W1875

Effect of Promoter Methylation of Multidrug Resistance 1 (MDR1) Gene inGastric CarcinogenesisTomomitsu Tahara, Tomoyuki Shibata, Ichiro Hirata, Tomiyasu Arisawa

Background and Aim: Altered expression of multidrug resistance 1 (MDR1) contributes tovarious cancers and its disease progression. DNA methylation is reported to present invarious non-cancerous tissue as age-related phenomenon, suggesting that it occurs early inthe process of tumorgenesis. We investigated the effect of MDR 1 gene promoter methylationon carcinogenesis in the stomach. Methods: In study A, we estimated the methylation ratio(MR) of the MDR1 gene promoter in both antral noncancerous mucosa and cancer lesionsin 83 patients with gastric cancer (GC). In study B, we also estimated the MR in non-cancerous gastric mucosa in 127 patients with GC and 82 cancer free (non-GC) subjects.Degree of histological chronic gastritis was scored according to updated Sydney systems in200 subjects. We employed methylation-specific PCR method. Results: In the study A, MRof cancer lesions (31.3±20.5) was significantly higher than that of non noncancerous mucosa(20.9±31.3, p= 0.0004). MR was especially higher in Lauren's intestinal cancer (p<0.0001).Higher MR was also observed in more advanced stage (p=0.005), and lymph vessel invasionpositive cases (p=0.002). In the study B, a significant association was found between highermethylation ratio (MR) and occurrence of GC (GC vs. non GC; 21.3±24.8 vs.10.3±14.3,H. pylori infection adjusted p = 0.001). Higher MR was also associated with occurrence ofGC in both H. pylori positive (p=0.006) and negative (p=0.03) subjects. Furthermore, higherMR was closely associated with H. pylori infection status (p<0.0001), and degree of intestinalmetaplasia (p=0.0008) Conclusion: The promotermethylation ofMDR1 have a significant rolein carcinogenesis and tumor progression in the stomach. Furthermore, promoter methylationstatus of MDR1 gene in non-cancerous gastric mucosa also seems to be associated with H.pylori infection, severity of intestinal metaplasia, and GC occurrence.

W1876

Stool Protein Markers for Colorectal Cancer: Identification of High-AbundanceCandidates By Mass SpectroscopyPatrick S. Quint, Jessica C. Albright, Jonathan J. Harrington, Douglas W. Mahoney, AnnL. Oberg, Mary E. Devens, H. R. Bergen, David A. Ahlquist

Stool testing approaches for occult blood and DNA are both endorsed by the AGA to screenfor colorectal cancer. Tumor-associated proteins represent an alternative class of potentialstool markers with the theoretical advantage of low assay cost. However, few data areavailable on the fecal proteome in patients with and without colorectal neoplasia. Aim: Toidentify candidate stool screening markers by comparing the high-abundance fecal proteomefrom healthy controls and patients with colorectal neoplasms. Methods: Subjects comprised43 healthy controls, 16 patients with adenomas >1cm, and 18 with colorectal cancer. Stoolsamples were collected prior to colonoscopy and frozen promptly at -80°C. From aqueoussupernatants of thawed fecal aliquots, proteins were separated by electrophoresis and digestedfor mass spectrometric analysis using an LTQ-Orbitrap. Shotgun-proteomic techniques wereused to identify proteins and their differential abundance. Data outputs were searched usinga composite ID from Mascot, Seaquest and X!Tandem against an updated Swissprot databasethat included all cataloged species. Protein abundance was estimated using spectral countdata from Scaffold. Normalization and differential expression analysis were performed usinga zero-inflated Poisson regression model. Results: In fecal supernatants, 209 human proteinswere identified; most were digestive enzymes from the proximal gut. A subset of 10 proteinswas exclusively present or substantially increased in stools from patients with neoplasms.Ranked according to p-value, the top 10 differentiating proteins were all blood-derivedincluding albumin,α-2-macroglobulin, compliment C3 protein, serotransferrin, haptoglobin,carbonic anhydrase 1, xaa-pro-dipeptidase, leukocyte elastase inhibitor, hemoglobin, andcatalase. A panel of 4 proteins (compliment C3 protein, serotransferrin, haptoglobin, andhemoglobin) was discriminating: >1, >2, and >3 markers from this panel were present in11%, 5.3%, and 0% of controls; 56%, 38%, and 25% for adenomas, and 88%, 83%, and50% for cancer. The mean total spectral abundance (MTSA) with this marker panel was 3for controls, 18 for adenomas, and 137 for cancers [25±10 for stage 1-2 (n=4), 88±30 forstage 3 (n=9), and 475±52 for stage 4 (n=3); p<0.01]. Conclusions: In stool supernatants,the high-abundance protein markers for colorectal neoplasia all originate from blood. Apanel of such markers provides greater diagnostic yield than does any single marker, anddetection rates are higher for cancer than for adenomas. Specific tumor-derived markers arelikely in much lower abundance and will require different search methods for discovery.

W1877

Changes in FXYD3 and NPM1 Expressions Are Early Markers of Adenomas inthe ColonJohn C. Wong, Simon K. Chan, David F. Schaeffer, Hagen Kennecke, Steven J. Jones,David A. Owen, Isabella T. Tai

Aims: In 2008, it is estimated there will be 170,000 new diagnoses of and 58,800 deathsfrom colorectal cancer (CRC) in Canada and the US. The significant burden of CRC has ledto searches for diagnostic and prognostic biomarkers that permit earlier disease detectionand identification of more aggressive disease. Previously, we performed a meta-analysis ofCRC expression profiling studies, identifying FXYD3 (FXYD domain containing ion transportregulator 3) and NPM1 (nucleolar phosphoprotein B23) as a consistently reported down-regulated and up-regulated gene, respectively. Our aim was to compare the expressions ofFXYD3 and NPM1 in normal colon, adenoma, CRC and metastatic lesions; and to correlatetheir expression in CRC with clinicopathological outcomes. Methods: A tissue microarrayrepresenting 309 tissue samples (64 normal colon; 42 adenomas; 137 CRC; 66 metastaticlesions), from 170 individuals (69M, 101F), was assembled. The staining intensities ofFXYD3 and NPM-1 were examined histologically and graded 0-3 by two independentpathologists. From the CRC Research Clinical Outcomes database at the British ColumbiaCancer Agency, data on AJCC stage, disease free and overall survivals, were collected. Byχ2 test, the expression profiles of FXYD3 and NPM1 in normal mucosa and the diseased

A-745 AGA Abstracts

groups were compared. Associations between the expression of both markers in CRC withdisease free and overall survivals were evaluated using the log-rank test. All tests were two-tailed and considered significant at a p<0.05. All statistical calculations were performedusing the SPSS statistical software package (version 16.0; SPSS Inc.). Results : FXYD3expression was downregulated in adenoma (p<0.03), CRC (p<0.005), lymph node anddistant metastasis (both p<0.005) compared to normal colon. FXYD3 expression was alsodownregulated in CRC compared to adenoma (p<0.03). NPM1 expression was upregulatedin adenoma, CRC, lymph node and distant metastasis (all p<0.005) versus normal colon.NPM1 expression did not differ in adenoma compared to CRC. There were no significantassociations between either markers with AJCC stage, disease free or overall survivals.Conclusions: The differential pattern of expression of FXYD3 and NPM1 in the normalcolon-adenoma-carcinoma sequence indicates that these changes occur in the early stagesof adenoma development and may implicate them as potential diagnostic markers of aden-omatous polyps and colon cancer.

W1878

Muc4: A Novel Tumor Suppressor Gene Impacts Upon Initiation andProgression Phases of Colorectal CarcinogenesisDhananjay Kunte, Christopher R. Weber, Surinder K. Batra, Srustidhar Das,Shantibhushan Senapati, Ramesh K. Wali, Jennifer Koetsier, Hemant K. Roy

Background: Mucins are a of family of ~20 heavily glycosylated proteins that have beenimplicated in GI carcinogenesis. In colorectal cancer (CRC), most mucins, except for Muc2,have been noted to be overexpressed. Muc4 is a transmembrane mucin that induces carcino-genesis in pancreas, lung and ovary, potentially via ErbB2 signaling. However, the studieson CRC has been inconclusive. We, therefore, wanted to systematically investigate the roleof Muc4 in colon carcinogesis. Methods: Immunohistochemistry (IHC):We used a previouslyvalidated monoclonal antibody for human IHC which included uninvolved mucosa (n=20), adenomas (n=14) or carcinomas (n=28). IHC were quantitated by a GI pathologist.Azoxymethane (AOM)-treated rat: Since the baseline for the IHC studies were uninvolvedmucosa, it is difficult to ascertain whether Muc4 expression had been altered at this earlystage. We, therefore, assessed the effect of a well-established chemopreventive agent on Muc4 levels in premalignant mucosa. 8 weeeks post AOM injection (prior to adenoma formation),10 rats were gavaged daily with sulindac or vehicle for 7 days. Animals were euthanizedand Muc4 expression was assessed through RT-PCR. Cell Culture: To further ascertain thebiological role of Muc4, we created a shRNA knockdown in HT-29 andmeasured proliferationby PCNA and S phase analysis via flow cytometry. Results: Muc 4 expression was progress-ively lost as the stage of colon carcinogenesis increased (see table). In the AOM-rats, shortterm treatment with sulindac resulted in a dramatic upregulation (133 % of control) ofMuc4 suggesting that even in the premalignant mucosa there was relative Muc4 loss. Tounderstand the cellular consequences of MUC4, we employed HT-29 with ~50% knockdownof MUC4. HT-29 cells with MUC4 shRNA showed ~29% increase in PCNA expressioncompared to empty-vector control (p<0.05). Further supporting the increased proiferationwas demonstration that the proportion of transfectants in S phase increased by ~24% whencompared to control (p<0.05). Conclusions: We report, for the first time, that Muc4 is atumor suppressor gene in colon carcinogenesis. Muc4 appears to impact both the initiation(as suggested by AOM-treated rat data) and progression phases (stepwise decrease in Muc4at later stages of malignancy). The importance of Muc4 loss is highlighted by the increasedproliferation in Muc 4 knockdown cells consonant with diffuse mucosal hyperproliferationevident in early colon carinogenesis.Muc4 Expression in Human CRC

Expressed as % of normal mucosa

W1879

Altered Local Expression of Proinflammatory and Anti InflammatoryCytokines in Normal Mucosa Predicts Risk of Spontaneous ColorectalAdenomasTemitope O. Keku, Janie Peacock, Samuel Nzewi, Xiang J. Shen, Wen Sui, Pauline K.Lund, Robert S. Sandler

Evidence from animal and human studies link inflammation with colorectal cancer. Therole of inflammation in risk of sporadic cancer in the absence of diagnosed inflammatorybowel disease has not been extensively addressed. The role of locally expressed of pro- andanti-inflammatory cytokines in adenoma risk is not defined. We hypothesized that animbalance in the expression of proinflammatory and anti-inflammatory cytokines in normalmucosa will be associated with elevated risk of adenomas. We examined the relationshipbetween local expression of cytokines and adenomas in normal colonic mucosa in a casecontrol study. Methods: Participants were consenting patients undergoing a screening colon-oscopy at UNC Hospitals with no history or diagnosis of inflammatory bowel disease.Subjects were defined as cases or controls based on the presence or absence of adenomatouspolyps. Expression of IL-6, IL-1β, TNF-α, IL-10 and IL-8 in biopsies of normal colonictissue were determined by real time RT-PCR using ABI Taqman commercial kits (AppliedBiosystems, Forster CA). Data were obtained as threshold cycle values (CT ). Expression ofeach cytokine relative to the housekeeping gene HMBS was calculated using the 2dd CTmethod. Logistic regression was used to compute odds ratio (OR) and 95% confidenceintervals (CI) while adjusting for body mass index, NSAIDs, waist-hip ratio, age, sex andrace. Results: Case subjects were more likely to be, male, have higher waist-hip ratio andelevated expression levels of IL-6, IL-1β, IL-8 and lower IL-10 expression. Compared tothe lowest quartile, subjects in the highest tertile of IL-6 and IL-8 were significantly morelikely to have adenomas (OR 2.06, 95% CI 1.2-3.5; OR 2.14, 95% CI 1.3-3.6 respectively).

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sCases also had high mean values for local expression of TNFα and IL-1β but the resultsdid not reach statistical significance. IL-10 mRNA expression was inversely related to aden-omas (OR 0.7, 95% CI 0.6-0.95). Conclusions: These results suggest that elevated expressionof local proinflammatory markers and reduced expression of antiinflammatory markers innormal mucosa may be unique biomarkers of adenoma risk.

W1880

A Shortened Peripheral Blood Lymphocyte Telomere Length Is Associatedwith Colorectal NeoplasiaDouglas Riegert-Johnson, Lisa Boardman

Background: Telomeres are repetitive DNA sequences that cap the ends of each chromosome.Telomere shortening is associated with chromosomal instability, the molecular pathway for80% of colorectal cancers (CRCs). Measurement of colorectal tissue telomere length ispossible, but difficult. Peripheral blood lymphocyte (PBL) telomere length has been proposedas a surrogate for tissue telomere length. Aim: The aim of this study was to evaluate andcompare PBL telomere length in 1.) Patients with advanced adenomas, 2.) Lynch syndromepatients with CRC and 3.) Control patients. Methods: All patients were enrolled retrospect-ively from a biobank. Patients in the control group had had a normal colonoscopy. Patientswith advanced adenomas had an adenomatous colorectal polyp with any of the following:size > 1 cm, high grade dysplasia, or villous component >20%. Lynch syndrome patientswith CRC met accepted diagnostic criteria. Determinations of PBL telomere length in basepairs (bp) were made using DNA extracted from peripheral blood lymphocytes by a PCRbased assay. Mean PBL telomere length between groups was compared using the student'st-test. Receiver operating curve (ROC) with area under the curve (AUC) calculations wereused to determine the point of maximum combined sensitivity and specificity for PBLtelomere length. Results: There was a stepwise decrease in PBL telomere length associatedwith increasing dysplasia. PBL telomere length was shorter in patients with advanced aden-omas and Lynch syndrome patients with CRC compared to controls (p<0.001) (Table).Mean PBL telomere length was also shorter in the Lynch syndrome CRC group comparedto the advanced adenoma group (p=0.051). A PBL telomere length measurement of 5,031bphad a sensitivity of 91% and specificity of 76% for advanced adenomas compared to controls(AUC 0.89). For Lynch syndrome patients with CRC, a PBL telomere length measurementof 4,645bp had a sensitivity of 90% and specificity of 99% (AUC 0.93). Conclusions: Thisexploratory study suggests that PBL telomere length is a promising blood biomarker foradvanced adenomas in a screening population and CRC in Lynch syndrome patients. Thestepwise decrease in PBL telomere length seen from normal controls to advanced adenomasto Lynch syndrome with CRC supports the hypothesis that progressive shortening of PBLtelomere length is associated with increasing dysplasia.

W1881

The Role of Bone Morphogenetic Protein Signaling and Transforming GrowthFactor β Signaling and Their Components in Colorectal CancerRutger Jacobs, Liudmila L. Kodach, Noel F. de Miranda, Daniel Hommes, Gijs R. van denBrink, Hans Morreau, James C. Hardwick

Background: Signaling pathways play a central role in tumorigenesis and may be used inthe future as independent survival prognosticators. Bone Morphogenetic Proteins (BMPs)are members of the TGFβ superfamily of multifunctional cytokines. We have shown thatintact BMP signaling is important for normal homeostasis of the intestinal epithelium andthat loss of BMP signaling correlates tightly with progression of adenoma to cancer andoccurs relatively early during cancer progression. Although in the early stages of cancerTGFβ exerts a tumor suppressor function, there is considerable evidence that in later stagesactivity of this pathway correlates with worse clinical outcome. Whether specific elementsof the BMP pathway can be used as predictive markers for survival of patients is not known.Aims and methods: In this study we aimed to investigate whether activity of BMP and TGFβpathways could be assessed by nuclear receptor phosphorylated SMADs and could predictsurvival outcome in patients with CRC. We used a large array of 250 colon cancers andperformed immunohistochemical (IHC) analysis of BMP pathway components by BMPreceptor 2 and pSMAD1/5/8, and a specific TGFβ signaling component: pSMAD2/3. Wealso assessed the expression of the key signal transduction element SMAD4, central to bothTGFb and BMP pathways. The IHC staining was scored blinded, quantifying activity of BMPsignaling by nuclear localization of SMAD4 and nuclear localization of pSMAD1/5/8 andactivity of TGFβ signaling by nuclear localization of SMAD4 and pSMAD2/3 . The correlationbetween receptor phosphorylated SMAD staining, Dukes classification and clinical outcomes,including disease-free survival and overall survival, was analyzed with SPSS version 16 forWindows. Results: The BMP pathway is inactivated, as judged by nuclear pSMAD1,5,8expression, in 65% of CRCs and this correlates with BMPR2 and SMAD4 loss (p<0.010,Fisher's Exact Test). Analysis of Kaplan-Meier survival curves for all BMP pathway compon-ents revealed a significant difference in nuclear SMAD4 expression (p<0.04) between patientgroups. At an early stage (Dukes A+B) loss of BMP signaling is associated with worse 5 yearoutcome, while, on the contrary, at a later stage (Dukes C+D) patients with active BMPsignaling have worse survival. Conclusion: Our data suggest that BMPs play a dual role incolon cancer progression similar to TGF-β.

A-746AGA Abstracts

W1882

Monitoring of Angiogenic Factors Cannot Replace Tumor Marker Analysis inBevacizumab-Based Cancer TherapyPatrick Starlinger, Claudia Nemeth, Sebastian F. Schoppmann, Irene Kuehrer, MichaelGnant, Birgit Gruenberger, Thomas Gruenberger, Christine Brostjan

Background: Access to the blood system is indispensable for tumor growth and involvesangiogenesis. Thus, the monoclonal antibody bevacizumab (Beva) has proven a promisingtool for cancer therapy by neutralizing vascular endothelial growth factor (VEGF). We aimedto compare established tumor markers such as CEA and CA19-9 to markers of angiogenesisin Beva therapy. Pro-angiogenic VEGF and platelet derived endothelial cell growth factorversus anti-angiogenic thrombospondin-1 (TSP-1) were chosen to reflect the angiogenicbalance. Study Design and Methods: The analyses were based on two ongoing Beva trials.In a study on stage IV colorectal cancer with resection of liver metastases, 24 patients havebeen investigated to date. Blood samples were taken at the beginning and the end of theirneoadjuvant as well as adjuvant treatment based on combined Beva and chemotherapy. Ina second study, blood samples of locally advanced pancreatic cancer patients (n=19) receivingneoadjuvant therapy were taken every other week. Treatment was based on gemcitabinewith or without the addition of Beva. Markers of angiogenesis were determined in plateletpoor plasma by enzyme linked immunosorbent assay. Results: Preliminary analyses oncolorectal cancer patients showed a striking difference in the course of tumor versus angiogen-esis markers. CEA levels dropped significantly (p=0.043) during neoadjuvant therapy andremained low thereafter. However, median VEGF levels rose significantly (p≤0.03) duringneoadjuvant (pre 21 vs. post 70 pg/ml) as well as adjuvant (pre 20 vs. post 62 pg/ml)treatment. Detailed analyses on the neoadjuvant treatment of pancreatic cancer patientsrevealed a further difference in the time course i.e. a rapid incline of VEGF levels after thefirst Beva administration (p=0.004) as opposed to the gradual and continuous decline oftumor markers during therapy. TSP-1 levels correlated inversely with VEGF plasma values(p=0.003) whereas platelet derived endothelial cell growth factor levels remained largelyunaffected by therapy. Conclusions: Tumor markers were shown to correlate with tumorresponse to therapy and the post-operative reduction in tumor load. In contrast, levels ofangiogenesis markers such as VEGF and TSP-1 seem to reflect the systemic response toVEGF inactivation by Beva resulting in a rapid feedback loop that elicits an increase inVEGF and decrease of TSP-1 production. Thus, monitoring of angiogenesis markers in Bevatherapy cannot replace tumor marker analysis but may provide insight into the changes ofthe systemic angiogenic balance upon VEGF neutralization.

W1883

Low Intraepithelial CD3+/FOXP3+ Regulatory T-Cell Ratio Predicts AdverseOutcome in Human Colon CarcinomasFrank A. Sinicrope, Rafaela L. Rego, Stephen Ansell, Keith Knutson, Nathan R. Foster,Daniel J. Sargent

Background & Aims: Regulatory T cells (Tregs) express the forkhead box transcriptionfactor (FoxP3) and function to suppress the anti-tumor immune response. We analyzed theintratumoral density of FoxP3+ andCD3+ lymphocytes and their relationship to clinicopatho-logical variables and clinical outcome. Methods: Immune marker expression and localizationwas determined in TNM stage II and III colon carcinomas (n= 160) and autologous normalmucosa (n= 25). FoxP3, CD3, CD8, CD25 were analyzed in relation to DNA mismatchrepair (MMR) status. Associations were sought with clinicopathological variables and withpatient survival using Cox proportional hazards models. Results: FoxP3+ and CD3+ Tlymphocytes were increased in tumors compared to autologous normal mucosa (p< 0.0001).The intraepithelial CD3+/FoxP3+ ratio exceeded that in tumor stroma (p<0.0001). MMR-deficient (vs. proficient) tumors showed increased T cell markers, a higher stromal CD8+/FoxP3+ T ratio, but a similar CD3+/FoxP3+ ratio. Colon cancers with low intraepithelialCD3+ T cells had worse disease-free survival (DFS) [5-year: 51.1 vs. 68.1%; HR 1.87 (1.10,3.16); p=0.018]. A low intraepithelial CD3+/FoxP3+ ratio was also associated with worseDFS [5 year: 46.2 vs. 66.7%; HR 2.17 (1.11, 4.23); p= 0.0205)] and OS (HR 1.76 (0.92,3.37, p= 0.0868). FoxP3+ T cell density alone was not prognostic. In a multivariate analysis,reduced intraepithelial CD3+ T cells (p= 0.0397) and a low CD3+/FoxP3+ ratio (p= 0.0318)were independently associated with worse DFS and were stronger variables than numberof metastatic lymph nodes. Conclusions. Reduced intraepithelial CD3+ T cells and a lowerCD3+/FoxP3+ ratio were associated with worse patient survival, suggesting the importanceof an effector to Treg ratio in colon cancer prognosis.

W1884

Polymorphisms in Cytokine and Cyclooxygenase Genes Influence Long-TermSurvival in Patients with Gastric AdenocarcinomaDavid Nicolás-Pérez, Enrique Quintero, Maria Asuncion Garcia-Gonzalez, Angel Lanas,Mark Strunk, Rafael Benito, Federico Sopena, Jesús Espinel, Rafael Campo, SantosSantolaria, Marisa Manzano, Luis Bujanda, Fernando Geijo, Maria Pellise, Miguel AngelSimón, Ferrán González-Huix, Jorge C. Espinós, Luis Barranco Priego, LLúcia Titó,Manuel Zaballa

Objective: to analyze the influence of genetic polymorphisms of pro- and antiinflammatorycytokines and ciclooxigenase 1 (COX1) and 2 (COX2) in the prognosis of gastric adenocarcin-oma. Patients and methods: 322 and 65 patients with distal gastric and cardial cancerrespectively, were enrolled from May-2002 to December-2003. DNA from blood sampleswas analysed for genotyping of twenty-four polymorphisms of proinflammatory (IL-1B,TNFA, LTA, IL-12p40), anti-inflammatory (IL-4, IL-1RN, IL-10, TGFB1) cytokines andCOX1 (PTGS1) and COX2 (PTGS2), through PCR, RFLP and Taqman probes. One hundredand seven patients were excluded for unknown stage (n=56), cancer non-related death (n=25) and missing follow-up (n=26). Finally, 290 patients were included. Median follow-upwas 282 days (range: 5-2221). For survival estimates, Kaplan-Meier and log-rank tests wereapplied. Cox regression was used to assess the prognostic importance of genotypes, withadjustment for age, gender, comorbidity (Charlson score), smoking habit, cancer location