Neoadjuvant Chemoradiation Therapy for Pancreatic Cancer is Enhanced by Early Placement of Uncovered Self-Expanding Biliary Metal Stents (SEMS)

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DEN-induced HCC tumors at 8 months after DEN injection. The number of BrdU-positive nuclei was significantly decreased in Sulf2-KO mice compared with WT mice. E-cadherin staining was stronger in tumors of Sulf2-KO mice than in tumors induced in WT mice. On the other hand, staining of the EMT markers Slug and Vimentin was stronger in tumors occurring in WT mice than in Sulf2-KO mice. In addition β-catenin immunostaining was stronger in nuclei of tumors occurring in WT mice than in Sulf2-KO mice. Conclusions: Knockout of Sulf2 reduced DEN-induced liver tumorigenesis in mice. The decrease in liver tumorigenesis in Sulf2-KO mice was associated with a more epithelial phenotype, suggesting that Sulf2 plays a role in the EMT during liver carcinogenesis.

AGA Abstracts

The Transient Receptor Potential Ca2+ Ion Channel TRPC1 Mediates TGFβInduced PTEN Downregulation and Pancreatic Cancer Cell Motility Jimmy Y. Chow, Chang-Whan Kim, Hui Dong, Tiffany A. Ornelas, Jenny M. Li, Peik L. Sia, Jeffrey Liu, John M. Carethers Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality - dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Mo1931 Clinical Outcome of Polypoid Lesion of Gallbladder 10 mm or Larger Won Jae Yoon, Yong-Tae Kim, Jaihwan Kim, Dong-Won Ahn, Ji Kon Ryu, Yong Bum Yoon Background and Aim: Polypoid lesions of the gallbladder (PLGs) are detected more frequently due to increased use of abdominal ultrasonography (US). The significance and appropriate management of PLGs are not well understood. Most reports are based on surgical series. The aims of this study was to evaluate the clinical outcome of PLGs with diameter ≥ 10 mm. Materials and Methods: We analyzed the data of patients who were diagnosed with PLGs with diameter ≥ 10 mm by US, endoscopic ultrasonography (EUS), and/or computed tomography between 2000 and 2008 at Seoul National University Hospital and were followed up for ≥ 6 months or underwent surgery. Size increase was defined as diameter increase > 3 mm. Data was collected until December 31st, 2009. Results: Two hundred sixty-nine patients (131 men, median age at PLG diagnosis 49 years) were collected. Mean diameter of the PLG was 13 mm. Solitary PLG was diagnosed in 153 patients; sessile PLGs were diagnosed in 76 patients. Coexisting gallstone was detected in 36 patients. One hundred eighty-one patients underwent surgery. In the 88 patients who did not undergo surgery (median follow-up period 23.8 months), only 3 patients demonstrated size increase of the PLG, with 2 patients developing malignancy; these patients showed size increase at 1 year. Of 188 patients who underwent surgery, 59 (31.4%) had neoplastic polyps (42 adenoma, 16 adenocarcinoma, and 1 lymphoma) In univariate analysis of factors associated with malignant PLG, age at diagnosis ≥ 50 years (P=0.035), initial PLG diameter ≥ 12 mm (P= 0.003), sessile PLG (P=0.016) were associated with malignancy; hyperechoic spots on US or endoscopic ultrasonography (EUS) was negatively associated with malignant PLG (P<0.001). In multivariate analysis, age at diagnosis ≥ 50 years (OR 3.72, 95% CI 1.0912.69; P=0.036) and initial PLG diameter ≥ 12 mm were associated with malignancy; hyperechoic spots on US or EUS was negatively associated with malignant PLG. In the 38 patients with initial diameter of PLG < 12 mm and hyperechoic spots on PLG, no malignant PLG was found. Conclusion: In patients with PLG diameter ≥ 10 mm, malignancy should be expected when PLG size increases (> 3 mm) at 1 year. Even in patients with PLG diameter ≥ 10 mm, the risk of malignancy seems low in patients with initial diameter of PLG < 12 mm and hyperechoic spots on PLG.

Mo1929 CITED2 is a Novel Direct Effector of Peroxisome Proliferator-Activated Receptor Gamma in Suppressing Hepatocellular Carcinoma Cell Growth Kin-Fai Cheung, Junhong Zhao, Alfred S. Cheng, Joseph J. Sung, Jun Yu Background: We previously reported that activation of peroxisome proliferator-activated receptor γ (PPARγ) by PPARγ agonist suppressed hepatocarcinogenesis both In Vitro and In Vivo [Yu et al, Hepatology 2006, 2010]. To elucidate the molecular basis of PPARγ action, we extended to explore PPARγ downstream effectors involved in the tumor suppressive effect in hepatocelullar carcinoma (HCC). Aim: To identify molecular targets and potential signaling pathways with anti-tumor effect regulated by PPARγ in HCC. Methods: PPARγ activation was induced by PPARγ agonist (rosiglitazone) in HCC cell line (Hep3B). Precise PPARγ binding activity was determined by enzyme-linked immunosorbent assay. The PPARγ downstream targets were identified through gene expression profiling using 44K oligo array. PPARγ-bound target was determined by chromosome immunoprecipitation (ChIP)-PCR. The anti-tumor effect of Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) was evaluated by colony formation assay and cell cycle analysis by flow cytometry. CITED2 target signal pathway was identified by human cancer pathway PCR array. Results: Optimal PPARγ binding activity was obtained with 100 μM rosiglitazone for 3 hours. Under PPARγ activation, 329 genes were up-regulated and 245 genes downregulated using oligo array. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database indicated that PPARγ-associated genes mainly involved in the pathways including MAPK, TGF-beta, p53, and ECM receptor interaction. CITED2 was found to be the most predominant PPARγ-bound target by ChIP-PCR. Induced expression of CITED2 was also observed after adenovirus-PPARγ transduction by Western blot. In nine primary HCCs, CITED2 mRNA was significantly down-regulated compared to the adjacent non-tumor tissues (P<0.05). The biological function of CITED2 was evaluated by loss of gene function assay. Knockdown of CITED2 was obtained in a nontumorigenic hepatocyte cell line (LO2) by sh-CITED2. Such knockdown dramatically increased the ability of LO2 to form colonies (P<0.001) and promoted S-phase DNA synthesis (22.6% vs 25.2%, P<0.01). Furthermore, knockdown CITED2 caused the upregulation of proliferative regulator (TEK), pro-invasion/ metastasis genes (MMP2, SERPINB5), anti-apoptotic genes (TNF, TERT), and downregulation of proliferative inhibitor (INFA1) and pro-apoptosis genes (TNFSF1A, TNFRSF25, CASP8, GZMA, PARP). Conclusions: CITED2 is a novel direct effector of PPARγ in inhibiting HCC growth and proliferation. The investigation of the regulation and function of CITED2 will reveal insights into the hepatocarcinogenesis and provide novel target for treatment of HCC.

Mo1932 Different Expression of E-Cadherin and β-Catenin in Intestinal Type and Pancreatobiliary Type of Ampulla Vater Cancer Seon Mee Park, Joung-Ho Han, Soon Man Yoon, Hee Bok Chae, Sei jin Youn, Eui Keun Seo, Young Shim Cho, Rohyun Sung Background and aims: Ampulla vater cancers(AVC) are classified histologically as an intestinal type and a pancreatobiliary type. The molecular pathogenesis of both histologic types are not well characterized. We aimed to investigate the expression patterns of E-cadherin and β-catenin in both histologic types and the relations with clinicopathologic features of AVC. Methos: Twenty-six resected AVC specimens were stained with antibodies to E-cadherin and β-catenin. Normal expression was defined as exclusive membraneous staining. Dysregulated expression was defined as cytoplasmic staining in more than 50% of tumor cells and/or nuclear staining. Demographics and histopathological data were collected by retrospective chart review. Results: Intestinal type was noted in 12(46%) cases and pancreatobiliary type in 14(54%) cases. Preserved membranous staining of E-cadherin was frequent in intestinal type than in pancreatobiliary type(83% vs. 29%; p=0.008). E-cadherin expression was well preserved in early stages(p=0.039), well differentiated type(p=0.034) and histologic features of intestinal type-pseudostratification(p=0.007) and carcinomas with adenoma component(p=0.014). Preserved membranous staining of β-cadherin was frequent in pancreatobiliary type than in intestinal type(75% vs. 25%, p=0.039). Aberrant cytoplasmic and/or nuclear staining of β-cadherin was prominent in the histopathologic features of intestinal type, such as pseudostratification(p=0.004) and carcinomas with adenoma component(p=0.027). Conclusions: Dysregulation of E-cadherin and β-cadherin expression may play a role differently in the carcinogenesis of AVC, E-cadherin in pancreatobiliary type and β-cadherin in intestinal type.

Mo1930 Decreased Liver Tumorigenesis in Sulfatase 2 (SULF2) Knockout Mice May Be Mediated Through Inhibition of the Epithelial-Mesenchymal Transition (EMT) Ikuo Nakamura, Michael K. Asiedu, Keith Knutson, Kadra H. Ahmed, Catherine D. Moser, Chunling Hu, Lewis R. Roberts

Mo1933 Neoadjuvant Chemoradiation Therapy for Pancreatic Cancer is Enhanced by Early Placement of Uncovered Self-Expanding Biliary Metal Stents (SEMS) John Y. Nasr, David Y. Lo, A. James Moser, Michael Sanders, Adam Slivka, Herbert Zeh, Georgios I. Papachristou, Jennifer Lewis, Eric Wright, Gennadiy Bakis, Ramesh Srinivasan, Abby Crume, Lisa A. Rutstein, Douglas A. Howell

Background and Aims: Sulfatase 2 (Sulf2) plays a critical role in tumorigenesis of hepatocellular carcinoma In Vivo. Analysis of genes expressed in association with SULF2 in human HCCs suggests that SULF2 is associated with the epithelial to mesenchymal transition (EMT) during liver carcinogenesis. We determined the effect of knockout of Sulf2 on liver carcinogenesis in mice. Methods: Liver carcinogenesis was induced in wild-type (WT) or Sulf2 knockout (Sulf2-KO) mice using the liver carcinogen diethylnitrosamine (DEN). We examined tumor size and number, DNA synthesis, the expression of cytokines and growth factors, BrdU staining as a measure of cell proliferation, E-cadherin staining as a marker of the epithelial cell phenotype, and Slug and Vimentin as markers of the mesenchymal cell phenotype. We also stained liver sections with β-catenin to study the relationship between SULF2 and Wnt signaling. Results: Knockout of Sulf2 decreased the size and volume of

AGA Abstracts

Background: Neoadjuvant chemoradiation (NeoRx) for pancreatic cancer obviates surgery for patients with unrecognized micrometastases and may improve 5 year survival (Evans, JCO 2008). Reports of frequent plastic biliary stent occlusion during NeoRx suggests a role for initial SEMS placement. Methods: We evaluated outcomes of pancreatic cancer patients treated with uncovered biliary SEMS and NeoRx at two referral centers since 2006. NeoRx consisted of 6-8 weeks of Gemcitabine-based RT and 4 weeks of recovery prior to planned surgical resection. Results: 77 patients (38 males: 39 females) with age range 37-86 were

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identified with the following clinical stages at diagnosis: 1B (n=9), IIA (n=32), IIB (n=31), stage III (n=5). Uncomplicated ERCP SEMS placement was successful in 100%. 6/77 patients (7.8%) did not complete NeoRx due to disease progression, poor tolerance, or were lost to follow-up. Of these 77 SEMS, 8 (10.4%) developed signs of stent dysfunction; recurrent jaundice (n=2), cholangitis (n=1), or stent occlusion (n=5), and all were managed successfully with repeat ERCP. 66 patients (85.7 %) completed NeoRx, of which 27 (40.9%) progressed. Among the remaining 39 patients, 7 (17.9 %) were unresectable due to occult metastases (n=3), vascular encasement (n=2), or medical comorbidities (n=2). No intraoperative complications were attributed to SEMS. 29 patients achieved R0 status, and 3 were R1. 4 minor and 3 major complications were observed within the first 60 days postoperatively. Conclusions: Early SEMS placement permits neoadjuvant therapy with a low complication and occlusion rate and no interference with surgery. Use of biliary SEMS eliminates the need for repeat ERCP to change from plastic stents. A multicenter trial of SEMS prior to neoadjuvant therapy is required.

HR hazard ratio, CI confidence interval, CRC colorectal cancer

Mo1934 Restoration of Death Receptor-Induced Apoptosis in Chemo-Resistant Pancreatic Cancer Stem Cells Inhibits Peritoneal Carcinomatosis Yi Shen, Ryan Herde, Courtney L. Scaife, Jill E. Shea, Scott K. Kuwada Cancer stem cells have been shown to be sufficient for new tumor formation and metastasis in mouse models. We recently found that pancreatic cancer stem cells express TNFalpha family death receptors, but are resistant to FASL- and TRAIL- induced apoptosis. AIMS: To determine how death receptor resistance can be overcome in pancreatic cancer stem cells (PCSC) In Vitro and In Vivo. METHODS: SU.86 and PL-45 human pancreatic cancer cell lines that express endogenous FAS, DR4, and DR5 death receptors were stably transfected with firefly luciferase to allow detection by bioluminescence. PCSC were enriched from SU.86 and PL-45 by FACS using CD133 antibodies. PCSC were co-cultured on primary human mesothelial cell monolayers, that express endogenous FASL and TRAIL. PCSC were injected intraperitoneally (IP) into athymic mice and peritoneal carcinomatosis observed and quantified by bioluminescent tumor detection (IVIS system) after IP administration of D-luciferin. RESULTS: PCSC were 1-1.5 log orders more efficient in generating peritoneal tumors at one week. Athymic mice received 2.5mg/kg BAY 11-7085 IP or equimolar concentrations of bortezomib or gemcitabine IP 4 hours prior to and every 3 days following IP injection of 40,000 PCSC for 3 weeks. BAY 11-7085 treatment resulted in 1000-, 86-, and 1,100-fold reductions in peritoneal tumor burden (photons/cm2) compared with vehicle, gemcitabine, and bortezomib, respectively (p<0.05 for all comparisons). In a large screen of cell survival proteins, 10μM BAY 11-7085, but not bortezomib or gemcitabine, inhibited FLIP expression in PCSC. PCSC were found to express FLIP protein while CD133- pancreatic cancer cells did not, which could explain why BAY 11-7085, but neither bortezomib or gemcitabine, inhibited peritoneal carcinomatosis. In the co-culture system, FAS or TRAIL receptor blocking antibodies rescued PCSC from apoptosis during adhesion to mesothelial cells. BAY 11-7085 induced JNK activation, which in turn caused FLIP degradation in PCSC. Inhibition of atypical but not classical or novel PKC isoforms inhibited BAY 11-7085induced JNK activation, FLIP degradation and PCSC death. CONCLUSIONS: The ability to pharmacologically inhibit FLIP represents a novel method of decreasing peritoneal seeding by allowing normal mesothelial cells lining the peritoneum to activate death receptor signaling in PCSC. This may represent a novel treatment to prevent peritoneal seeding during pancreatic cancer resection.

Mo1936 A Combination of Intratumoral Interferon-Alpha Gene Transfer and Syngeneic Hematopoietic Stem Cell Transplantation Induces an Effective Antitumor Immunity for Solid Cancers Kenta Narumi, Toshihide Okada, Masakazu Yamagishi, Takahiro Ochiya, Kazunori Aoki In an autologous hematopoietic stem cell transplantation, the transfused T cells recognize low-affinity self antigens including tumor-associated antigens under the condition of lymphopenia-induced homeostatic proliferation (HP) and lead to a break in tolerance against tumors. HP-driven antitumor responses, however, decay gradually since they are vulnerable to a development of tolerance. Type I interferon (IFN) has important roles in regulating the innate and adaptive immune system. In this study, we examined whether HP-induced antitumor activity can be enhanced by IFN-alpha gene transfer during a physiologic immune reconstitution and investigated mechanisms of the enhancement. First, to examine whether HP of T cells induces antitumor immunity in lymphopenic hosts, lethally irradiated BALB/ c mice were injected subcutaneously with CT26 colon cancer cells shortly after irradiation, and syngeneic bone marrow and T cells were transfused (syngeneic hematopoietic stem cell transplantation; SynHSCT). The growth of CT26 subcutaneous tumors was significantly suppressed in the SynHSCT recipients. Then, to examine whether a combination of intratumoral IFN-alpha gene transfer enhances antitumor immunity after SynHSCT, the IFN-alphaexpressing plasmid complexed with cationic liposome was injected into the tumors 7 days after HSCT. The combination of IFN-alpha gene transfer and SynHSCT resulted in a synergistic tumor suppression. The suppression was evident even in distant tumors that were not injected with the IFN-alpha plasmid in the liver metastasis models. In the treated mice, the proliferation and activation of tumor-specific T lymphocytes were confirmed by ELISpot assay. There was no significant toxicity in the treated animals. To analyze antitumor immune mechanisms of the combination therapy, we isolated CD11c+ cells from the tumors treated by IFN-alpha gene transfer in SynHSCT mice. An ELISpot assay showed that a culture of lymphocytes with the CD11c+ cells resulted in a higher number of IFN-gamma spots than cultures with control CD11c+ cells isolated from tumors treated with SynHSCT alone, demonstrating that IFN-alpha gene transfer effectively augmented antitumor immunity mediated by CD11c+ cells. An analysis of cytokine profile showed that the CD11c+ cells secreted a significant amount of IL-6. It is known that IL-6 suppresses the proliferation and differentiation of regulatory T cells. In fact, the inhibitory activity of regulatory T cells was significantly suppressed by co-culture with the CD11c+ cells treated by a combination therapy, suggesting that the CD11c+ cells suppress the regulatory T cells. A combination of the intratumoral IFN-alpha gene transfer with SynHSCT is a promising immunotherapy for solid cancers, based on the activation of tumor-specific immunity, suppression of the immunotolerant environment and an excellent safety features.

Mo1935 The Effects of Metformin on Survival After Diagnosis of Colorectal Cancer in Diabetic Patients Jin Ha Lee, Tae Il Kim, Soung Min Jeon, Sung Pil Hong, Jae Hee Cheon, Won Ho Kim Background & Aims: Metformin use has been associated with decreased cancer risk and mortality. However, the effects of metformin on clinical outcomes after colorectal cancer (CRC) diagnosis are not defined. This study aimed to evaluate the relationship between prediagnosis metformin use and mortality after CRC diagnosis in diabetic patients. Methods: We identified 676 consecutive patients who were diagnosed both CRC and diabetes mellitus between 2000 and 2008. Pateints were compared by two groups; 258 diabetic patients taking metformin and 337 diabetic patients not taking metformin. Patients demographics, clinical characteristics, relapse free survival, overall mortality, and CRC-specific mortality were analyzed. Results: After a median follow-up of 41 months, there were 71 total deaths (27.5%) and 55 CRC-specific deaths (21.3%) among 258 patients who used metformin, compared with 136 total deaths (40.4%) and 104 CRC-specific deaths (30.9%) among 337 patients who did not use metformin. Metformin use was associated with decreased overall mortality (P=0.018) and CRC-specific mortality (P=0.042) by univariate analysis. After adjustment for clinically relevant factors, including age at diagnosis, sex, stage of cancer, BMI, family history of CRC, smoking status, use of aspirin prediagnosis, use of insulin and use of thiazolidinedione, metformin use showed lower risk of overall mortality (HR, 1.448; 95% CI, 1.084-1.934; P=0.016) and CRC-specific mortality (HR, 1.436; 95% CI, 1.0262.0609; P=0.035) in CRC patients with diabetes. In subgroup analysis, these effects were shown in stage 3 CRC patients, not in other stages of CRC. Conclusions: CRC patients with diabetes receiving metfornin have lower mortality than do diabetics not receiving metformin. This is the first study to demonstrate an antitumor effect of metformin against CRC in diabetic patients. Further studies to evaluate the potential of metformin as an antitumor agent are warranted. Univariate and multivariate adjusted overall survival and CRC-specific survival analysis

Mo1937 DARPP-32 Mediates Activation of PI3K Signaling and Resistance to Gefitinib in Gastric Cancer Shoum Zhu, Abbes Belkhiri, Alexander Zaika, Wael El-Rifai Background: Gastric cancer is an aggressive tumor that is often resistant to chemotherapeutics. The 17q amplicon genes, including dopamine and cAMP regulated phosphoprotein MW 32 kDa (DARPP-32), are overexpressed in two-thirds of gastric adenocarcinomas and may induce chemotherapy resistance via abrogation of apoptosis. EGFR plays an important role in tumor growth and activation of the PI3K pathway. Gefitinib (Iressa) is a specific and effective EGFR inhibitor that has shown antitumor activity in clinical trials against several gastrointestinal cancers, including gastric cancers. However, molecular mechanisms that mediate resistance to gefitinib remain poorly understood. Methods and Results: The expression of DARPP-32 in the multi-step carcinogenesis cascade was examined using IHC analysis on 533 samples. The composite expression score, calculated from immunostaining patterns, progressively increased significantly from normal or gastritis to metaplasia, dysplasia, and adenocarcinoma (p<0.001). Using clonogenic survival assays, and ATP-GLO cell viability assays, we found that overexpression of DARPP-32 leads to a 3-fold increase in gefitinib

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