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M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients – A Prospective, Controlled Study
AGA Abstracts
(14%) cases. Fourteen (40%) patients had stage I or II disease and 16 (46%) had stage III or IV. Staging was not available in 5 (14%) patients. Dysplasia was found in 18 surgical specimens of SBA (51%). It was adjacent to SBA in 9 cases, distant in 2 cases and both contiguous and distant in 7 cases. Dysplasia was flat in 12 cases, elevated (DALM) in 5 cases and both flat and elevated in 1 case. High grade dysplasia was observed in 14 of 18 (78%) cases. All lesions of dysplasia without cancer were found in the ileum. It was flat dysplasia in 3 patients (low grade in 2 cases and high grade in 1 case) and one serrated lesion with low and high grade dysplasia in one patient. All carcinomatous and dysplastic lesions, except one, occurred in inflammatory areas. Conclusion: Dysplasia can be found in the vicinity or at distance from SBA in half of the cases. The dysplasia-carcinoma sequence seems valid in SBA associated with CD. Endoscopic surveillance of the small bowel aimed to detect dysplasia should be considered in patients with CD of the small bowel with risk factors associated with SBA (Am J Gastroenterol 2008;103:1). M1950 Protein Kinase D Mediates Synergistic COX-2 Expression Induced by CrossTalk Between TNF-α and G-Protein Coupled Receptor Agonists in Human Colonic Myofibroblasts James Yoo, Citlali E. Rodriguez-Perez, James Sinnett-Smith, Enrique Rozengurt
A = NK-kB gene target; B = NK-1R gene target
Background: Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-α (TNF-α)-associated colitis-associated colon cancer but the precise mechanism(s) involved remains incompletely understood. Myofibroblasts are a major source of the inducible isoform of cyclooxygenase (COX-2) and of prostaglandins (e.g. PGE2) in the gastrointestinal tract. We hypothesize that TNF-α signaling cross talks with pro-inflammatory mediators, including bradykinin (BK) and lysophosphatidic acid (LPA), to elicit COX-2 expression and PGE2 production that are essential for colon cancer development. Results: Treatment of 18Co cells, a model of human colonic myofibroblasts, with TNF-α and either the G protein-coupled receptor agonists (GPCR) BK or LPA induced striking synergistic COX-2 protein expression that was paralleled by increases in the production of PGE2. Synergistic COX-2 expression was also induced by sequential exposure to TNF-α followed by BK or LPA. COX-2 expression in 18Co cells treated with BK and TNF-α was prevented by the B2 BK receptor antagonist HOE140, and the preferential protein kinase C (PKC) inhibitors Ro31-8220, GF109203X, and Go6976, suggesting that COX-2 expression in response to TNF-α and BK was mediated through a PKC/protein kinase D (PKD)-dependent pathway. Indeed, TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser744) and auto-phosphorylation site (Ser916). Transfection of 18Co cells with siRNA targeting PKD1 completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-α demonstrating, for the first time, a critical role of PKD1 in the pathways leading to synergistic expression of COX-2. We also found that exposure of 18Co cells to the combination of TNF-α and BK produced synergistic stimulation of ERK activation. Cell treatment with U0126, at a concentration that completely prevented ERK activation, markedly reduced COX-2 expression in response to stimulation with the combination of TNF-α and BK implying that the ERK pathway contributes to PKD signaling leading to COX-2 expression. Conclusion: Our results indicate that crosstalk between TNFα and GPCR agonists amplifies a PKC/PKD1/ERK phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. We propose that PKD1 mediates COX-2 expression in colonic myofibroblasts to create a microenvironment that supports tumor growth.
M1952 Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms Marco Scarpa, Marina Bortolami, Diego Faggian, Andromachi Kotsafti, Cesare Ruffolo, Filippo Navaglia, Anna Pozza, Renata D'Incà, Mario Plebani, Giacomo C. Sturniolo, Imerio Angriman Background The inconsistency between dysplasia rate and incidence of cancer in ulcerative colitis (UC)suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of costimulatory molecules CD80 and CD86, the lamina propria mononuclear cells populations and the cytokines network at the different stages of carcinogenesis in UC compared to the non inflammatory omologues. Patients and methods Five groups of patients affected by UC (19 pts), UC with colonic dysplasia (10 pts), UC and cancer (7 pts), colonic adenoma (11 pts), sporadic colonic cancer (15 pts) and a group of healthy control (11 pts) were enrolled. Mucosal CD80 and CD86 mRNA levels were quantified with Real Time PCR. Mucosal expression of CD80, CD86, CD3, CD20, CD68 and p53 was analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and TNFα were measured with immunometric assays. Kruskal-Wallis ANOVA, Mann-Whitney U test and Kendall's tau correlation test were used. Results In UC, CD80 expression was higher in patients with dysplasia (p=0.017). This difference was not evident in the non inflammatory carcinogenesis (fig 1). CD80 expression directly correlated with IL-2 expression and with lamina propria T and B lymphocytes and monocytes populations. In UC, CD86 expression resulted significantly higher than in non inflammatory carcinogenesis without any differences between the steps of the carcinogenesis. CD86 expression correlated with inflammation severity and with IFNgamma expression. Conclusion In UC, CD80 is significantly more expressed in the dysplastic colonic tissue and it seems to be down regulated in cancer. CD80 mucosal levels correlate with T cell population and with IL-2 expression suggesting its involvement in the immunosurveillance mechanism in UC that prevent that all the dysplasia progress to cancer.
M1951 Gene Expression of the Truncated Neurokinin-1 Receptor as Well as Two NFkB Subunits is Altered in the Transition of Colonic Epithelial Cells From Quiescent Colitis to High Grade Dysplasia to Carcinoma in Colitis Associated Colorectal Cancer Earl Gillespie, Jeremy T. Hetzel, Jennifer Coukos, Francis A. Farraye, Sandra R. Cerda, Michael J. O'Brien, Susan E. Leeman, Arthur F. Stucchi, James M. Becker INTRODUCTION Patients with long-standing ulcerative colitis (UC) have an increased risk of developing colorectal cancer (CRC). Although chronic inflammation is a key risk factor, the mechanisms that initiate the transition of normal epithelia to dysplasia to cancer remain unclear. It is known that the neurokinin-1 receptor (NK-1R) is pro-inflammatory and is overexpressed in many cancers. The NK-1R exists in two forms: A full-length and truncated form, but the differences in their expression in colitis-associated CRC have not been previously studied. The aim of this study was to examine changes in gene expression in the progression of colitis-associated CRC. METHODS Archived formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 5 colectomy specimens of CRC patients with pre-existing UC. From each colon, samples were identified and graded by pathologists (MO, SC) as carcinoma (CA), high-grade dysplasia (HGD), or quiescent colitis with no evidence of dysplasia or cancer (QC). Colonic crypt epithelial cells were microdissected using laser capture microscopy, and RNA was extracted and purified using MDS Analytical Technologies' Paradise Plus kit. Reverse transcription and pre-amplification of 88 target genes related to cancer and inflammation was performed using RT2 FFPE Pre-Amp kit; and gene expression was assayed by RT2 Profiler PCR Array (SABiosciences). The data was analyzed by paired t-test using R (HTqPCR Bioconductor package). RESULTS The expression of several genes downstream of the NK1R and NF-kB is significantly changed in the progression from QC to HGD to CA in colitisassociated CRC. Of particular interest are the increased expression of the truncated NK-1R gene, TACR1S, the lack of change in the full-length NK-1R gene, and the increased expression of the NF-kB subunits REL and NFKB2 (see table). CONCLUSION The genes identified in this study, especially the truncated NK-1R, further our understanding of the mechanisms by which chronic inflammation promotes the progression to CRC.
AGA Abstracts
M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients - A Prospective, Controlled Study Alex Vilkin, Doron Boltin, Zohar Levi, Ori Elkayam OBJECTIVES: JC virus (JCV), a polyoma virus, is the etiological agent of progressive multifocal leukoencephalopathy in immunosuppressed patients. JCV T-Ag has proven oncogenic potential and is expressed in colonic polyps and carcinomas. We proposed that the prevalence of JCV T-Ag is higher in the normal gastrointestinal (GI) mucosa of immunosuppressed patients compared to their immune competent counterparts. METHODS: Morphologically normal samples of upper and lower GI mucosa were obtained from 38 immunosuppressed patients. A control group included samples from 19 immune competent inflammatory bowel disease (IBD) and 29 non-IBD cases. DNA was extracted and PCR was performed using primers specific for T-Ag. RESULTS: JCV T-Ag was found in 9 of the immunosuppressed patients (23.7%) and in 3 of the controls (6.3%), P=0.02. Transplant recipients had a particularly high prevalence of JCV T-Ag (35.3%). Patients with IBD receiving immunosuppressive drugs had a higher prevalence of JCV T-Ag in comparison with IBD patients who did not receive immunosuppression, 22.2% versus 10.5% respectively, but this difference was not statistically significant (P=0.574). CONCLUSION: JCV T-Ag is more prevalent in
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from tumor development even in the presence of a strong Th-1-mediated colonic inflammation. Aim: Since IFN-gamma expression results under direct control of the TGF-beta signaling, aim of this study was to investigate the role of IFN-gamma in the protection against CACC. Material and Methods: Wt, Smad7tg, IFN-gamma -/- and mice carrying the double mutation (Smad7tg IFN-gamma -/-) underwent the Azoxymethane/Dextran Sulphate Sodium protocol of CACC. Tumor development was screened by conventional endoscopy and inflammation was graded by histology. Immune cell infiltration in tumoral and peritumoral areas was evaluated by flow cytometry. Expression of cytotoxicity markers (i.e. Perforin A, Granzyme B an FasL) was quantified by qPCR. Results: As previously observed, Smad7tg mice resulted protected from tumors even the presence of strong colonic Th-1 mediated inflammation in comparison to the Wt. In contrast, the loss of IFN-gamma in the Smad7tg restored tumor susceptibility being tumor incidence and tumor score similar to Wt and IFN-gamma -/groups. Inflammation was stronger in Smad7tg mice in comparison to all the other groups. Analysis of the immune cell infiltrate showed that the loss of IFN-gamma expression in the Smad7tg mice was associated with a lower number of CD8+ T cells infiltrating the tumors and to a reduced number of CD4+ and NKT cells in the tumor surrounding areas. Finally, mice carrying the double mutation had a reduced expression of cytotoxicity markers in both tumoral and peritumoral areas. Conclusions: IFN-gamma mediates, at least in part, the antitumoral immune response against CACC generated in the presence of the T cell-specific block of the TGF-beta signaling operated by Smad7 expression.
M1954 In PSC With Colitis, Long-Time UDCA is Associated With a Reduction of Colonic Cacrcinomas Gerda Rudolph, Daniel Gotthardt, Petra Kloeters-Plachky, Hasan Kulaksiz, Adolf Stiehl Introduction: Patients with PSC and IBD have a high incidence of colonic carcinomas and the annual incidence of colonic carcinomas increases with duration of the disease. In previous controlled studies with observation times of up to 4 years, UDCA treatment has been suggested to reduce dysplasias and carcinomas in patients with IBD. In this study the effect of UDCA long-time treatment on the incidence of colorectal carcinomas was evaluated. Results: Altogether 120 of 171 PSC patients included had IBD. Of the patients with IBD, 108 had ulcerative coltis and 12 had Crohn's disease. All patients have been treated with UDCA for a median treatment time of 6.9 years. Five patients with ulcerative colitis had undergone previous colectomy with ileoanal pouch and 16 further patients underwent colectomy in the course of the study. 69 patients with ulcerative colitis and 7 with Crohn's disease received 5-aminosalicylic acid (3g/d) and 39 with ulcerative colitis and the 5 patients with Crohn's disease received intermittently corticosteroids (methyl prednisolone, 10-50 mg/ d). Seven patients with PSC and IBD developed a colorectal carcinoma yielding a prevalence of colo-rectal carcinomas in PSC with IBD of 5.8 %. In years 0-3 after start of UDCA treatment (n=120), the annual incidence rate of colo-rectal carcinomas was 0.62 per 100 patient years, in years 3-6 (n=93) it increased to 1.28 and decreased thereafter in years 6-9 (n=67) to 1.17, in years 9-12 (n=42) to 0 and after >12 years (n=24) it remained 0. In patients with IBD and colo-rectum preserved (no recto-colectomy) in years 0-3 on UDCA (n=115), the annual incidence rate of colo-rectal carcinomas was 0.68 per 100 patient years, in years 36 (n=82) it increased to 1.47 and decreased thereafter in years 6-9 (n=58) to 1.38, in years 9-12 (n=36) to 0 and after >12 years (n=18) it remained 0. In PSC with IBD the Kaplan Meier estimate of colonic carcinoma formation increased with time in the first years of treatment and reached a plateau after 9 years; after treatment for 9 years or longer, no further colorectal carcinomas were observed. Summary and conclusion: After start of UDCA, the annual incidence of colonic carcinomas increased up to six years and subsequently decreased. In PSC with IBD, long-time treatment with UDCA of over 6 years may reverse the carcinogenic potential in the intestine.
M1957 Evolving Patterns of Colonic Dysplasia in Inflammatory Bowel Disease (IBD) 1994-2009: Increasing Detection of Low Grade Dysplasia Jana G. Hashash, Marwa El Mourabet, Marc Schwartz, Melissa I. Saul, Leonard Baidoo, Miguel Regueiro, Scott R. Owens, Jason M. Swoger, Arthur M. Barrie, Michael A. Dunn, David G. Binion Background: Chronic inflammatory damage in IBD colitis predisposes to the development of dysplasia and colonic adenocarcinoma (CA). Low grade dysplasia (LGD) and high grade dysplasia (HGD) have been targeted for colonoscopic detection in order to identify “at risk” patients who may undergo prophylactic colectomy prior to the development of CA. Patterns of dysplasia/cancer in IBD may be influenced by treatment options, surveillance regimens, endoscopic equipment and identification of high risk subgroups of patients, which have evolved over recent years. Aims: Characterize rates of LGD, HGD and CA detected on colonoscopic sampling over a 15 year time period in a cohort of IBD patients. Methods: Retrospective review of all the colonoscopy/histology reports of IBD patients (UC, CD and IC) that included the term ‘dysplasia' and that were performed between 9/1994 and 9/2009 in a hospital system. Abnormal pathology included the following: HGD, LGD, CA, indefinite for dysplasia (ID) and adenoma (Ad) (including tubular adenoma, tubulo-villous adenoma, and villous adenoma). In patients with multiple biopsy abnormalities, the diagnosis was categorized as the most advanced lesion (CA>HGD>LGD>Ad>ID). The diagnosis of ID was excluded from the analysis because of changes in diagnostic nomenclature over the course of the study period. Patients were divided into 3 groups based on years of detection: Group 1 (1994-1999), Group 2 (2000-2004) and Group 3 (2005-2009). Pearson Chi square was used to test for the difference between the rates of pathologies encountered over time. A pvalue<0.05 was considered significant. Results: Out of 1401 IBD colonoscopy reports reviewed, 356 had abnormal pathology (25.4%). There were 55 (15.4%) colonoscopy reports between the years 1994 and 1999 (group 1), 141 (39.6%) colonoscopy reports from group 2, and 160 (44.9%) from group 3. Table 1 summarizes the difference in dysplasia frequency before and after 2004. Conclusion: Patterns of IBD dysplasia have evolved, with a significant increase in detection of LGD, while HGD has fallen. Rates of Ad in IBD have remained stable. Factors leading to the increased detection of LGD and the reduction in HGD are not defined, but may reflect changes in diagnosis and treatment. Studies are warranted to define factors the underlying the evolution of dysplasia patterns in IBD colitis. Table 1
M1955 Tumor Growth in an Experimental Model of Ulcerative Colitis Depends on TLR2-Stimulated Epithelial Cells That Mediate Induction of IL-6 Stefan Fichtner-Feigl, Warren Strober, Hans J. Schlitt One of the major long-term complications of inflammatory bowel disease, in particular ulcerative colitis, is the development of inflammation-induced colorectal cancer. In these experimental studies we tried to elucidate the role of the innate immune system on tumor immunosurveillance and tumor growth in the “AOM-Oxa-model”. This newly developed new animal model for colitis-associated tumorigenesis is based on a chronic oxazolonecolitis. The AOM-Oxa-model was performed in female BALB/c mice. After an initial intraperitoneal injection of azoxymethane (AOM) and repeated intra-rectal administrations of oxazolone (Oxa) dissolved in ethanol, mice developed a chronic intestinal inflammation lasting for 12 weeks. In addition we could observe colonic tumor growth 5 weeks after initiation of the disease. To determine the role of the innate immune system, in particular the TLR signaling pathway, we performed the AOM-Oxa-model in MyD88-deficient animals. We could show that the basis for the chronic intestinal inflammation was an upregulation of IL-13 produced by NKT-cells in the colon of animals with Oxa-colitis. Tumor growth in this model was highly dependent on the presence of IL-6 in the colon. The loss of the main signaling TLR-signaling pathway in MyD88-deficient animals unexpectedly did not interfere with the chronic phase of the intestinal inflammation during the AOM-Oxa-model. However, tumor growth was dramatically reduced in MyD88-deficient animals. The reason for this reduction in colonic tumor formation was a reduction of CD4+ T-cell derived IL-6 production, which is acting as a stimulus for tumor cell proliferation in the AOM-Oxa-model. This defective activation of tumor growth in MyD88-deficient animals was traced to epithelial cells that could not be stimulated via TLR2 to produce versican to induce IL-6 production by resident CD4+ T cells. In this newly developed animal model of inflammation-induced colonic tumorigenesis resembling immunologic features of ulcerative colitis we could elucidate a central role of MyD88 for tumor growth. Intact TLR-signaling through the MyD88 is indispensable for inflammation-induced intestinal tumorigenesis.
M1958 Regulation of GLI1 and Snail in Helicobacter pylori Infected Gastric Epithelial Cells SangHun Lee, Jie-Hyun Kim, Yeo Song Lee, Sung-Hwa Sohn, Kyung-Ran Park, Sung Kwan Shin, Sang-Kil Lee, Yong Chan Lee
M1956 High IFN-Gamma Expression is Required for Tumor Protection in a Model of Colitis-Associated Colorectal Cancer Massimo C. Fantini, Angelamaria Rizzo, Daniele Fina, Roberta Caruso, Massimiliano Sarra, Carmine Stolfi, Christoph Becker, Thomas T. MacDonald, Francesco Pallone, Markus F. Neurath, Giovanni Monteleone
Background/Aims: Helicobacter pylori (H. pylori) with various virulence factors cause mucosal damage and may participate in gastric carcinogenesis. Sonic hedgehog plays a key role in a variety of processes, such as embryogenesis, tissue repair during chronic persistent inflammation, and carcinogenesis and leads to pathological consequences in esophageal cancer, gastric cancer, pancreatic cancer, breast cancer and other tumors. The Hedgehog pathway transcription factor Glioma-associated oncogene (Gli1) induces transformation of epithelial cells via induction of Snail, the zinc finger transcription factor, functions as a potent repressor of E-cadherin expression. The aim of this study was to examine the changes snail and Gli1 in gastric epithelial cells in response to H. pylori infection. Methods: AGS and MKN45, gastric epithelial cells were infected by strains 60190 (cagPAI positive), 8822 (cagPAI negative) H. pylori. The expression and transcriptional regulation of Gli1 and Snail were evaluated by RT-PCR, immunoblot and promoter assay. Results and Conclusion: The gli1 and Snail expression in H. pylori-infected gastric cancer cell higher than those of uninfected gastric cancer cell. Infection of gastric epithelial cells by cagA positive H. pylori strains significantly increased transactivity of Gli1 and Snail, but cagA negative H. pylori
Background: Patients affected by inflammatory bowel diseases (IBDs) have a higher risk to develop colorectal cancer (CC). In IBDs intestinal inflammation is sustained, at least in part, by the block of the TGF-beta signaling operated by the intracellular inhibitor Smad7 and chronic inflammation of the gut is thought to promote tumor onset primarily by inducing genomic damage in epithelial cells but also by releasing cytokines and growth factors which have been shown to be crucial in tumor progression. However IFN-gamma, a proinflammatory cytokine highly expressed in Th-1 mediated colitis, such as Crohn's disease, a major form of IBD, has been shown to activate the immune-surveillance leading to tumor protection in many experimental settings. We have previously observed that in a model of colitis-associated colorectal cancer (CACC) mice over-expressing Smad7 in T cells (Smad7tg) are protected
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AGA Abstracts
AGA Abstracts
the upper and lower GI mucosa of immunosuppressed patients, possibly indicating that the virus undergoes reactivation in these patients. This may account for the higher prevalence of GI carcinomas in immunosuppressed patients.
(14%) cases. Fourteen (40%) patients had stage I or II disease and 16 (46%) had stage III or IV. Staging was not available in 5 (14%) patients. Dysplasia was found in 18 surgical specimens of SBA (51%). It was adjacent to SBA in 9 cases, distant in 2 cases and both contiguous and distant in 7 cases. Dysplasia was flat in 12 cases, elevated (DALM) in 5 cases and both flat and elevated in 1 case. High grade dysplasia was observed in 14 of 18 (78%) cases. All lesions of dysplasia without cancer were found in the ileum. It was flat dysplasia in 3 patients (low grade in 2 cases and high grade in 1 case) and one serrated lesion with low and high grade dysplasia in one patient. All carcinomatous and dysplastic lesions, except one, occurred in inflammatory areas. Conclusion: Dysplasia can be found in the vicinity or at distance from SBA in half of the cases. The dysplasia-carcinoma sequence seems valid in SBA associated with CD. Endoscopic surveillance of the small bowel aimed to detect dysplasia should be considered in patients with CD of the small bowel with risk factors associated with SBA (Am J Gastroenterol 2008;103:1). M1950 Protein Kinase D Mediates Synergistic COX-2 Expression Induced by CrossTalk Between TNF-α and G-Protein Coupled Receptor Agonists in Human Colonic Myofibroblasts James Yoo, Citlali E. Rodriguez-Perez, James Sinnett-Smith, Enrique Rozengurt
A = NK-kB gene target; B = NK-1R gene target
Background: Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-α (TNF-α)-associated colitis-associated colon cancer but the precise mechanism(s) involved remains incompletely understood. Myofibroblasts are a major source of the inducible isoform of cyclooxygenase (COX-2) and of prostaglandins (e.g. PGE2) in the gastrointestinal tract. We hypothesize that TNF-α signaling cross talks with pro-inflammatory mediators, including bradykinin (BK) and lysophosphatidic acid (LPA), to elicit COX-2 expression and PGE2 production that are essential for colon cancer development. Results: Treatment of 18Co cells, a model of human colonic myofibroblasts, with TNF-α and either the G protein-coupled receptor agonists (GPCR) BK or LPA induced striking synergistic COX-2 protein expression that was paralleled by increases in the production of PGE2. Synergistic COX-2 expression was also induced by sequential exposure to TNF-α followed by BK or LPA. COX-2 expression in 18Co cells treated with BK and TNF-α was prevented by the B2 BK receptor antagonist HOE140, and the preferential protein kinase C (PKC) inhibitors Ro31-8220, GF109203X, and Go6976, suggesting that COX-2 expression in response to TNF-α and BK was mediated through a PKC/protein kinase D (PKD)-dependent pathway. Indeed, TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser744) and auto-phosphorylation site (Ser916). Transfection of 18Co cells with siRNA targeting PKD1 completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF-α demonstrating, for the first time, a critical role of PKD1 in the pathways leading to synergistic expression of COX-2. We also found that exposure of 18Co cells to the combination of TNF-α and BK produced synergistic stimulation of ERK activation. Cell treatment with U0126, at a concentration that completely prevented ERK activation, markedly reduced COX-2 expression in response to stimulation with the combination of TNF-α and BK implying that the ERK pathway contributes to PKD signaling leading to COX-2 expression. Conclusion: Our results indicate that crosstalk between TNFα and GPCR agonists amplifies a PKC/PKD1/ERK phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. We propose that PKD1 mediates COX-2 expression in colonic myofibroblasts to create a microenvironment that supports tumor growth.
M1952 Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms Marco Scarpa, Marina Bortolami, Diego Faggian, Andromachi Kotsafti, Cesare Ruffolo, Filippo Navaglia, Anna Pozza, Renata D'Incà, Mario Plebani, Giacomo C. Sturniolo, Imerio Angriman Background The inconsistency between dysplasia rate and incidence of cancer in ulcerative colitis (UC)suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of costimulatory molecules CD80 and CD86, the lamina propria mononuclear cells populations and the cytokines network at the different stages of carcinogenesis in UC compared to the non inflammatory omologues. Patients and methods Five groups of patients affected by UC (19 pts), UC with colonic dysplasia (10 pts), UC and cancer (7 pts), colonic adenoma (11 pts), sporadic colonic cancer (15 pts) and a group of healthy control (11 pts) were enrolled. Mucosal CD80 and CD86 mRNA levels were quantified with Real Time PCR. Mucosal expression of CD80, CD86, CD3, CD20, CD68 and p53 was analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and TNFα were measured with immunometric assays. Kruskal-Wallis ANOVA, Mann-Whitney U test and Kendall's tau correlation test were used. Results In UC, CD80 expression was higher in patients with dysplasia (p=0.017). This difference was not evident in the non inflammatory carcinogenesis (fig 1). CD80 expression directly correlated with IL-2 expression and with lamina propria T and B lymphocytes and monocytes populations. In UC, CD86 expression resulted significantly higher than in non inflammatory carcinogenesis without any differences between the steps of the carcinogenesis. CD86 expression correlated with inflammation severity and with IFNgamma expression. Conclusion In UC, CD80 is significantly more expressed in the dysplastic colonic tissue and it seems to be down regulated in cancer. CD80 mucosal levels correlate with T cell population and with IL-2 expression suggesting its involvement in the immunosurveillance mechanism in UC that prevent that all the dysplasia progress to cancer.
M1951 Gene Expression of the Truncated Neurokinin-1 Receptor as Well as Two NFkB Subunits is Altered in the Transition of Colonic Epithelial Cells From Quiescent Colitis to High Grade Dysplasia to Carcinoma in Colitis Associated Colorectal Cancer Earl Gillespie, Jeremy T. Hetzel, Jennifer Coukos, Francis A. Farraye, Sandra R. Cerda, Michael J. O'Brien, Susan E. Leeman, Arthur F. Stucchi, James M. Becker INTRODUCTION Patients with long-standing ulcerative colitis (UC) have an increased risk of developing colorectal cancer (CRC). Although chronic inflammation is a key risk factor, the mechanisms that initiate the transition of normal epithelia to dysplasia to cancer remain unclear. It is known that the neurokinin-1 receptor (NK-1R) is pro-inflammatory and is overexpressed in many cancers. The NK-1R exists in two forms: A full-length and truncated form, but the differences in their expression in colitis-associated CRC have not been previously studied. The aim of this study was to examine changes in gene expression in the progression of colitis-associated CRC. METHODS Archived formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 5 colectomy specimens of CRC patients with pre-existing UC. From each colon, samples were identified and graded by pathologists (MO, SC) as carcinoma (CA), high-grade dysplasia (HGD), or quiescent colitis with no evidence of dysplasia or cancer (QC). Colonic crypt epithelial cells were microdissected using laser capture microscopy, and RNA was extracted and purified using MDS Analytical Technologies' Paradise Plus kit. Reverse transcription and pre-amplification of 88 target genes related to cancer and inflammation was performed using RT2 FFPE Pre-Amp kit; and gene expression was assayed by RT2 Profiler PCR Array (SABiosciences). The data was analyzed by paired t-test using R (HTqPCR Bioconductor package). RESULTS The expression of several genes downstream of the NK1R and NF-kB is significantly changed in the progression from QC to HGD to CA in colitisassociated CRC. Of particular interest are the increased expression of the truncated NK-1R gene, TACR1S, the lack of change in the full-length NK-1R gene, and the increased expression of the NF-kB subunits REL and NFKB2 (see table). CONCLUSION The genes identified in this study, especially the truncated NK-1R, further our understanding of the mechanisms by which chronic inflammation promotes the progression to CRC.
AGA Abstracts
M1953 JC Virus T-Antigen Expression in Gastrointestinal Mucosa of Immunosuppressed Patients - A Prospective, Controlled Study Alex Vilkin, Doron Boltin, Zohar Levi, Ori Elkayam OBJECTIVES: JC virus (JCV), a polyoma virus, is the etiological agent of progressive multifocal leukoencephalopathy in immunosuppressed patients. JCV T-Ag has proven oncogenic potential and is expressed in colonic polyps and carcinomas. We proposed that the prevalence of JCV T-Ag is higher in the normal gastrointestinal (GI) mucosa of immunosuppressed patients compared to their immune competent counterparts. METHODS: Morphologically normal samples of upper and lower GI mucosa were obtained from 38 immunosuppressed patients. A control group included samples from 19 immune competent inflammatory bowel disease (IBD) and 29 non-IBD cases. DNA was extracted and PCR was performed using primers specific for T-Ag. RESULTS: JCV T-Ag was found in 9 of the immunosuppressed patients (23.7%) and in 3 of the controls (6.3%), P=0.02. Transplant recipients had a particularly high prevalence of JCV T-Ag (35.3%). Patients with IBD receiving immunosuppressive drugs had a higher prevalence of JCV T-Ag in comparison with IBD patients who did not receive immunosuppression, 22.2% versus 10.5% respectively, but this difference was not statistically significant (P=0.574). CONCLUSION: JCV T-Ag is more prevalent in
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from tumor development even in the presence of a strong Th-1-mediated colonic inflammation. Aim: Since IFN-gamma expression results under direct control of the TGF-beta signaling, aim of this study was to investigate the role of IFN-gamma in the protection against CACC. Material and Methods: Wt, Smad7tg, IFN-gamma -/- and mice carrying the double mutation (Smad7tg IFN-gamma -/-) underwent the Azoxymethane/Dextran Sulphate Sodium protocol of CACC. Tumor development was screened by conventional endoscopy and inflammation was graded by histology. Immune cell infiltration in tumoral and peritumoral areas was evaluated by flow cytometry. Expression of cytotoxicity markers (i.e. Perforin A, Granzyme B an FasL) was quantified by qPCR. Results: As previously observed, Smad7tg mice resulted protected from tumors even the presence of strong colonic Th-1 mediated inflammation in comparison to the Wt. In contrast, the loss of IFN-gamma in the Smad7tg restored tumor susceptibility being tumor incidence and tumor score similar to Wt and IFN-gamma -/groups. Inflammation was stronger in Smad7tg mice in comparison to all the other groups. Analysis of the immune cell infiltrate showed that the loss of IFN-gamma expression in the Smad7tg mice was associated with a lower number of CD8+ T cells infiltrating the tumors and to a reduced number of CD4+ and NKT cells in the tumor surrounding areas. Finally, mice carrying the double mutation had a reduced expression of cytotoxicity markers in both tumoral and peritumoral areas. Conclusions: IFN-gamma mediates, at least in part, the antitumoral immune response against CACC generated in the presence of the T cell-specific block of the TGF-beta signaling operated by Smad7 expression.
M1954 In PSC With Colitis, Long-Time UDCA is Associated With a Reduction of Colonic Cacrcinomas Gerda Rudolph, Daniel Gotthardt, Petra Kloeters-Plachky, Hasan Kulaksiz, Adolf Stiehl Introduction: Patients with PSC and IBD have a high incidence of colonic carcinomas and the annual incidence of colonic carcinomas increases with duration of the disease. In previous controlled studies with observation times of up to 4 years, UDCA treatment has been suggested to reduce dysplasias and carcinomas in patients with IBD. In this study the effect of UDCA long-time treatment on the incidence of colorectal carcinomas was evaluated. Results: Altogether 120 of 171 PSC patients included had IBD. Of the patients with IBD, 108 had ulcerative coltis and 12 had Crohn's disease. All patients have been treated with UDCA for a median treatment time of 6.9 years. Five patients with ulcerative colitis had undergone previous colectomy with ileoanal pouch and 16 further patients underwent colectomy in the course of the study. 69 patients with ulcerative colitis and 7 with Crohn's disease received 5-aminosalicylic acid (3g/d) and 39 with ulcerative colitis and the 5 patients with Crohn's disease received intermittently corticosteroids (methyl prednisolone, 10-50 mg/ d). Seven patients with PSC and IBD developed a colorectal carcinoma yielding a prevalence of colo-rectal carcinomas in PSC with IBD of 5.8 %. In years 0-3 after start of UDCA treatment (n=120), the annual incidence rate of colo-rectal carcinomas was 0.62 per 100 patient years, in years 3-6 (n=93) it increased to 1.28 and decreased thereafter in years 6-9 (n=67) to 1.17, in years 9-12 (n=42) to 0 and after >12 years (n=24) it remained 0. In patients with IBD and colo-rectum preserved (no recto-colectomy) in years 0-3 on UDCA (n=115), the annual incidence rate of colo-rectal carcinomas was 0.68 per 100 patient years, in years 36 (n=82) it increased to 1.47 and decreased thereafter in years 6-9 (n=58) to 1.38, in years 9-12 (n=36) to 0 and after >12 years (n=18) it remained 0. In PSC with IBD the Kaplan Meier estimate of colonic carcinoma formation increased with time in the first years of treatment and reached a plateau after 9 years; after treatment for 9 years or longer, no further colorectal carcinomas were observed. Summary and conclusion: After start of UDCA, the annual incidence of colonic carcinomas increased up to six years and subsequently decreased. In PSC with IBD, long-time treatment with UDCA of over 6 years may reverse the carcinogenic potential in the intestine.
M1957 Evolving Patterns of Colonic Dysplasia in Inflammatory Bowel Disease (IBD) 1994-2009: Increasing Detection of Low Grade Dysplasia Jana G. Hashash, Marwa El Mourabet, Marc Schwartz, Melissa I. Saul, Leonard Baidoo, Miguel Regueiro, Scott R. Owens, Jason M. Swoger, Arthur M. Barrie, Michael A. Dunn, David G. Binion Background: Chronic inflammatory damage in IBD colitis predisposes to the development of dysplasia and colonic adenocarcinoma (CA). Low grade dysplasia (LGD) and high grade dysplasia (HGD) have been targeted for colonoscopic detection in order to identify “at risk” patients who may undergo prophylactic colectomy prior to the development of CA. Patterns of dysplasia/cancer in IBD may be influenced by treatment options, surveillance regimens, endoscopic equipment and identification of high risk subgroups of patients, which have evolved over recent years. Aims: Characterize rates of LGD, HGD and CA detected on colonoscopic sampling over a 15 year time period in a cohort of IBD patients. Methods: Retrospective review of all the colonoscopy/histology reports of IBD patients (UC, CD and IC) that included the term ‘dysplasia' and that were performed between 9/1994 and 9/2009 in a hospital system. Abnormal pathology included the following: HGD, LGD, CA, indefinite for dysplasia (ID) and adenoma (Ad) (including tubular adenoma, tubulo-villous adenoma, and villous adenoma). In patients with multiple biopsy abnormalities, the diagnosis was categorized as the most advanced lesion (CA>HGD>LGD>Ad>ID). The diagnosis of ID was excluded from the analysis because of changes in diagnostic nomenclature over the course of the study period. Patients were divided into 3 groups based on years of detection: Group 1 (1994-1999), Group 2 (2000-2004) and Group 3 (2005-2009). Pearson Chi square was used to test for the difference between the rates of pathologies encountered over time. A pvalue<0.05 was considered significant. Results: Out of 1401 IBD colonoscopy reports reviewed, 356 had abnormal pathology (25.4%). There were 55 (15.4%) colonoscopy reports between the years 1994 and 1999 (group 1), 141 (39.6%) colonoscopy reports from group 2, and 160 (44.9%) from group 3. Table 1 summarizes the difference in dysplasia frequency before and after 2004. Conclusion: Patterns of IBD dysplasia have evolved, with a significant increase in detection of LGD, while HGD has fallen. Rates of Ad in IBD have remained stable. Factors leading to the increased detection of LGD and the reduction in HGD are not defined, but may reflect changes in diagnosis and treatment. Studies are warranted to define factors the underlying the evolution of dysplasia patterns in IBD colitis. Table 1
M1955 Tumor Growth in an Experimental Model of Ulcerative Colitis Depends on TLR2-Stimulated Epithelial Cells That Mediate Induction of IL-6 Stefan Fichtner-Feigl, Warren Strober, Hans J. Schlitt One of the major long-term complications of inflammatory bowel disease, in particular ulcerative colitis, is the development of inflammation-induced colorectal cancer. In these experimental studies we tried to elucidate the role of the innate immune system on tumor immunosurveillance and tumor growth in the “AOM-Oxa-model”. This newly developed new animal model for colitis-associated tumorigenesis is based on a chronic oxazolonecolitis. The AOM-Oxa-model was performed in female BALB/c mice. After an initial intraperitoneal injection of azoxymethane (AOM) and repeated intra-rectal administrations of oxazolone (Oxa) dissolved in ethanol, mice developed a chronic intestinal inflammation lasting for 12 weeks. In addition we could observe colonic tumor growth 5 weeks after initiation of the disease. To determine the role of the innate immune system, in particular the TLR signaling pathway, we performed the AOM-Oxa-model in MyD88-deficient animals. We could show that the basis for the chronic intestinal inflammation was an upregulation of IL-13 produced by NKT-cells in the colon of animals with Oxa-colitis. Tumor growth in this model was highly dependent on the presence of IL-6 in the colon. The loss of the main signaling TLR-signaling pathway in MyD88-deficient animals unexpectedly did not interfere with the chronic phase of the intestinal inflammation during the AOM-Oxa-model. However, tumor growth was dramatically reduced in MyD88-deficient animals. The reason for this reduction in colonic tumor formation was a reduction of CD4+ T-cell derived IL-6 production, which is acting as a stimulus for tumor cell proliferation in the AOM-Oxa-model. This defective activation of tumor growth in MyD88-deficient animals was traced to epithelial cells that could not be stimulated via TLR2 to produce versican to induce IL-6 production by resident CD4+ T cells. In this newly developed animal model of inflammation-induced colonic tumorigenesis resembling immunologic features of ulcerative colitis we could elucidate a central role of MyD88 for tumor growth. Intact TLR-signaling through the MyD88 is indispensable for inflammation-induced intestinal tumorigenesis.
M1958 Regulation of GLI1 and Snail in Helicobacter pylori Infected Gastric Epithelial Cells SangHun Lee, Jie-Hyun Kim, Yeo Song Lee, Sung-Hwa Sohn, Kyung-Ran Park, Sung Kwan Shin, Sang-Kil Lee, Yong Chan Lee
M1956 High IFN-Gamma Expression is Required for Tumor Protection in a Model of Colitis-Associated Colorectal Cancer Massimo C. Fantini, Angelamaria Rizzo, Daniele Fina, Roberta Caruso, Massimiliano Sarra, Carmine Stolfi, Christoph Becker, Thomas T. MacDonald, Francesco Pallone, Markus F. Neurath, Giovanni Monteleone
Background/Aims: Helicobacter pylori (H. pylori) with various virulence factors cause mucosal damage and may participate in gastric carcinogenesis. Sonic hedgehog plays a key role in a variety of processes, such as embryogenesis, tissue repair during chronic persistent inflammation, and carcinogenesis and leads to pathological consequences in esophageal cancer, gastric cancer, pancreatic cancer, breast cancer and other tumors. The Hedgehog pathway transcription factor Glioma-associated oncogene (Gli1) induces transformation of epithelial cells via induction of Snail, the zinc finger transcription factor, functions as a potent repressor of E-cadherin expression. The aim of this study was to examine the changes snail and Gli1 in gastric epithelial cells in response to H. pylori infection. Methods: AGS and MKN45, gastric epithelial cells were infected by strains 60190 (cagPAI positive), 8822 (cagPAI negative) H. pylori. The expression and transcriptional regulation of Gli1 and Snail were evaluated by RT-PCR, immunoblot and promoter assay. Results and Conclusion: The gli1 and Snail expression in H. pylori-infected gastric cancer cell higher than those of uninfected gastric cancer cell. Infection of gastric epithelial cells by cagA positive H. pylori strains significantly increased transactivity of Gli1 and Snail, but cagA negative H. pylori
Background: Patients affected by inflammatory bowel diseases (IBDs) have a higher risk to develop colorectal cancer (CC). In IBDs intestinal inflammation is sustained, at least in part, by the block of the TGF-beta signaling operated by the intracellular inhibitor Smad7 and chronic inflammation of the gut is thought to promote tumor onset primarily by inducing genomic damage in epithelial cells but also by releasing cytokines and growth factors which have been shown to be crucial in tumor progression. However IFN-gamma, a proinflammatory cytokine highly expressed in Th-1 mediated colitis, such as Crohn's disease, a major form of IBD, has been shown to activate the immune-surveillance leading to tumor protection in many experimental settings. We have previously observed that in a model of colitis-associated colorectal cancer (CACC) mice over-expressing Smad7 in T cells (Smad7tg) are protected
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AGA Abstracts
AGA Abstracts
the upper and lower GI mucosa of immunosuppressed patients, possibly indicating that the virus undergoes reactivation in these patients. This may account for the higher prevalence of GI carcinomas in immunosuppressed patients.