of ZIP4 (zinc transporter) has been shown in PDA which may contribute to pancreaticcancer pathogenesis and progression, and it is a target for molecular therapy. The objectiveof this study is to assess whether or not the ZIP4 expression by immunohistochemistry study(IHC) in pre-operative EUS-guided fine needle aspiration (FNA) specimens can diagnosis PDAbefore surgery and provide FNA samples to direct neo-adjuvant, targeted therapy. Methods.We compared the expression of ZIP4 by IHC in specimens obtained by EUS-FNA and thecorresponding surgically resected pancreatic tumors as parallel control. Cases with visiblemalignant cells on both FNA and surgical specimens were selected. Adjacent normal ductalglands served as internal negative control. Staining intensity (0, no staining; 1+, weak; 2+,moderate; 3+, strong) and percentage of positive cells (negative, less than 10%; 1+, 10-25%;2+, 26- 75%; 3+, greater than 75%) were assessed. Positive staining was defined as morethan 10% cells with at least 1+ intensity. Samples were reported in terms of sensitivity,specificity and predictive values as well as 95% confidence intervals (CI). Results. A totalof twenty three cases with both cytology and surgical specimens in a period of two years(2004-06) were evaluated. ZIP4 is significantly overexpressed in tumor cells in both sets ofsamples. The sensitivity, specificity, positive predictive value (PPV) and negative predictivevalue (NPV) of ZIP4 in pre-operative EUS-FNA samples are 73.9%, 73.7%, 77.3% and70.0%, respectively. While the sensitivity, specificity, PPV and NPV of ZIP4 in surgicalspecimens are 100%, 66.7%, 85.2% and 100%, respectively. Conclusion. ZIP4 could serveas a novel diagnostic marker of pancreatic cancer through IHC staining in pre-operative EUS-FNA samples. Pre-operative EUS-FNA is capable of detecting ZIP4 positivity in most cases.

ZIP4 IHC staining positive in pancreatic cancer FNA specimen

ZIP4 IHC staining negative in pancreatic cancer FNA specimen

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Clinical Significance of NIBP Expression in Human Colon Cancer TissuesHong Wang, Shu Liu, Xinmin Zhang, Yonggang Zhang, Fang Li, Wenhui Hu

Background. NIBP (NIK and IKK2-Binding Protein) is a prototype member of a novelprotein family. It forms a novel subcomplex of NIK-NIBP-IKK2 without IKK1 and IKK γand enhances cytokine-induced IKK2-mediated NFκB activation. It is also a key memberof trafficking particle protein complex II which is essential in trans-Golgi networking (TGN).Both NFκB and TGN are critical in inflammation-linked tumorigenesis and cancer metastasis.Bioinformatics studies have shown that NIBP is highly expressed in various tumor tissuesand cancer cell lines. We previously reported that NIBP retains constitutive and inducibleNFκB activation in human gastrointestinal cancer cell line, and stable knockdown of NIBPeffectively inhibits cell proliferation and colony formation (DDW 2011). Aim. To evaluateNIBP expression in human colon tissues and to explore the clinical correlation of its expressionwith tumor grades and stages. Methods. The expression of NIBP mRNA was determinedby real-time polymerase chain reaction (PCR) using TissueScan human colon cancer cDNAarray (Origene). Levels of NIBP protein were measured by semi-quantitative immunohisto-chemical analysis of a progression tissue microarray (NCI-cancer diagnosis program) contain-ing 367 cases of invasive colon carcinoma with 4 stages, 34 cases of adjacent normal colonepithelium, 40 cases of diverticulitis patients with no cancer and 37 cases of adenomatouspolyps. Each core was assessed by two pathologists blind to the clinical information andwas scored as negative (0), weekly positive (1+), moderately positive (2+) or strongly positive

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(3+). Results. NIBP mRNA was highly expressed in 80% colon cancer patients, with thehighest in stage I. NIBP-like immunoreactivity was found significantly increased in invasivecolon carcinoma compared with non-cancer colon tissues (p ,0.0001 by Kruskal Wallistest). In univariate and multivariate analyses, NIBP was a significant predictor of survivaland recurrence. However, there was no significant correlation of NIBP expression levelswith the stage, grade, age, sex, types, locations and node invasion. Conclusion. NIBP ishighly expressed in human colon cancer tissues. NIBP may serve as a biomarker for thedevelopment and prognosis of colon cancer.

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Distinct Subcellular Localization of Phosphorylated ErbB2, ErbB3 and ErbB4in Colorectal Cancer Cells and Their Relevance to Clinical SignificanceAtsushi Tatsuguchi, Keigo Mitsui, Masaoki Yonezawa, Shu Tanaka, Shunji Fujimori, KatyaGudis, Choitsu Sakamoto

Background: The various members of the erbB family of receptors, including epidermalgrowth factor receptor (EGFR), erbB2, erbB3, and erbB4, play different and distinctivephysiological and pathological roles. Pharmacological EGFR blockers have had modestefficacy against colorectal cancers in the clinical setting. In colon cancer cell lines, erbB2and erbB3 have been shown to be expressed at high levels and to be involved in theregulation of invasiveness and resistance of apoptosis. However, the clinical significance oferbB4 and its phosphorylation has yet to be explored in colorectal cancer. Aims: To investigatephosphorylated erbB2 (pErbB2), erbB3 (pErbB3) and erbB4 (pErbB4) protein expressionin human colorectal cancer and determine the relationship between their expression andclinicopathological factors and patient prognosis. Patients and Methods: We analyzed theeffects of heregulin (HRG)-induced phosphorylation on erbB2, erbB3, erbB4 in Caco-2,DLD-1, and HCT116 colon cancer cell lines by western blot. We examined 155 surgicalresections from colorectomy patients. Cellular localization of pErbB2, pErbB3, pErbB4 andheregulin protein in colorectal cancer surgical resections was analyzed by immunohistoche-mistry. Immunohistochemical results were compared with clinicopathological factors andpatient prognosis. Results: We found evidence in a previous study of exogenous HRG-induced phosphorylation of erbB2 and erbB3 in both the nuclear and cytosolic fractions ofCaco-2 and DLD-1 cells, suggesting that HRG stimulates translocation of erbB2 and erbB3into the nucleus. In contrast, HRG-induced phosphorylation of erbB4 is detected only inthe cytosolic fraction of HCT116 cells, suggesting that erbB4 fails to translocate into nucleus.Nuclear pErbB2 and pErbB3 immunoreactivity was predominantly observed in cancer cellsin 15 (10%) and 36 (23%)cases, respectively. In contrast, pErbB4 immunoreactivity waspredominantly observed in the cell membrane and cytoplasm in 15 (10%) cases and pErbB4expression was significantly higher in advanced cancer than in the early stages. In univariateanalysis, pErbB2, pErbB3, pErbB4 and HRG correlated with worse prognosis in colorectalcancer patients. In multivariate analysis, pErbB2, pErbB3, pErbB4 and HRG expressionretained independent prognostic significance. Conclusions: ErbB2 and erbB3 phosphorylatedby HRG translocate into the nucleus of colorectal cancer cells. In contrast, erbB4 phosphory-lated by HRG retains its location in the cell membrane of cancer cells. This difference insubcellular localization suggests distinct functions for the different members of the phosphor-ylated EGFR family. Moreover, since the translocation of erbB2 and erbB3 into the nucleusmay contribute to molecular targeting therapy resistance, erbB4 may serve as a novel targetfor therapeutic intervention.

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Role of Isoprostanes in Colorectal CancerDzifaa Lotsu, Martha Shrubsole, Qiuyin Cai, Walter E. Smalley, Ginger L. Milne, Qi Dai,Reid Ness, Wei Zheng, Harvey Murff

BACKGROUND: Epidemiological studies suggest a westernized diet may be associated withincreased colorectal neoplasm risk; however, the mechanism through which this is mediatedis unknown. N3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA) have been extensively studied in heart disease for theiranti-inflammatory properties and recently found to have anti-tumor properties. We proposedthat higher n6 are associated with increased risk of colorectal neoplasms mediated througheffects of lipid peroxidation. The n6 PUFA, arachidonic acid, is oxidized to F2-isoprostanes(IsoPs), while EPA, an n3 PUFA, is oxidized to F3-IsoPs. METHODS: Data source for thisstudy was the TN Colorectal Polyp Study (TCPS), a case-control study, designed to evaluategenetic and lifestyle factors in colorectal cancer. Our study included 1050 adenoma casesand 1050 polyp-free controls. Cases were matched to controls in a 1:1 ratio. Blood andurine samples were collected prior to colonoscopy. Primary exposure of our study wasurinary levels of F2- and F3-IsoPs. Levels of F2- and F3-IsoPs were quantified using gasliquid chromatography and tandem mass spectrometry. We compared the distribution ofdemographics such as age, race, gender, education, income, BMI, and other lifestyle factors.We used Student's T-test or Wilcoxon Rank-Sum test for continuous factors according to theirdistribution and Chi-square tests for categorical variables. We used conditional multinomiallogistic regression models to analyze F2- and F3-IsoPs with advanced adenomas and non-advanced adenoma adjusting for demographics and study site. RESULTS: Preliminary resultsincluded 411 total subjects. We included 221 cases and 190 controls in our analysis. Thesample was predominately male (65%), Caucasian (94%), and primarily from Vanderbilt(78%). Urinary IsoPs levels were subdivided into quartiles. Odd-ratios (OR) of F2-IsoPs,F3-IsoPs, and ratio of F3:F2-IsoPs were calculated with the first quartile as reference value.The adjusted (aOR) of F2-IsoPs and adenoma risk was 1.26, 95% CI 0.72-2.23(Q2-Q1);1.06, 95% CI 0.60-1.87(Q3-Q1); and 1.07; 95% CI 0.57-2.04(Q4-Q1). aOR of F3-IsoPsand adenoma risk was 0.69; 95% CI 0.32-1.47(Q2-Q1), 0.54; 95% CI 0.25-1.15(Q3-Q1),and 0.84, 95% CI 0.38-1.91(Q4-Q1). The aOR of F3:F2-IsoPs and adenoma risk was 0.48,95% CI 0.28-0.98(Q2-Q1), 0.57, 95% CI 0.28-1.16(Q2-Q1); and 0.61, 95% CI 0.29-1.28(Q2-Q1). Conclusion: Our preliminary results suggest that there is lack of associationbetween F2- and F3-IsoPs and colorectal neoplasm risk; however, the ratio of F3/F2 IsoPsappears to have an association to colorectal cancer risk although this relationship does notappear linear. These results suggest that a higher F3:F2-IsoPs ratio may be protective;however, the mechanism is unclear and will require further investigation.

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