDF15, IL9R) with"immune cell processes" being the most highly associated gene grouping. Oncomine databaseand transcriptomic studies demonstrated that LIF (BO: FC=42.6, EAC: FC=96.7; P<0.0001),GDF15 (BO: FC=35, EAC: FC=74; P<0.0001) and C1Qa (in progress) may be dysregulatedin EAC and were validated via RT-PCR in patient cohorts. In functional work, inhibitionof complement component C1Qa by siRNA, followed by addition of exogenous C1q furtherdecreased cell viability by 55% (P=0.0005). Conversely, exposure of EAC cells to C1 inhibitor(C1INH) increased cell growth by 41% (P<0.05) further supporting a role for complementbiology and EAC survival and growth. Recombinant LIF was observed to rescue changes incell viability elicited by siRNA mediated inhibition of LIF transcript indicating the LIF-STAT3 signaling axis may be vital in EAC survival. Inhibition of GDF15 was observed toresult in altered wound closure in scratch wound assays which could be rescued by recombi-nant GDF15 treatment. GDF15 was also discovered to play a role in Th17 cell differentiationusing in-vitro assays. Conclusion: Genes regulating EAC proliferation have been defined bysiRNA library screening. We have identified immune related genes, not previously associatedwith EAC biology, capable of regulating EAC cell survival. Further functional validation ofthese genes may prove their potential as future therapeutic targets.

935

The Timing of Oncogenic Events in the Evolution of the OesophagealAdenocarcinoma Genome and Implications for Clinical DiagnosticsJamie M. Weaver, Caryn Ross-Innes, Nicholas B. Shannon, Andy Lynch, Tim Forshew,Mariagnese Barbera, Chin-Ann J. Ong, Muhammed Murtaza, Pierre Lao-Sirieix, Mark J.Dunning, Laura Smith, Mike Smith, Benilton Carvalho, Timothy Underwood, AndrewMay, Nicola Grehan, Charlotte Anderson, Jim Davies, Richard H. Hardwick, MariaODonovan, Arusha Oloumi, Sam Aparicio, Carlos Caldas, Matthew D. Eldridge, Paul A.Edwards, Nitzan Rosenfeld, Simon Tavaré, Rebecca Fitzgerald

Presented on behalf of the OCCAMS Consortium Background A series of clonal expansionsare thought to underlie the progression of Barrett's oesophagus (BE) to oesophageal adenocar-cinoma (OAC). Each expansion carries with it somatic driver mutation(s) fixing it within a

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larger population and therefore increasing the likelihood of acquiring a second mutation.However, the precise order in which somatic variants occur remains unknown. Aims Todetermine the order of somatic mutations in the BE-dysplasia-adenocarcinoma sequence inorder to inform molecular diagnostic tests. Methods We performed whole genome sequencingin 25 cases of OAC and 3 matched cases of BE. Findings were validated in a larger cohortof OACs (n=90), metaplastic never-dysplastic BE (NDBE, n= 66 with a median follow-upof 58 months) and high-grade dysplasia (n = 43) using amplicon resequencing. Mutationalsignatures and gene-centric somatic mutations were determined using an in-house pipelineincorporating standard statistical methods and the publically available EMu pipeline. ResultsThere were 7 distinct mutational signatures present in both early (BE) and late disease (OAC)including 3 novel signatures not previously described. Fifteen genes were determined to bepotential novel drivers of OAC development. Surprisingly in 53% of NDBE tissue sampleswe identified clonal expansion of cells (>10% mutant fraction) harbouring mutations in oneor more of 13/15 of these putative driver genes. No difference in the frequency of mutationof these genes was observed between any of the disease stages studied. TP53 mutationsclearly delineate between HGD/OAC and benign NDBE (p<0.001). Whilst SMAD4 mutationsare only observed in OAC (p<0.001) demonstrating for the first time a clear genetic differencebetween the two. Conclusions Mutagenic processes active in OAC are also active in theearliest stages of BE. Recurrent driver mutations identified in cancer may be acquired veryearly in the disease and may provide little or no progression advantage. Molecular diagnosticapproaches must account for this.

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Evaluation of a Four-Protein Biomarker Panel (Biglycan, Annexin-A6,Myeloperoxidase and Protein S100-A9; B-AMP ©) for Detection of EsophagealAdenocarcinomaAli H. Zaidi, Vanathi Gopalakrishnan, Pashtoon M. Kasi, Usha Malhotra, JeyaBalasubramanian, Shyam Visweswaran, Xuemei Zeng, Mai Sun, Jacques J. Bergman,William L. Bigbee, Blair A. Jobe

Background: Esophageal Adenocarcinoma (EAC) is associated with a dismal prognosis, witha five-year survival of less than 15%. The identification of cancer biomarkers advances thepossibility for early detection and better monitoring of tumor progression and/or responseto therapy. The current study presents results of the development of a serum based four-protein (biglycan, myeloperoxidase (MPO), annexin-A6 (ANXA6), and protein S100-A9; B-MAP ©) biomarker-panel for EAC that are noted to be clinically relevant and followed adistinct pattern of expression along the sequence of disease progression. Methods: A verticallyintegrated proteomics-based biomarker approach was used to identify serum biomarkersfor detection of EAC. Initially Laser capture micro dissection (LCM) dissection of formalin-fixed, paraffin-embedded (FFPE) samples that were collected from across the Barrett's esopha-gus (BE) carcinogenesis spectrum was performed followed by nanoflow liquid chromatogra-phy-tandem mass spectrometry (LC-MS/MS). MS-based tissue discovery data was used toguide the selection of candidate serum biomarkers. We hypothesized that serum proteinabundances would mirror the differential protein expression we observed between BE, HGD,and EAC tissues. The serum biomarker data was then validated in an independently assembledexternal cohort. Subsequently the data was used to develop a multi-parametric risk assessmentmodel to predict the presence of disease. Results: With a minimum threshold of 10 spectralcounts 352 proteins were differentially abundant along the spectrum of BE-, HGD- andEAC (p-value < 0.05 by Kruskal - Wallis test). Eleven highly significant and functionallyrelevant proteins from this data were tested in serum samples from patients with non-BEGERD (normal control) and those with EAC (cases). This yielded five proteins that weresignificantly dysregulated in the serum of EAC patients and also mirrored trends across thedisease spectrum which were reported by the tissue data. Using serum data a BRL predictivemodel with four biomarkers was developed to accurately classify disease class; the cross-validation results for the merged dataset analyzed by BRL yielded accuracy of 87%, BACCof 86% and AUROC of 93 %. Conclusions: Serum biomarkers hold significant promise forearly non-invasive detection of EAC. With ease of administration and relatively low cost,our B-AMP © panel for EAC was able to identify patients with EAC with an accuracy of87%. Further testing in larger group of patients prospectively would further characterizethe effectiveness of the B-AMP © panel for detection and/or monitoring of EAC therapy inthese patients.

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Randomized Comparison of Surveillance Intervals After ColonoscopicRemoval of Adenomatous Polyps: Results From the Japan Polyp StudyTakahisa Matsuda, Takahiro Fujii, Yasushi Sano, Shin-ei Kudo, Yasushi Oda, KazuhiroKaneko, Kinichi Hotta, Tadakazu Shimoda, Yutaka Saito, Nozomu Kobayashi, KazuoKonishi, Hiroaki Ikematsu, Hiroyasu Iishi, Kiyonori Kobayashi, Yuichiro Yamaguchi,Kiwamu Hasuda, Tomoaki Shinohara, Hideki Ishikawa, Yoshitaka Murakami, HirokazuTaniguchi, Takahiro Fujimori, Yoichi Ajioka, Daizo Saito, Shigeaki Yoshida

Background & Aim: The National Polyp Study (NPS) Workgroup recommended an intervalof at least 3 years between colonoscopic removal of newly diagnosed adenomatous polypsand the follow-up examination in 1993. However, the study was conducted prior to therecent epidemiologic studies documenting the importance of nonpolypoid colorectal neo-plasms (NP-CRNs). NP-CRNs are easily overlooked, and some of them are more likely tocontain advanced histology. The aim of this study was to assess whether follow-up colonos-copy using high-definition colonoscope at 3 years as well as at both 1 and 3 years woulddetect important lesions including NP-CRNs. Methods: The Japan Polyp Study (JPS), amulticenter randomized control trial conducted at 11 participating centers was initiated in2003. Patients were eligible if they have had two complete colonoscopies (1st- and 2nd-TCS: interval; 1 year) with removal of all neoplastic lesions. Following this they wererandomly assigned to have follow-up colonoscopy at 1 and 3 years (two-exam group) or at3 years only (one-exam group). Index lesions (ILs) were defined as any low-grade dysplasia(LGD) ≥10mm, high-grade dysplasia (HGD) or invasive cancer. Results: 4752 patients withno history of FAP, HNPCC, IBD or personal history of polypectomy with unknown histology,no history of colectomy were referred for colonoscopy. 3926 patients mean age 57.3 years

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