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sSa1984

Lewis Biomarkers for Detection of Dysplasia in Barrett's EsophagusElizabeth L. Bird-Lieberman, Maria O'Donovan, Pierre Lao-Sirieix, Laurence Lovat,Rebecca Fitzgerald

Background: Biomarkers are required to improve the problems associated with poor interand intra-observer agreement in the diagnosis of dysplasia in endoscopic biopsies of Barrett'sesophagus (BE). Glycosylation changes associated with the development of cancer have beenused clinically as tissue biomarkers in other contexts. Hypothesis: Glycosylation changesthat take place as dysplasia develops within BE may act as biomarkers to more accuratelypredict propensity for cancer. Aims: (i) Empirically identify, using gene expression data, theglycan groups with greatest alteration of expression in progression from BE to AC. (ii)Validate findings in an Early Detection Research Network (EDRN) Phase 2 biomarker study.Methods: Gene set enrichment analysis and unsupervised clustering of gene expressionprofiles was used to identify glycan genes with altered expression in the development ofoesophageal cancer. Automated validation immunohistochemistry was undertaken in anindependent cohort (n=86) of patients from University College Hospital. Variance betweengroups and multivariable logistic regression was undertaken using SPSS. ANOVA analysiswas performed using the Bonferroni correction. Results: A list of glycan genes which hadaltered expression in progression to cancer were identified. 5 of the 6 genes at the top ofthis list are involved in the synthesis of Lewis (Le) antigens. E-selectin gene expression alsoincreased in the progression towards cancer (P,0.05) Histochemistry confirmed that sialylLea expression did increase in progression to cancer within multiple epithelial compartments(apical epithelial membrane P,0.00001, pan-membranous P,0.0001, diffusely within thecytoplasm P=0.001). Lex similarly validated (apical epithelial membrane P ,0.00001, dif-fusely within the cytoplasm P,0.0001) and in a pan-membranous distribution P,0.00001.The sialylated form of the Lex antigen failed to validate. Multivariable logistic regressionconfirmed that the following were independent predictors of dysplasia: sLea diffuse cyto-plasmic staining (P=0.028, 95%CI 1.030-1.681), Lex pan membranous staining (P=0.044,95%CI 1.008-1.886), Lex apical membrane staining (P=0.008 95%CI 1.095-1.806) and Lexdiffuse cytoplasmic staining (P=0.018, 95%CI 0.499-0.938). Conclusions: This phase 2biomarker study has shown that sLea and Lex are able to act as biomarkers for the detectionof dysplasia and staining for this could be performed in an automated manner in the clinicalsetting. A Phase 3 study is warranted.

Sa1985

Leukotriene B4 Receptor-1 (Blt-1) and Cysteinyl Leukotriene Receptor-2(Cyslt-2) Are Deregulated in Both Transformed and Non-TransformedEpithelium of Patients With Esophageal Squamous Cell CarcinomaMarino Venerito, Christoph Helmke, Doerthe Kuester, Thomas Wex, Peter Malfertheiner

Background: A role for leukotriene B4 receptors (BLT-1 and BLT-2) and cysteinyl leukotrienereceptors (CysLT-1 and CysLT-2) is suggested in malignant cell transformation and prolifera-tion. The expression of these receptors in esophageal squamous cell carcinoma (ESCC) hasnot been investigated. Aim: To investigate the expression of BLT-1, BLT-2, CysLT-1 andCysLT-2 in ESCC and adjacent non-transformed squamous epithelium of the esophagus(NTSE), as well as in control biopsy samples from esophageal squamous epithelium (CSE)of patients with functional dyspepsia. Methods: Nineteen consecutively diagnosed patientswith ESCC were prospectively enrolled between March 2010 and January 2011. UICC stagewas calculated based on the 7th AJCC/UICC edition. Furthermore, 9 sex- and age-matchedpatients with functional dyspepsia were included as controls. Expression levels of BLT-1,BLT-2, CysLT-1 and CysLT-2 were analyzed by immunohistochemistry (IHC) and quantita-tive reverse transcription-polymerase chain reaction (qRT-PCR) in biopsy samples. Spearmancorrelation, Friedman test, Wilcoxon rang sum test and Mann-Whitney-U-test (2-sided)were used for statistical analysis. Results: Most patients with ESCC were at AJCC/UICC stageIV (11/19, 58%), whereas only 2, 2 and 4 patients were in stage I, II and III, respectively.In general, BLT-1, BLT-2, CysLT-1 and CysLT-2 were found to be ubiquitously expressedin all biopsy samples. Protein and transcript levels of BLT-1 were significantly increased inboth ESCC and NTSE compared to CSE (p , 0.05). ESCC showed a significantly increasedBLT-2 protein expression compared to NTSE and CSE (p , 0.05), whereas BLT-2 transcriptlevels did not differ among the three groups. The protein expression of CysLT-1 and CysLT-2 was significantly increased in ESCC compared to NTSE and CSE (p , 0.05). CysLT-1mRNA expression was significantly decreased (3.2 fold) in ESCC compared to CSE (p ,0.05), but not to NTSE. A significant decrease of CysLT-2 mRNA expression was observedin both ESCC (17 fold) and NTSE (16.1 fold) compared to CSE (p , 0.05). Gene expressionlevels of BLT-1, BLT-2, CysLT-1 and CysLT-2 were not correlated with AJCC/UICC stage.Conclusions: The expression of LT receptors is deregulated in ESCC. Deregulation of BLT-1 and CysLT-2 gene expression occurs already in NTSE of patients with ESCC suggestinga potential role of these receptors in early steps of esophageal carcinogenesis.

Sa1986

Prognostic Impact of Body Mass Index in Patients With Squamous CellCarcinoma of the EsophagusMasayuki Watanabe, Yoshifumi Baba, Naoya Yoshida, Hideo Baba

Objective: Although recent studies have revealed that obesity might influence the prognosisof cancer patients, the prognostic significance of BMI for patients undergoing esophagectomyfor esophageal squamous cell carcinoma (ESCC) remains unknown. Aim of this study is toclarify the prognostic impact of body mass index (BMI) in patients with esophageal squamouscell carcinoma (ESCC). Methods: Two hundred and forty-three patients who underwentesophagectomy for ESCC from April 2005 through December 2010 were eligible. Prognosesof the patients were compared between groups stratified according to BMI. Univariate andmultivariate analyses were performed to identify factors affecting disease-free survival. Wealso analyzed the survival difference using propensity score-matching to adjust differencesin staging and treatment. Results: Low, normal, and high BMI groups had 35, 177, and 31patients, respectively. The low BMI group included more advanced cases than did the normal

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BMI group, while tumor stage was equivalent in the normal and high BMI groups. Disease-free survival of the low and high BMI groups was significantly worse than that of the normalBMI group (P,0.0001 between the low and normal BMI groups; P=0.0076 between thenormal and high BMI groups). Multivariate analysis revealed that high BMI was an indepen-dent risk factor for recurrence in patients who underwent curative esophagectomy (HR2.227, 95% confidence interval, 1.104 to 4.189). Disease-free survival of the high BMI groupwas significantly worse than that of the normal BMI group in the propensity score-matchedcohort (P=0.0020). Multivariate analysis in this cohort demonstrated that high BMI was anindependent prognostic factor (HR 2.949, 95% confidence interval, 1.132-7.683). Conclu-sions: Both low and high BMI are associated with poor prognosis for ESCC, through differentmechanisms. Advanced tumors are the reason for poor prognosis in patients with a lowBMI, while biological aggressiveness of tumors is possibly the determining factor in patientswith a high BMI. Although further analysis is required to clarify the influence of overweighton the biological features of ESCC, glucose metabolism may be a therapeutic target for ESCC.

Sa1987

AMACR Immunohistochemistry for Prediction of Neoplastic Progression inPatients With Barrett's Esophagus: Results From a Large MulticentreProspective CohortFlorine Kastelein, Katharina Biermann, Sophie van Olphen, Ewout W. Steyerberg,Leendert Looijenga, Ernst J. Kuipers, Manon C. Spaander, Marco J. Bruno

Background and aims: Surveillance is recommended for Barrett's esophagus (BE) to detectesophageal adenocarcinoma (EAC) at an early stage, but is problematic given the overalllow incidence of EAC and lack of discriminative tests for risk stratification. Histologicaldiagnosis of low-grade dysplasia (LGD) is currently the only accepted predictor for progres-sion and therefore crucial for defining surveillance intervals. However, the predictive valueof LGD is low due to sample error and interobserver variation. Use of biomarkers mayimprove risk stratification. The aim of this study was therefore to evaluate the value ofalpha-methylacyl-CoA racemase (AMACR) immunohistochemistry for predicting neoplasticprogression in BE patients. Methods: We conducted a case-control study within a multicenterprospective cohort of 720 BE patients. Patients were followed according to the ACG guidelinesand incident cases of high-grade dysplasia (HGD) and EAC were identified during follow-up. Patients who developed HGD or EAC were classified as cases and patients withoutneoplastic progression as controls. AMACR expression was determined by immunohistoche-mistry in more than 12.000 biopsies from 635 patients and was scored (no expression, mildoverexpression, strong overexpression) independently by two expert pathologists who wereblinded for long-term outcome. Data were analyzed in loglinear regression models adjustedfor age, gender, BE length and esophagitis. Results: 635 BE patients (73% male, median age60 years (IQR 53-69)) were included in this study and followed during surveillance for amedian duration of 6.6 years (IQR 5.1-7.3). Forty-nine (8%) patients developed HGD orEAC during follow-up and were classified as cases. The remaining 586 (92%) patients wereclassified as controls. AMACR overexpression was more common with higher grades ofdysplasia and was seen in 49% of samples without dysplasia, 63% of samples with LGD,91% of samples with HGD and 77% of samples with EAC (P ,0.001). Mild AMACRoverexpression was associated with a trend towards a higher risk of neoplastic progression(RRa 1.6; 95%CI 0.9-3.1), but the risk was especially elevated with strong AMACR overex-pression (RRa 4.8; 95%CI 1.9-12.6). The sensitivity of mild AMACR overexpression forpredicting progression was 67% with a specificity of 50%. The sensitivity of strong AMACRoverexpression for predicting progression was 10% with a specificity of 96%. The positivepredictive value increased from 11% with mild AMACR overexpression to 19% with strongAMACR overexpression. Interobserver agreement was moderate ( κ = 0.44; 95%CI 0.41-0.48). Conclusion: Strong AMACR overexpression is associated with an increased risk ofneoplastic progression in BE patients. However, AMACR expression is not sensitive norspecific enough to be used as a single biomarker for prediction of neoplastic progression.

Sa1988

Recovery of Gastric Function After Helicobacter pylori Eradication andAcetium Administration: A 6 Years Study in Atrophic Gastritis SubjectsFrancesco Di Mario, Massimo Rugge, Francesco Ferrara, Nadia Dal Bò, Tiziana Slongo,Roberto Marcello, Helena Heras Salvat, Giovanni Guarnieri, Alessandro Caroli, RobertoRigoli, Angelo Paolo Dei Tos, Carmelo Scarpignato

INTRODUCTION: The relationship between Helicobacter pylori (H. pylori) eradication andatrophic changes in the gastric mucosa has not yet been fully defined. Athough studiesreport a partial restoration of serum pepsinogen I (sPGI) levels after eradication, it is notclear if this finding reflects gastric mucosal ealing on a morphological level. Recently a newcompound (L-cystein Acetium, Biohit, Finland) has been proposed for prevention of gastriccarcinogenesis in patients with atrophic gastritis, by reducing acetaldehyde production afterfood intake. AIMS: To assess alteration in gastric function after H. pylori eradication onmoderate-severe body atrophiv gastritis by determination of sPGI levels and Gastrin 17 (sG17). METHODS:.74 dyspeptic patients, selected from 738 consecutive H. pylori positivepatients, with histological features of moderate-severe body atrophic gastritis and sPGI , 25microg/L (34 men, mean age 58.4 yrs, range 26-83 yrs), underwent an upper gastrointestinal

endoscopy with gastric biopsies and sPGI and sG 17 determination at baseline. All patientsunderwent eradication therapy. Serum sPGI and sG 17 were measured againa after 6 months,and at 1, 2, 3, 5 and 6 yrs after eradication therapy. RESULTS: Mean sPGI levels prior toeradication were 13,4 microgr/L (range: 1,5-24 microgr/L). 6 months after eradicationtherapy, mean sPGI levels significantly increase to 16,6microgr/L (p=0.05). At the completionof the study, 6 yrs after eradication, sPGI levels increased to 27,3 microgr/L (p=0.01).Conversely, the sG 17 dropped out from 84,8 at baseline to 67,6 pmol/L after the 6 yrsfollow up period (p,0.01). 5 patients (4 female, mean age 56, range 49-66 yrs) out of the74 sample atrophic gastritis subjects experienced a 3 month period of treatment with Acetium100 mg at dosage of 3 capsules daily before eating. In all, both sPGI and sG 17 were reducedat baseline and after 3 months period of treatment. The mean levels of sPGI increases from5,3 microgr/L at baseline to 7,8 after 90 days of Acetium intake as well as sG 17 drop outfrom 74,6 to 79,8 pmol /L. CONCLUSION: After H.Pylori eradication subjects with bodyatrophic gastritis showed long-lasting improvement of physiological gastric function, reflectedby significantly and stabile increasing of sPGi levels and a parallel decreased of sG 17 overa 6-year period of follow up.

Sa1989

Novel Genes Mutation and Methylation Targets in Colon Cancer Using WholeExome SequencingHassan Ashktorab, Hamed Rahi, Mohammad Daremiporan, Edward L. Lee, Wayne A.Frederick, Adeyinka O. Laiyemo, Mehdi Nouraie, Hassan Brim

Background: Several driver mutations and methylated genes have been discovered in colo-rectal cancer (CRC) progression. This includes KRAS, APC and BRAF that have practicalsignificant therapeutic and prognostic values. Sequencing technologies have advanced andnow provide a tool for high throughput discovery of novel driver mutations. In addition,the sequencing of sodium bisulfite modified DNA coupled with enrichment for high GCareas allowwhole genomemethylation analysis. Here, we performed whole exome sequencing(WES) and whole genome methylation analysis to elucidate the involvement of novel candi-date genes in the path to colon carcinogenesis. Patients and Methods: WES was carried outon genomic DNA extracted from 8 normal-tumor pairs of frozen biopsies from AfricanAmericans patients with CRC. WES and Reduced Representation Bisulfite Sequencing (RRBS)were performed. Pyrosequencing and Sanger sequencing were used for the validation ofmethylation and single nucleotide variants (SNV) respectively. For WES, base call qualityrecalibration, realignment around indels, SNV calling and variant call recalibration werecarried out using Genome Analysis Tool Kit. Variants were then annotated using Annovar.Forty-four paired colorectal tumors and normal adjacent colonic tissue samples were usedfor validation. Results: WES uncovered somatic mutations alterations in many genes thatare known be mutated in CRC including APC, BRAF, KRAS, Notch1, PIK3C2A, and NDRG4.We discovered a number of Novel SNVs in EID3, RGS3, HNRNPF, and GNAS in tumorsamples. One GNAS missense mutation was discovered among KRAS mutated tumors. TheRRBS analysis and validation revealed the methylation status of 355 CpG sites located in16 gene promoter regions associated with CpG islands. Fifty-nine CpG sites located on CpGislands of ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), NDRG4 (2), Sept9(6), and TNFAIP2 (1), were significantly methylated in tumor vs. normal (p ,0.05). Mostof these genes showed significant hypermethylation in tumors compared to normal mucosa.According to gene ontology analysis, GAS7 methylation is associated with the cadherinsignaling (WNT) pathway.In addition, the tumor with the GNAS mutation was significantlyhypomethylated (P,0.05) in the CpG sites of GNAS promoter. Ingenuity pathway analysis(IPA) showed that GNAS was involved in CRC metastasis signaling via APC, BRAF, GSK3A,KRAS, MLH1, MLH2, MLH3, Notch family, and PIK3C family. Conclusion:This work pro-vides insight into the intricacies between genetic and epigenetic processes in the path tocancer. Such is the case for GAS7, a novel gene, that was hypermethylated in our validationset and for which functional analysis is underway.

Sa1990

Association Between EBV Infection and HER2 Expression in Gastric CancerKei Mitsuhashi, Yasutaka Sukawa, Mayumi Nakazawa, Takafumi Naito, Miki Ito,Hisayoshi Igarashi, Hiroaki Kunimoto, Katsuhiko Nosho, Yasushi Adachi, HiroyukiYamamoto, Kenzo Takada, Yasuhisa Shinomura

Background: It is well known that Epstein-barr virus (EBV) infection is presented in 10%of gastric cancer cases and plays a role in carcinogenesis through the methylator phenotype.HER2 expression has been receiving increasing attention since the efficacy of an anti-HER2antibody, Trastuzumab (Tmab), in gastric cancer with HER2 overexpression was proved.EBV reportedly suppresses and upregulates HER2 expression in ovarian cancer and breastcancer, respectively. However, the relationship between EBV infection and HER2 expressionin gastric cancer is not known. Aim: The aim of this study was to clarify the associationbetween EBV infection and HER2 expression in gastric cancer. Method: We used HER2-positive JRST and HER2-negative NUGC3 gastric cancer cell lines. These cell lines wereinfected with EBV by the cell-cell contact method and they were designated as JRST-EBVand NUGC3-EBV, respectively. We analyzed the effects of EBV infection on HER2 expressionin gastric cancer cell lines by real-time RT-PCR, Western blotting, MTT assay, scratch assayand caspase 3 assay. We also analyzed 373 gastric cancer tissues for EBV infection andHER2 expression by in situ hybridization of EBER1 and immunohistochemistry using aHERCEPTEST kit, respectively. Results: HER2 mRNA expression levels were similar in JRST-EBV and JRST cells. In contrast, HER2 expression levels analyzed by Western blotting werelower in JRST-EBV cells than in JRST cells. Expression of pAkt and pMAPK, both ofwhich are downstream signal molecules of HER2, was also suppressed in JRST-EBV cells.Proliferation activity analyzed by the MTT assay was significantly suppressed in JRST-EBVcells compared with that in JRST cells. Cell migration analyzed by the scratch assay wassimilar in NUGC3-EBV and NUGC3 cells, while it was significantly suppressed in JRST-EBV cells compared with that in JRST cells (p ,0.001). Tmab-induced apoptosis analyzedby the caspase 3 assay was similar in JRST-EBV and JRST cells. Tmab suppressed migrationof JRST cells but not that of JRST-EBV cells. EBV infection and HER2 expression werepresent in 24 (6.4%) and 54 (14.5%) cases, respectively. Only two cases were positive for

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both EBV infection and HER2 expression. One case was HER2 FISH-positive and pAkt-negative. In contrast, the other case was HER2 FISH-negative and pAkt-positive. Conclusion:EBV appears to suppress HER2 expression at the protein level in gastric cancer cell lines,resulting in reduced cell proliferation and migration. EBV may also be involved in theregulation of HER2 expression in gastric cancer tissues. Furthermore, EBV may play a rolein resistance of gastric cancer cells to Tmab therapy.

Sa1991

Volatile Markers Can Discriminate Between Gastric Cancer and BenignConditionsHaitham Amal, Konrads Funka, Inta Liepniece-Karele, Roberts Skapars, Marcis Leja,Hossam Haick

BACKGROUND: Most of the gastric cancers are diagnosed at advanced stages when thesurvival results are unsatisfactory. Therefore, there is a need for non-invasive markers todiagnose the disease at early stage. Here we report the initial results of volatile markerdiagnostic development approach program. AIMS: To identify potential volatile organiccompounds (VOCs) present in exhaled breath characteristic to gastric adenocarcinoma.METHODS: Altogether 364 patients (148 men, 216 women; median age 62; range 22-87)were included to the study. Out of them 82 patients had morphologically confirmed gastricadenocarcinoma prior to surgery or chemotherapy; 45 patients had peptic ulcer disease(either active or a scar), 21 patients were diagnosed low-grade dysplasia in the gastricmucosa, while 216 patients were serving as group of control (all with documented stomachmucosa status according to Updated Sydney system; this group included also patients withatrophy and/or intestinal metaplasia). Exhaled breath samples were collected in adsorbenttubes and stored until analyzed. The analysis was performed by using gas chromatographycoupled with mass spectrometry (GC-MS) in order to identify the chemical composition ofthe samples. Student t-test was used to judge the difference in VOCs between the groups;the significance level was set at 0.05. RESULTS: Altogether 133 volatile components wereidentified; 17 of them revealed differences between some of the comparisons. Eleven VOCswere found in significantly higher concentrations in the cancer group when compared tothe controls (see Table). Six compounds (2- methyl-1-propene; 2,4 dimethyl-Heptane; 4-methyl-Octane; 1,2,3 trimethyl-Benzene; 4- methyl-Decane; Naphtalene) were higher incancer than in dysplasia cases. Four compounds (o-Xylene; alpha-Methylstyrene; 1,2,3trimethyl-benzene; 1,2,3,4 tetramethyl-benzene) were higher in peptic ulcer than in controls.However, no difference was revealed between the dysplasia and control as well as betweenthe cancer and peptic ulcer groups. CONCLUSIONS: Specific volatile components in exhaledbreath can discriminate between gastric cancer and benign conditions. This finding providesbackground for further new developments of volatile component applications in diagnostics,including for nanosensor based applications.Control vs Cancer

VOCs from exhaled breath samples identified in Gastric cancer and control patients whichshow significant (p , 0.05) statistical difference between the two groups.

Sa1992

SFRS2 and CDC5L Interaction: An insight Into Downstream Events FollowingAPC Deletion During Colorectal CarcinogenesisShahram A. Ibrahim, Karen R. Reed, Alan R. Clarke, D. M. Pritchard, John R. Jenkins

Colonic adenocarcinoma develops via the premalignant adenoma stage as a result of theaccumulation of multiple genetic changes. Inactivation of the Adenomatous Polyposis Coli(Apc) tumor suppressor gene is a key early step in this process and results in overactivationof the Wnt/Beta catenin signalling pathway. However, the molecular changes downstreamof Apc inactivation have not yet been fully elucidated. We have previously assessed theproteomic profile of intestinal epithelial cells from a tissue specific inducible mouse modelof Apc deletion (AhCre+Apcfl/fl) using iTRAQ protein-tagging mass spectrometry. Splicingfactor arginine/serine-rich 2 (SFRS2), a protein involved in cell cycle modulation and pre-mRNA splicing was found to be significantly up-regulated in AhCre+Apcfl/fl intestine. Wehave now investigated the role of this protein in colorectal tumorigenesis, in particular itsinteraction with Cell division cycle 5-like protein (CDC5L), a regulator of the cell cycleand pre-mRNA splicing. Methods Formalin fixed, paraffin embedded tissue sections fromAhCre+Apcfl/fl, ApcMin/+ and ApcMin/+Pten-/- mice were used to investigate the expression ofSFRS2 and CDC5L by immunohistochemistry. SFRS2 and CDC5L were knocked down inHCT116 and HT29 colon cancer cell lines using siRNA. Knockdown efficiency was assessedby western blotting and the consequences on cell proliferation were assessed using sulforho-damine B (SRB) and clonogenic survival assays. Sub-cellular localisation of CDC5L wasassessed in cell lines by immunocytochemistry. Results Weak SFRS2 expression and strong

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