Sa1893

Annexina2 is Increasingly Expressed and Released Into the Serum of PatientsPositive for Colonic Growths/Tumors in Relation to Disease Progression:Diagnostic ImplicationsShubhashish Sarkar, Carrie A. Maxwell, Gurinder Luthra, Ashwani Singal, Suimin Qiu,Valerie P. Bauer, Anthony O. Okorodudu, Pomila Singh

Background. Overexpression of AnnexinA2 (ANXA2) has been reported in several epithelialcancers, including colorectal cancers (CRC) (Cancer Letters, 2007). Increasing expressionof gastrin gene, and hence progastrin (PG), has also been reported in CRCs. We recentlydiscovered that ANXA2 serves as a non-conventional receptor for PG (Oncogene, 2007).We additionally reported that binding of PGwith extracellular, membrane associated, ANXA2results in endocytosis and co-localization of PG/ANXA2 intracellularly (Gastro, 2010), andthat ANXA2 is required for mediating mitogenic and anti-apoptotic effects of PG on coloncancer cells. ANXA2 is normally involved with intracellular trafficking. During the courseof our studies we discovered that ANXA2 is also present on outer membranes of CRC cells,bound to a 29KDa transmembrane protein, and also released into the medium. To furtherunderstand physiological significance of these unexpected findings, we examined if ANXA2is present in serum of patients positive for colorectal growth and if ANXA2 co-localizes withPG in tumor sections, in relation to disease status. Methods, Results and Conclusions.Secretion of ANXA2 (10-50ng/106 cells) was confirmed in conditioned medium (CM) ofCRC cells using quantitative Western Blot assay using standard curve with rhANXA2; non-transformed cells were negative. Serum from CRC tumor bearing mice were positive forANXA2, while control mice were negative. Next, serum samples were obtained from con-sented patients at time of endoscopy or as discarded samples from CRC patients on day ofsurgery, as per our IRB protocols. Serum from patients, negative for colonic growths (Normal,N) had low levels of ANXA2 (<1ng/ml), while serum from patients with hyperproliferativegrowths (Hp), Adenomas (Ad) and adenocarcinomas (AdCA) were on an average positivefor 42, 80, and 490 ngs/ml, respectively, suggesting elevation of serum ANXA2 in relationto disease status. Paraffin blocks of Hp, Ads, AdCAs and adjoining N mucosa were obtainedas discarded specimens from Department of Pathology and stained immunofluorescently forPG/ANXA2. Hp, Ads and AdCAs were increasingly positive for ANXA2 and PG expressioncompared to N specimens; ~ 10, 25 and 60 % of respective specimens were positive forintracellular co-localization of PG/ANXA2, suggesting presence of functional PG/ANXA2axis, confirmed by progressive elevation of nuclear β-catenin and stem cell markers(DCAMKL+1, Lgr5) in relation to disease status. It remains to be determined if, 1) presenceof co-localized PG/ANXA2 intracellularly represents a prognostic marker for predictingtreatment/recurrence, and 2) if serum ANXA2 levels can be used as a non-invasive diagnosticmarker for predicting presence of colorectal growths, stage of disease and/or relapse. Sup-ported by NCI grants CA97959 and CA114264 to PS.

Sa1894

MAb DAS-1, a Monoclonal Antibody Reactive Against a Colonic Phenotype,Identifies Pancreatic Adenocarcinoma and High-Grade PancreaticIntraepithelial Neoplasm (PanIN) With High SpecificityKoushik K. Das, Kiron M. Das, Mari Mino-Kenudson

BACKGROUND: Pancreatic intraepithelial neoplasms (PanIN) are microscopic precursorlesions to pancreatic ductal adenocarcinoma (PDAC) and are classified into three grades,based on the degree of architectural and nuclear atypia present. mAb Das-1 is a murinemonoclonal antibody that reacts specifically to a colonic epithelial phenotype. It is bothsensitive and specific in identifying various pre-malignant and malignant lesions of the upperGI tract including Barrett's esophagus, and incomplete gastrointestinal metaplasia associatedwith gastric cancer. AIM: To assess the ability of mAb Das-1 to identify high-grade PanINlesions and associated PDAC. METHODS: We examined paraffin embedded surgical tissuesamples from 84 patients with 99 distinct PanIN lesions, 75 pancreatic adenocarcinomas,and 31 associated lymph node metastases. A representative section from each lesion wasstained with Das-1 by immunohistochemistry with positive (normal colon) and negative(normal duodenum) controls. The cytoplasmic expression of Das-1 in greater than 5% ofabnormal glands was considered to be positive. Statistical significance was calculated by theFisher exact test when compared to internal controls of normal pancreatic ducts observedin 44 cases. RESULTS: All of the normal pancreatic duct controls were negative for Das-1,0/44 (0%). Of the low-grade lesions, 0/48 (0%) of PanIN 1 and 4/36 (11%) PanIN 2 lesionswere reactive to Das-1. Compared to normal pancreatic duct controls and PanIN 1 & 2lesions, Das-1 expression amongst PanIN 3 was significantly higher 10/15 (67%) (p<0.001and p<0.001, respectively). Among invasive carcinomas, when compared to normal controlsand PanIN 1& 2 lesions, Das-1 was expressed in 56/75 (75%, p<0.001 and p<0.001) PDACsand 22/31 (71%, p<0.001 and p<0.001) associated metastatic lymph nodes. Reactivity toDas-1 was notably less frequent in poorly differentiated lesions, both in the primary neoplasmand in metastatic sites. Overall, the sensitivity and specificity of Das-1 in segregating PanIN3/Adenocarcinoma from low-grade PanIN lesions were 73% and 95%, respectively. CON-CLUSION: mAb Das-1 reacts with high specificity to high-grade/risk PanIN lesions andpancreatic adenocarcinoma as compared to normal pancreatic ducts and low-grade lesions.The expression of this marker may be a useful tool to identify the progression of PanINlesions to PDAC, and to aid the diagnosis of high-risk PanIN lesions and PDAC in limitedtissue samples such as FNA cytology and biopsy.

PanIN = Pancreatic Intraepithelial Neoplasms, PDAC = Pancreatic Ductal Adenocarcinoma,LN = Lymph Node Metastasis

S-341 AGA Abstracts

Sa1895

Fluorescence Detection of Colonic Neoplasia Using a Novel PeptideBishnu P. Joshi, Sharon J. Miller, Christine M. Komarck, D. K. Turgeon, Henry D.Appelman, Zhongyao Liu, Thomas D. Wang

Background: Colorectal cancer is a major cause of morbidity and mortality. Despite therecent improvements in preventive approaches, screening techniques and development ofchemotherapy, the median overall endurance period for patients with metastatic colorectalcancer is about two years. Early detection of colorectal neoplasm with improved methodswould have important clinical applications to increase the survival rate. Peptides are promisingfor clinical use as a novel imaging agent for the detection of flat and depressed lesions andfor monitoring tumor margins during resection. Aims: We aim to select and validate anaffinity peptide that binds to colorectal neoplasia for future clinical studies. Methods: Thecandidate peptide was selected using techniques of phage display against HT29 cells asthe biopanning substrate. Negative selection was performed against non-malignant humanintestinal cells (Hs738.st/int.). Validation of the peptide was performed In Vitro on cells inculture by bound phage counts, enzyme-linked immunosorbent assay (ELISA), flow cytome-try, competitive inhibition, fluorescence microscopy and immunohistochemistry. Fluores-cence staining using this peptide was also assessed on human surgical tumor tissues andnormal tissues from colon. Results: The target peptide was selected and showed preferentialbinding to HT29 cells In Vitro with ELISA, flow cytometry analysis, immunohistochemistry,and fluorescence microscopy. Reduced binding was observed on a competition assay withunlabeled peptide in a dose-dependent manner. A binding affinity of Kd =65 nmol/L wasmeasured, and peptide binding to the HT29 cells was validated on fluorescence microscopy.On human surgical specimens (n = 10), the fluorescence intensity (mean ± SEM) in serial10 μm sections classified histologically as dysplasia (n = 4), carcinoma (n = 6), non-neoplastic(n = 10), was 25.0±6.5, 38.0±13.0 and 7.4±3.75 arb. units, respectively, resulting in atarget-to-background ratio of 3.4 and 5.1 to dysplasia and adenocarcinoma, respectively.Conclusions: A target peptide was selected and found to bind selectively to pre-malignant(dysplastic) and malignant (adenocarcinoma) human colonic mucosa compared to normal.This peptide can be labeled with fluorescence for targeted imaging of pre-malignant andmalignant mucosa on clinical imaging.

Sa1896

Role of Reprimo, a p53-Dependent G2 Arrest Mediator Candidate, in thePathogenesis and Early Diagnosis of Human Gastric CarcinomaAlejandro H. Corvalan, Maria J. Maturana, Veronica Torres, Cynthia Villarroel, EudociaSantibañez, Jose Diaz, Francisco Aguayo, Alfonso Calvo, Catterina Ferreccio

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death worldwide. This scenario indicates the necessity of developing biomarkers forearly diagnosis. We have identifed a potential biomarker for massive screening, the DNAmethylation of the promoter region of Reprimo, a p53-dependent G2 arrest mediator candid-ate (Clin Cancer Res 2008;14:6264-9). Here, we aim to characterize the role of Reprimo ingastric cancer pathogenesis and to develop a quantitative assay for early detection. DNAmethylation status of the promoter region of Reprimo was evaluated by Bisulfite Sequencingmethod in four gastric carcinoma cell lines (AGS, NCI-N87, MKN-28, MKN-45). Three celllines (AGS, MKN-45, NCI-N87) contain dense/uniform DNA methylation patterns. MKN-28 contains a heterogeneous pattern. After 5-aza-cytidine treatment, a demethylating drug,AGS, MKN-45 and NCI-N87 cells showed >30% reversion of DNA methylation. RT-PCRfor Reprimo expression was negative in two cell lines (AGS andMKN-45) before the treatmentbut became positive after the treatment. Sequence of full coding region of RPRM reveal nomutation in all cell lines tested. Stable transfection of a recombinant construct expressingReprimo in AGS cell line up-regulated mRNA of Reprimo and implicated a decrease in theability of cells to grow in semi solid-medium. Finally by RealTime-PCR (Methylight), cut-off of 15.125 copies/ml correctly distinguish healthy donors vs gastric cancer by ROC curveanalysis (sensitivity: 87%, specificity: 97.6%, AUC: 0,974, 95% CI: 0,829-0,974, p value:0,0001). Our data confirms an epigenetic regulation by DNA methylation of promoter regionof Reprimo in gastric cancer. Methylight is able to correctly distinguish gastric cancer vshealthy donors in plasma samples.

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