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476 Intestinal Inflammation is Not Sufficient to Promote the Development of Colitis Associated Colorectal Cancer: Role of the Enteric Microbiota
AGA Abstracts
mortality during admission and 3 months follow-up. Secondary endpoints included other morbidity and total costs at 6 months follow-up. RESULTS: Infected necrosis was present in 81 patients (92%). The primary endpoint occurred in 31 of 45 patients (69%) assigned to open necrosectomy and in 17 of 43 patients (40%) assigned to the step-up approach (risk ratio 0.57; 95% confidence interval 0.38 to 0.87; P=0.006). In the step-up approach group, 35% of patients were successfully treated with percutaneous drainage only and did not require necrosectomy. New onset multiple organ failure occurred less often in the stepup approach group (12% versus 40%; P=0.002). At 6 months follow-up, patients in the step-up approach group had a lower rate of incisional hernias (7% versus 24%; P=0.03), use of pancreatic enzymes (7% versus 33%; P=0.002) and new onset diabetes (16% versus 38%; P=0.02). Mean total costs per patient were $15,963 (12%) lower in the step-up approach group. CONCLUSIONS: A minimally invasive step-up approach, as compared to open necrosectomy, reduced the rate of the composite endpoint of major morbidity and mortality as well as long-term morbidity and costs in patients with (suspected) infected necrotizing pancreatitis. In these patients, percutaneous drainage should be considered as the first step of treatment.
475q Tm4sf1 is Highly Expressed in Pancreatic Cancer and Promotes Cancer Cell Migration, Invasion and Survival Jia Cao, Vijaya Ramachandran, Thiruvengadam Arumugam, Friso Nast, Zhaoshen Li, Craig D. Logsdon Introduction: Pancreatic cancer is a major oncological challenge. Our microarray data showed over-expression of TM4SF1 in human pancreatic cancer tissues and cell lines.TM4SF1 is a member of the four-transmembrane-domain family of cell surface molecules. It is highly expressed and has been implicated in the regulation of metastasis in several human epithelial cell carcinomas. However, little is known about the functions of TM4SF1 in pancreatic cancer. Therefore, in the current study we examined the expression and functions of TM4SF1 in pancreatic cancer cells. Methods: TM4SF1 was detected in human pancreatic cancer tissues by immunohistochemistry (IHC).The expression of TM4SF1 mRNA in pancreatic cell lines, pancreatic ductal adenocarcinoma (PDAC) tissues, adjacent normal pancreatic (NP) tissues and chronic pancreatitis (CP) tissues were quantitated by qRT-PCR. The effects of TM4SF1 in pancreatic cancer cells were evaluated by siRNA and shRNA techniques in control or TM4SF1 silenced MPanc96, MIA PaCa-2, PANC-1 and HPAC cells. And the effects on cell migration and invasion were also analyzed by Boyden's chamber and BioCoat Matrigel invasion chamber. Cell growth was assessed by the MTS method. Sensitivity to gemcitabine treatment was analyzed by flow cytometry (FACS analysis).Results: TM4SF1 was mainly found distributed in ductal luminal epithelial cancer cells. Compared with NP or CP, the expression of TM4SF1 was highly elevated in PDAC as accessed by IHC. TM4SF1 mRNA expression was also found to be 5~10 fold high in PDAC compared with NP or CP. The migration and invasion of pancreatic cancer cell lines MPanc96, MIA PaCa-2, PANC1 and HPAC cells were significantly decreased when TM4SF1 was silenced by either siRNA or shRNA in comparison with their respective controls. In contrast, TM4SF1 silencing had no effect on cell proliferation. Flow cytometry data showed that the gemcitabine mediated apoptosis percentage of MPanc96, MIA PaCa-2 and PANC-1 cells were significantly increased after silencing TM4SF1 in comparison with its respective controls. Conclusions: These findings suggest that TM4SF1 is a surface membrane antigen expressed highly in pancreatic cancer and promotes migration, invasion and survival of the pancreatic cancer cells. Thus, TM4SF1 may be a useful target for future development of novel therapies for this disease.
475o Resistin, a Potent Adipokine, is Associated With Acute Pancreatitis: Assessment of Functional Genetic Polymorphisms and Serum Levels Venkata Muddana, Anna C. Evans, Christopher J. Langmead, Gilles Clermont, Michael M. Barmada, Georgios I. Papachristou, David C. Whitcomb Background: Patients who are obese are more susceptible to acute pancreatitis (AP), with increased risk of major local and systemic complications linked to the inflammatory response. Adipokines are a class of cytokines produced by visceral fat. We tested the hypothesis that pathologic effects of obesity in AP are mediated, in part, by the adipokine resistin. Methods: AP patients and controls were enrolled from the University of Pittsburgh Medical Center; those with a BMI>30 kg/m2 were classified as obese. Severe AP (SAP) was defined by >1 organ dysfunction of cardiovascular, pulmonary and/or renal systems persisting for > 48 hours. Functional resistin single nucleotide polymorphisms (SNP) rs1862513 C>G, rs35547567 C>T, rs35139946 C>T, rs3745367 G>A and rs3745369 G>C were evaluated by TaqMan assay. In a subgroup of patients with early serum samples, resistin levels were measured using a commercial Luminex assay. Results: Resistin SNPs were assessed in 188 AP patients [mild AP (MAP): n=148; SAP: n=40] and controls (n=163), of which 67 patients (36%) and 34 controls (22%) were obese (p=0.01). Genotype and haplotype analysis suggested that the primary genetic effect was through the resistin promoter SNP rs3745367 A allele. The A allele was associated with overall susceptibility to AP (p=0.0009, OR=1.8, CI= 1.3-2.6), and the effect was stronger in lean subjects (p=0.0001, OR=2.4, CI=1.5-3.9). Serum was available in 46 patients (27 MAP, 19 SAP) and 58 controls. At admission, resistin levels were significantly higher in SAP patients (median=51316 pg/ml; IQR =30769-110451) than in MAP patients (7504 pg/ml; 5879-11548) and controls (6,574 pg/ml; 4,965-8225) (p<0.001). Conclusions: The resistin rs3745367 A allele increased susceptibility to AP, with enhanced effect in patients with BMI < 30kg/m2. Resistin levels on admission may be useful in predicting persistent organ failure in AP.
475r Surgical Versus Endoscopic Drainage in Obstructive Chronic Pancreatitis: A Cost-Effectiveness Analysis Philippe Laramee, David Wonderling, Djuna L. Cahen, Marco J. Bruno, Marcel G. Dijkgraaf, Dirk J. Gouma, Stephen P. Pereira Two published randomized controlled trials (RCTs) [1,2] have reported that surgical drainage of the pancreatic duct was more effective than endoscopic drainage for patients with chronic pancreatitis. The objective of this analysis was to assess the cost-effectiveness of surgical versus endoscopic drainage in obstructive chronic pancreatitis. Methods: This analysis was conducted mainly based on one RCT [1], from a UK National Health Service perspective, and over a 24-month time horizon for the base-case analysis (median follow-up time in the trial). A lifetime horizon was used in the sensitivity analysis. The health outcome was the Quality-Adjusted Life Year (QALY), which was generated using EQ-5D data collected during the trial. Also collected as part of the trial were details of the use of diagnostic and therapeutic procedures, time in hospital and the treatment of pancreatic exocrine and endocrine insufficiency. This resource use was combined with UK unit costs. Key elements of resource use, such as complication rates and conversion to surgery were varied in sensitivity analyses. The trial reported one death in the endoscopy group (5%), which was not clearly related to the intervention. For the base-case analysis, we assumed no mortality in either group and applied a mortality rate of up to 2% to surgical drainage in the sensitivity analysis. Results: The result of the base-case analysis was that surgical drainage of the pancreatic duct dominated endoscopic drainage, being both more effective and less costly. The sensitivity analysis showed that the surgical option remains dominant in a majority of scenarios. When compared with a threshold of £20,000 per QALY gained, all analyses indicated that the probability that surgical drainage is cost-effective compared to endoscopic drainage exceeds 95%. Conclusion: In patients with obstructive chronic pancreatitis, surgical drainage is highly cost-effective compared to endoscopic drainage. This economic analysis was developed from a UK perspective, but considering the magnitude of the improvement in quality of life, the substantial cost savings and the robustness of the results to sensitivity analyses, it is likely that this conclusion can be generalised to other healthcare systems. References: [1] Cahen DL, Gouma DJ, Nio Y et al. Endoscopic versus surgical drainage of the pancreatic duct in chronic pancreatitis. N Engl J Med 2007; 356: 676-684. [2] Dite P, Ruzicka M, Zboril V et al. A prospective, randomized trial comparing endoscopic and surgical therapy for chronic pancreatitis. Endoscopy 2003; 35: 553-558.
475p Prognostic Value of Blood Urea Nitrogen (BUN) in the Early Assessment of Acute Pancreatitis: An International Study Bechien U. Wu, Olaf J. Bakker, Georgios I. Papachristou, Kathryn Repas, Marc G. Besselink, Hjalmar C. van Santvoort, Venkata Muddana, Vikesh K. Singh, David C. Whitcomb, Hein G. Gooszen, Peter A. Banks Background: An accurate and reliable approach to monitor early disease progression is a critical step to improve treatment in acute pancreatitis (AP). A recent retrospective cohort study identified BUN at admission and change in BUN at 24 hours as independent predictors of mortality in AP. Aim: To evaluate the accuracy of early changes in BUN for prediction of mortality in a series of rigorously defined prospective AP cohort studies. Patients & Methods: Data were collected from 3 prospective AP cohort studies: Brigham and Women's Hospital (BWH, June 2005 - May 2009), University of Pittsburgh Medical Center (UPMC, June 2003 - September 2007) and from 15 Dutch hospitals (Dutch Pancreatitis Study Group [DPSG] March 2004 - March 2007). All centers utilized the same set of standardized criteria for diagnosis of AP. Early BUN trajectories were compared between survivors and fatal cases of AP (univariate analysis). Subsequent multivariate logistic regression was used to obtain age- and gender-adjusted estimates of risk of in-hospital mortality associated with elevation in BUN at admission and/or rise in BUN at 24 hours. Accuracy of BUN was compared with APACHE II for prediction of in-hospital mortality using area under ROC (AUC) curves. Results: Data from 1,385 AP cases were included from the 3 study cohorts (620 BWH, 186 UPMC, 576 DPSG). There were a total of 61 (4.4%) deaths. Early rise in BUN was associated with mortality in both center-specific and pooled analysis (BUN change t-test, p<0.0001). In multivariate analysis, after adjusting for age- and gender, both admission BUN (wald χ2 p<0.0001) and change in BUN (wald χ2 p<0.0001) were independent predictors of mortality. Accuracy of BUN (admission and change in 24 hours) within individual study cohorts ranged from AUC=0.78 to AUC=0.91. Overall accuracy of BUN for prediction of mortality was comparable to APACHE II in each of the 3 study cohorts. Conclusions: In this prospective multicenter international study, an elevated BUN at admission and rise in BUN during the initial 24 hours of hospitalization proved to be independent predictors of mortality. Serial BUN measurement is a simple and accurate method for predicting mortality in acute pancreatitis.
AGA Abstracts
476 Intestinal Inflammation is Not Sufficient to Promote the Development of Colitis Associated Colorectal Cancer: Role of the Enteric Microbiota Joshua M. Uronis, Marcus Muehlbauer, Temitope O. Keku, Hans H. Herfarth, Tara C. Rubinas, Janelle C. Arthur, Christian Jobin Introduction: Although the etiology of colitis-associated colon cancer (CAC) remains elusive, the intestinal microbiota has been implicated in the pathogenesis of this disease. We recently developed a novel experimental model of CAC in which the colon-specific carcinogen azoxymethane (AOM) is used to initiate colorectal cancer in Il10-/- mice. Aim: Determine the composition of the microbiota and its role in the development of CAC. Methods: Wildtype (WT) and Il10-/- germ-free mice (n=19) were conventionalized under specific pathogen-free (SPF) conditions or mono-associated with Enterococcus faecalis (E. faecalis). Tumors were induced by intraperitoneal injection of azoxymethane (AOM) once a week for 6 weeks and then followed for 14 weeks. Inflammation and tumor grade were evaluated histologically using a scale of 0-4. Tumors were classified as low-, high-grade or invasive carcinoma. We
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differences among each bacteria family is in progress between N and AD or CA individuals. Conclusion : This is the first large series showing potential role of intestinal microflora in CRC. Increase of Bacteroidetes may be account for helper T IL17 producing cell recruitment previously reported in colon mucosa in CRC (Nat Med 2009; Gut 2008). Whether this is the cause of consequence of colon carcinogenesis remains further investigation. Microflora may be used as screening test. 479 Effect of High Fat Dietary on Colon Tumor Development in Azoxymethane and Dextran Sulfate Sodium Treated Mice In-Wha Kim, Seung-Jae Myung, Mi Young Do, Yeon Mi Ryu, Mi Jung Kim, Sehan Park, Eun Joo Do, Byong Duk Ye, Jeong-Sik Byeon, Suk-Kyun Yang, Jin-Ho Kim Backgrounds/Aims High fat dietary is known to play important roles in inflammatory bowel disease and colon carcinogenesis. The purpose of this study was to understand the mechanisms by which fat dietary can regulate susceptibility to intestinal inflammation and carcinogenesis. Methods A high fat diet (HFD) was based on a western-style diet that contained 20% fat, while a normal diet (ND) contained 5% fat with the additional calories provided by cornstarch. Male Balb/c mice were fed either an HFD (n=52) or an ND (n=52) for 4 weeks, and exposed to azoxymethane (AOM) followed by 2% dextran sulfate sodium (DSS) in drinking water for 7 days. We measured the expression of COX-2 and 15-PGDH using real-time PCR and western blot. Immunohistochemistry analysis for F4/80 and βcatenin were carried out. Colon tissues PGE2 and serum tumor necrosis factor-α (TNF-α) levels were determined by enzyme immunoassay. Results Body weight gain and food intake were comparable in mice between HFD and ND groups. The mean length of large bowel was significantly shorter in an HFD group than an ND group at 8 weeks after cessation of the administration with DSS (9.71. ± 0.90 vs 11.26 ± 1.03 cm, p<0.05). The incidence of colon tumor in mice fed an HFD were 100% with a multiplicity of 16.75 ± 2.37, while that in mice fed an ND were 20% with a multiplicity 0.80 ± 1.93 (p<0.05). The development of colitis proceeded faster in mice fed an HFD compared to an ND (Inflammation score 2.51 ± 1.34 vs 1.53 ± 0.88, p<0.05 at day 4 after DSS initiation). Immunohistochemical staining revealed that the number of infiltrating F4/80-positive macrophages increased progressively during the development of the colitis induced by DSS. Feeding of HFD accelerated the infiltration of macrophages in the colon (macrophages number 67.4 ± 9.9 vs 37.8 ± 7.2 /field, p<0.05, at day 6 after DSS initiation). Mice given HFD had higher expression levels of COX-2 and PGE2, and a lower level of 15-PGDH compared with an ND group at day 1 after cessation of the DSS treatment. An HFD also elevated the serum concentration of TNF-α and the nuclear translocation of nuclear factor-kappa B. Regardless of whether they were given an ND or an HFD, colon cancer had higher expression of COX-2 and PGE2 but lower expression of 15-PGDH compared to normal tissues. In addition, expression of unphosphated (active) β-catenin was undetectable in normal tissues, however, its accumulation was significantly enhanced in the cytoplasmic and nuclear of colon carcinomas. Conclusions We concluded that high fat dietary strongly facilitated inflammation, thereby resulting in promoting AOM/DSS-induced colon carcinogenesis.
477 Effect of the Probiotic VSL#3 on the Transition From Chronic Inflammation to Dysplasia in a Model of Colitis-Associated Colon Cancer Caroline B. Appleyard, Idamary Passalacqua, Angel A. Isidro, Myrella L. Cruz, Raymond A. Isidro, Victor A. Rodriguez Rapale, Claudio De Simone Patients with long-standing colitis have a significantly increased risk of developing colorectal cancer. It is not yet clear how the presence of chronic inflammation results in neoplastic transformation and progression, but there is much evidence to support an important role for the microflora. Probiotics, through modulation of both immune and barrier mechanisms, may alter this process. Aim: To investigate the protective efficacy of the probiotic VSL#3 in preventing the transition from chronic inflammation to dysplasia in a novel model of colitis-associated colorectal cancer. Methods: Chronic colitis was induced in male Sprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for three days. To induce colitis-associated dysplasia and cancer the animals then received TNBS (iv) twice a week for ten weeks. One group of rats received the probiotic in drinking water (VSL#3; ~5 billion cfu /100 gram body weight) from one week prior to initial colitis induction until the time of sacrifice (n=22-23/group). After sacrifice the colons were removed and analyzed for total macroscopic damage, microscopic damage and presence of dysplasia or cancer. Cell proliferation was assessed by immunohistochemical staining. Results: Those animals receiving the probiotic had significantly less macroscopic (p<0.05) and microscopic damage (p<0.05) than the vehicle treated controls in all areas of the colon. Pathological analysis revealed that 29.4% of the vehicle treated animals developed carcinoma, which was confined to the proximal region. None of the probiotic treated animals developed carcinoma. Although there was no significant difference in the amount of dysplasia found in any region of the colon between the groups, treatment with the probiotic caused a shift to a less severe diagnosis with no high-grade dysplasia found in either the proximal or mid colon. Animals receiving the probiotic had more non-specific inflammation in any region of the colon with less diagnosis of IBD compared to vehicle treated. Treatment with the probiotic reduced crypt cell proliferation by 22% as assessed by measurement of BrdU incorporation in comparison with the vehicle treated group. Fecal alkaline phosphatase activity in the probiotic treated animals was half that found in those treated with vehicle. Conclusion: Our results suggest that treatment with the probiotic VSL#3 can attenuate the development of colonic damage and possibly delay the transition to dysplasia and cancer, offering its potential therapeutic use in patients with long-standing colitis.
480 Colorectal Cancer (CRC), Adenoma and IBD Detection in Blood Cells by Array PCR - Role of Circulating Free DNA as Mononuclear Cell Activator Béla Molnár, Kinga Tóth, Sándor Spisák, Orsolya Galamb, Ottó Jász, Alexandra Kalmar, Ferenc Sipos, Pál Miheller, Zsolt Tulassay Background: Whole genomic(WG) gene expression analysis of peripheral blood(PBL) and colon migrating cell populations could contribute to the identification of diagnostic mRNA markers of colorectal diseases. Aims: Validation and verification of WG blood based biomarkers on an independent test set were performed by RT-PCR. Identification of circulating free DNA (cFDNA) as activator molecule of the mononuclear cell fraction causing their local colon alteration specific mRNA expression changes was aimed. Materials and methods: Total RNA was isolated from PBL samples of 16 healthy(HE), 19 CRC, 12 adenoma(AD) and 11 IBD patients. After globin reduction, WG expression profile analysis was done. For classification of samples Prediction analysis of microarrays(PAM) was used. Array results were confirmed by sequence specific array RT-PCR on independent sample set (40 HE, 40 CRC, 40 AD, 40 IBD) in PBL and in selected cases in mononuclear (MNC) and polymorphonuclear cell compartments(PMNC). A linear logistic regression based model was built for the classification of random test set samples. In parallel cfDNA was isolated from 12 IBD patients using from 4 ml blood, each. The isolated cfDNA was added to Ficoll separated MNC fraction of HE subjects and the IBD related mRNA expression pattern analysis was performed. A CRC related DNA segment was artificially synthesized and injected into the tail vein of healthy mice(3). The analysis of cell type changes in colonic stroma and lymphoid follicles(LF) was performed using IHC for CD34, and CD133. Results: CRC could be classified from all of the other groups by 85% sensitivity(SE) and 86% specificity(SP) depending on the levels of 34 genes including septin 5, CD36, nuclear coactivator 1. ADs were detected by 95% SE and 91% SP in relation to HE on the expression of 21 genes including EGFR and MMP8. IBD and HE could be discriminated using 13 genes by 94% SE and 93% SP. Array RT-PCR confirmation yielded a highly reproducible gene set that worked on an independent set of patients (CRC SE:82%, AD SE:85%, IBD SE: 91% at 95% SP) and was present in MNC cells. IBD related cfDNA increased the expression of IBD related genes in MNCs of healthy subjects. Injection of CRC related cfDNA fragments into the tail of healthy mice, increased the volume of LF in the colon and the number of CD34 and CD133 cells, locally in the colon. Discussion: PBL based detection of local colorectal diseases can be performed by array RT-PCR after WG whole blood mRNA expression analysis with high sensitivity and specificity. One of the local alteration reporting molecules can be the cfDNA that can cause local stem cell infiltration in a mouse model.
478 Intestinal Microbiota: New Investigation Field in Colorectal Cancer (CRC) Iradj Sobhani, Jean-Pierre Furet, Francoise Roudot-Thoraval, Gérard Corthier Overweight is a risk factor of CRC. Commensal intestinal microbiota is changed in obese individuals while Bacteroides fragilis, is shown to favor colon cancer growth in animals (Nat Med, Oct 2009). The aim was to analyze dysbiosis, in average and higher risk individuals for CRC. Patients & Methods. After informed consent was obtained, stool samples were collected in 223 consecutive individuals 1-2 wks prior to colonoscopy. Stool bacteria DNAs were extracted after homogenization and mechanic destruction and quality of samples was verified. Bacteria DNA samples from 6 individuals with normal colonoscopy and 6 with invasive carcinoma were submitted to complete DNA pyrosequencing procedure by targeting V3-V4 region of 16S rDNA. Second, quantitative PCR (qPCR) measurements in separate sets, targeted specific sequences for all bacteria, and for those species with trends to difference between normal and tumoral subjects as revealed by full pyrosequencing. Appropriate statistical tests were used with P<0.05 as significant. Results. Stool DNAs of 119 normal subjects (N) were compared to those of 104 with colon or rectal tumours (Advanced adenomas-AD, n=44; invasive carcinoma, CA, n=60). These groups were balanced for all characteristics except for age (57 versus 67 yrs; p=0.01). Pyrosequencing results of two determinations per sample were reproducible and showed (mean of two values) 8 to 12% of no hits sequences according to 200 nucleotides minimal sequence reads and 98% matching rate. Significant and trends of differences regarding dominant and subdominant bacteria families were found. Consequently quantitative and qualitative comparisons were performed: qPCR expressed as mean±SD of log10 (bactéries/g stool) between N, AD and CA showed no difference (P=0.16) in All bacteria (11.88 ± 0.35, 11.97 ± 0.44 and 11.80 ± 0.56, respectively); among dominant or sub dominant bacteria families, those of Bacteroidetes were higher (All bacteria-Bacteriodetes; P=0 .009) in CA (1.04±0.55) than in AD (1.43±0.75) or N (1.40±0.83). ROC curve showed AUC of 0.656 [0.579 - 0.734] with a threshold of 1.2 that corresponded to 75 % sensitivity and 51.5 % specificity to identify cancers. Qualitative
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AGA Abstracts
AGA Abstracts
determined the composition of adherent bacteria in distal colons using PCR and terminal restriction fragment length polymorphism (TRFLP) of 16S rRNA genes. Terminal restriction fragment (TRF) peak height- and statistical analyses were performed using PRIMER VI and STATA respectively. Results: SPF Il10-/- mice developed colitis (ave. score = 2.6) with an average of 1.7 colorectal tumors/mouse compared to WT mice, which showed no signs of colitis and developed only 0.2 tumors/mouse. To further study the relationship between inflammation and colorectal cancer, we mono-associated Il10-/- germ-free mice with E. faecalis, a known inducer of colitis. Surprisingly, although E. faecalis-monoassociated germ free Il10-/- mice developed severe colitis (ave. score = 3.4), tumor development was strongly diminished (0.05 tumors/mouse) suggesting that inflammation alone is not sufficient to drive tumorigenesis. Principal component analysis showed that adherent bacterial communities present in mice with colorectal tumors cluster independently of those in non-tumor bearing mice (R = 0.189; P = 0.027). Strikingly, bacterial profiles in mice harboring invasive carcinomas cluster independently from those observed in mice with non-invasive tumors. We identified 8 TRFs each representing a bacterial community that showed significant variance between tumor- and non-tumor-bearing mice (P = 0.002). Conclusions: These findings indicate that bacterial composition strongly influences the development of CAC in Il10-/- mice and suggest that chronic inflammation is not the sole component leading to CAC development. Further investigation of the composition and influence of therapeutic modulation of the microbiota in patients with IBD and a high risk for CAC are warranted.
mortality during admission and 3 months follow-up. Secondary endpoints included other morbidity and total costs at 6 months follow-up. RESULTS: Infected necrosis was present in 81 patients (92%). The primary endpoint occurred in 31 of 45 patients (69%) assigned to open necrosectomy and in 17 of 43 patients (40%) assigned to the step-up approach (risk ratio 0.57; 95% confidence interval 0.38 to 0.87; P=0.006). In the step-up approach group, 35% of patients were successfully treated with percutaneous drainage only and did not require necrosectomy. New onset multiple organ failure occurred less often in the stepup approach group (12% versus 40%; P=0.002). At 6 months follow-up, patients in the step-up approach group had a lower rate of incisional hernias (7% versus 24%; P=0.03), use of pancreatic enzymes (7% versus 33%; P=0.002) and new onset diabetes (16% versus 38%; P=0.02). Mean total costs per patient were $15,963 (12%) lower in the step-up approach group. CONCLUSIONS: A minimally invasive step-up approach, as compared to open necrosectomy, reduced the rate of the composite endpoint of major morbidity and mortality as well as long-term morbidity and costs in patients with (suspected) infected necrotizing pancreatitis. In these patients, percutaneous drainage should be considered as the first step of treatment.
475q Tm4sf1 is Highly Expressed in Pancreatic Cancer and Promotes Cancer Cell Migration, Invasion and Survival Jia Cao, Vijaya Ramachandran, Thiruvengadam Arumugam, Friso Nast, Zhaoshen Li, Craig D. Logsdon Introduction: Pancreatic cancer is a major oncological challenge. Our microarray data showed over-expression of TM4SF1 in human pancreatic cancer tissues and cell lines.TM4SF1 is a member of the four-transmembrane-domain family of cell surface molecules. It is highly expressed and has been implicated in the regulation of metastasis in several human epithelial cell carcinomas. However, little is known about the functions of TM4SF1 in pancreatic cancer. Therefore, in the current study we examined the expression and functions of TM4SF1 in pancreatic cancer cells. Methods: TM4SF1 was detected in human pancreatic cancer tissues by immunohistochemistry (IHC).The expression of TM4SF1 mRNA in pancreatic cell lines, pancreatic ductal adenocarcinoma (PDAC) tissues, adjacent normal pancreatic (NP) tissues and chronic pancreatitis (CP) tissues were quantitated by qRT-PCR. The effects of TM4SF1 in pancreatic cancer cells were evaluated by siRNA and shRNA techniques in control or TM4SF1 silenced MPanc96, MIA PaCa-2, PANC-1 and HPAC cells. And the effects on cell migration and invasion were also analyzed by Boyden's chamber and BioCoat Matrigel invasion chamber. Cell growth was assessed by the MTS method. Sensitivity to gemcitabine treatment was analyzed by flow cytometry (FACS analysis).Results: TM4SF1 was mainly found distributed in ductal luminal epithelial cancer cells. Compared with NP or CP, the expression of TM4SF1 was highly elevated in PDAC as accessed by IHC. TM4SF1 mRNA expression was also found to be 5~10 fold high in PDAC compared with NP or CP. The migration and invasion of pancreatic cancer cell lines MPanc96, MIA PaCa-2, PANC1 and HPAC cells were significantly decreased when TM4SF1 was silenced by either siRNA or shRNA in comparison with their respective controls. In contrast, TM4SF1 silencing had no effect on cell proliferation. Flow cytometry data showed that the gemcitabine mediated apoptosis percentage of MPanc96, MIA PaCa-2 and PANC-1 cells were significantly increased after silencing TM4SF1 in comparison with its respective controls. Conclusions: These findings suggest that TM4SF1 is a surface membrane antigen expressed highly in pancreatic cancer and promotes migration, invasion and survival of the pancreatic cancer cells. Thus, TM4SF1 may be a useful target for future development of novel therapies for this disease.
475o Resistin, a Potent Adipokine, is Associated With Acute Pancreatitis: Assessment of Functional Genetic Polymorphisms and Serum Levels Venkata Muddana, Anna C. Evans, Christopher J. Langmead, Gilles Clermont, Michael M. Barmada, Georgios I. Papachristou, David C. Whitcomb Background: Patients who are obese are more susceptible to acute pancreatitis (AP), with increased risk of major local and systemic complications linked to the inflammatory response. Adipokines are a class of cytokines produced by visceral fat. We tested the hypothesis that pathologic effects of obesity in AP are mediated, in part, by the adipokine resistin. Methods: AP patients and controls were enrolled from the University of Pittsburgh Medical Center; those with a BMI>30 kg/m2 were classified as obese. Severe AP (SAP) was defined by >1 organ dysfunction of cardiovascular, pulmonary and/or renal systems persisting for > 48 hours. Functional resistin single nucleotide polymorphisms (SNP) rs1862513 C>G, rs35547567 C>T, rs35139946 C>T, rs3745367 G>A and rs3745369 G>C were evaluated by TaqMan assay. In a subgroup of patients with early serum samples, resistin levels were measured using a commercial Luminex assay. Results: Resistin SNPs were assessed in 188 AP patients [mild AP (MAP): n=148; SAP: n=40] and controls (n=163), of which 67 patients (36%) and 34 controls (22%) were obese (p=0.01). Genotype and haplotype analysis suggested that the primary genetic effect was through the resistin promoter SNP rs3745367 A allele. The A allele was associated with overall susceptibility to AP (p=0.0009, OR=1.8, CI= 1.3-2.6), and the effect was stronger in lean subjects (p=0.0001, OR=2.4, CI=1.5-3.9). Serum was available in 46 patients (27 MAP, 19 SAP) and 58 controls. At admission, resistin levels were significantly higher in SAP patients (median=51316 pg/ml; IQR =30769-110451) than in MAP patients (7504 pg/ml; 5879-11548) and controls (6,574 pg/ml; 4,965-8225) (p<0.001). Conclusions: The resistin rs3745367 A allele increased susceptibility to AP, with enhanced effect in patients with BMI < 30kg/m2. Resistin levels on admission may be useful in predicting persistent organ failure in AP.
475r Surgical Versus Endoscopic Drainage in Obstructive Chronic Pancreatitis: A Cost-Effectiveness Analysis Philippe Laramee, David Wonderling, Djuna L. Cahen, Marco J. Bruno, Marcel G. Dijkgraaf, Dirk J. Gouma, Stephen P. Pereira Two published randomized controlled trials (RCTs) [1,2] have reported that surgical drainage of the pancreatic duct was more effective than endoscopic drainage for patients with chronic pancreatitis. The objective of this analysis was to assess the cost-effectiveness of surgical versus endoscopic drainage in obstructive chronic pancreatitis. Methods: This analysis was conducted mainly based on one RCT [1], from a UK National Health Service perspective, and over a 24-month time horizon for the base-case analysis (median follow-up time in the trial). A lifetime horizon was used in the sensitivity analysis. The health outcome was the Quality-Adjusted Life Year (QALY), which was generated using EQ-5D data collected during the trial. Also collected as part of the trial were details of the use of diagnostic and therapeutic procedures, time in hospital and the treatment of pancreatic exocrine and endocrine insufficiency. This resource use was combined with UK unit costs. Key elements of resource use, such as complication rates and conversion to surgery were varied in sensitivity analyses. The trial reported one death in the endoscopy group (5%), which was not clearly related to the intervention. For the base-case analysis, we assumed no mortality in either group and applied a mortality rate of up to 2% to surgical drainage in the sensitivity analysis. Results: The result of the base-case analysis was that surgical drainage of the pancreatic duct dominated endoscopic drainage, being both more effective and less costly. The sensitivity analysis showed that the surgical option remains dominant in a majority of scenarios. When compared with a threshold of £20,000 per QALY gained, all analyses indicated that the probability that surgical drainage is cost-effective compared to endoscopic drainage exceeds 95%. Conclusion: In patients with obstructive chronic pancreatitis, surgical drainage is highly cost-effective compared to endoscopic drainage. This economic analysis was developed from a UK perspective, but considering the magnitude of the improvement in quality of life, the substantial cost savings and the robustness of the results to sensitivity analyses, it is likely that this conclusion can be generalised to other healthcare systems. References: [1] Cahen DL, Gouma DJ, Nio Y et al. Endoscopic versus surgical drainage of the pancreatic duct in chronic pancreatitis. N Engl J Med 2007; 356: 676-684. [2] Dite P, Ruzicka M, Zboril V et al. A prospective, randomized trial comparing endoscopic and surgical therapy for chronic pancreatitis. Endoscopy 2003; 35: 553-558.
475p Prognostic Value of Blood Urea Nitrogen (BUN) in the Early Assessment of Acute Pancreatitis: An International Study Bechien U. Wu, Olaf J. Bakker, Georgios I. Papachristou, Kathryn Repas, Marc G. Besselink, Hjalmar C. van Santvoort, Venkata Muddana, Vikesh K. Singh, David C. Whitcomb, Hein G. Gooszen, Peter A. Banks Background: An accurate and reliable approach to monitor early disease progression is a critical step to improve treatment in acute pancreatitis (AP). A recent retrospective cohort study identified BUN at admission and change in BUN at 24 hours as independent predictors of mortality in AP. Aim: To evaluate the accuracy of early changes in BUN for prediction of mortality in a series of rigorously defined prospective AP cohort studies. Patients & Methods: Data were collected from 3 prospective AP cohort studies: Brigham and Women's Hospital (BWH, June 2005 - May 2009), University of Pittsburgh Medical Center (UPMC, June 2003 - September 2007) and from 15 Dutch hospitals (Dutch Pancreatitis Study Group [DPSG] March 2004 - March 2007). All centers utilized the same set of standardized criteria for diagnosis of AP. Early BUN trajectories were compared between survivors and fatal cases of AP (univariate analysis). Subsequent multivariate logistic regression was used to obtain age- and gender-adjusted estimates of risk of in-hospital mortality associated with elevation in BUN at admission and/or rise in BUN at 24 hours. Accuracy of BUN was compared with APACHE II for prediction of in-hospital mortality using area under ROC (AUC) curves. Results: Data from 1,385 AP cases were included from the 3 study cohorts (620 BWH, 186 UPMC, 576 DPSG). There were a total of 61 (4.4%) deaths. Early rise in BUN was associated with mortality in both center-specific and pooled analysis (BUN change t-test, p<0.0001). In multivariate analysis, after adjusting for age- and gender, both admission BUN (wald χ2 p<0.0001) and change in BUN (wald χ2 p<0.0001) were independent predictors of mortality. Accuracy of BUN (admission and change in 24 hours) within individual study cohorts ranged from AUC=0.78 to AUC=0.91. Overall accuracy of BUN for prediction of mortality was comparable to APACHE II in each of the 3 study cohorts. Conclusions: In this prospective multicenter international study, an elevated BUN at admission and rise in BUN during the initial 24 hours of hospitalization proved to be independent predictors of mortality. Serial BUN measurement is a simple and accurate method for predicting mortality in acute pancreatitis.
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476 Intestinal Inflammation is Not Sufficient to Promote the Development of Colitis Associated Colorectal Cancer: Role of the Enteric Microbiota Joshua M. Uronis, Marcus Muehlbauer, Temitope O. Keku, Hans H. Herfarth, Tara C. Rubinas, Janelle C. Arthur, Christian Jobin Introduction: Although the etiology of colitis-associated colon cancer (CAC) remains elusive, the intestinal microbiota has been implicated in the pathogenesis of this disease. We recently developed a novel experimental model of CAC in which the colon-specific carcinogen azoxymethane (AOM) is used to initiate colorectal cancer in Il10-/- mice. Aim: Determine the composition of the microbiota and its role in the development of CAC. Methods: Wildtype (WT) and Il10-/- germ-free mice (n=19) were conventionalized under specific pathogen-free (SPF) conditions or mono-associated with Enterococcus faecalis (E. faecalis). Tumors were induced by intraperitoneal injection of azoxymethane (AOM) once a week for 6 weeks and then followed for 14 weeks. Inflammation and tumor grade were evaluated histologically using a scale of 0-4. Tumors were classified as low-, high-grade or invasive carcinoma. We
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differences among each bacteria family is in progress between N and AD or CA individuals. Conclusion : This is the first large series showing potential role of intestinal microflora in CRC. Increase of Bacteroidetes may be account for helper T IL17 producing cell recruitment previously reported in colon mucosa in CRC (Nat Med 2009; Gut 2008). Whether this is the cause of consequence of colon carcinogenesis remains further investigation. Microflora may be used as screening test. 479 Effect of High Fat Dietary on Colon Tumor Development in Azoxymethane and Dextran Sulfate Sodium Treated Mice In-Wha Kim, Seung-Jae Myung, Mi Young Do, Yeon Mi Ryu, Mi Jung Kim, Sehan Park, Eun Joo Do, Byong Duk Ye, Jeong-Sik Byeon, Suk-Kyun Yang, Jin-Ho Kim Backgrounds/Aims High fat dietary is known to play important roles in inflammatory bowel disease and colon carcinogenesis. The purpose of this study was to understand the mechanisms by which fat dietary can regulate susceptibility to intestinal inflammation and carcinogenesis. Methods A high fat diet (HFD) was based on a western-style diet that contained 20% fat, while a normal diet (ND) contained 5% fat with the additional calories provided by cornstarch. Male Balb/c mice were fed either an HFD (n=52) or an ND (n=52) for 4 weeks, and exposed to azoxymethane (AOM) followed by 2% dextran sulfate sodium (DSS) in drinking water for 7 days. We measured the expression of COX-2 and 15-PGDH using real-time PCR and western blot. Immunohistochemistry analysis for F4/80 and βcatenin were carried out. Colon tissues PGE2 and serum tumor necrosis factor-α (TNF-α) levels were determined by enzyme immunoassay. Results Body weight gain and food intake were comparable in mice between HFD and ND groups. The mean length of large bowel was significantly shorter in an HFD group than an ND group at 8 weeks after cessation of the administration with DSS (9.71. ± 0.90 vs 11.26 ± 1.03 cm, p<0.05). The incidence of colon tumor in mice fed an HFD were 100% with a multiplicity of 16.75 ± 2.37, while that in mice fed an ND were 20% with a multiplicity 0.80 ± 1.93 (p<0.05). The development of colitis proceeded faster in mice fed an HFD compared to an ND (Inflammation score 2.51 ± 1.34 vs 1.53 ± 0.88, p<0.05 at day 4 after DSS initiation). Immunohistochemical staining revealed that the number of infiltrating F4/80-positive macrophages increased progressively during the development of the colitis induced by DSS. Feeding of HFD accelerated the infiltration of macrophages in the colon (macrophages number 67.4 ± 9.9 vs 37.8 ± 7.2 /field, p<0.05, at day 6 after DSS initiation). Mice given HFD had higher expression levels of COX-2 and PGE2, and a lower level of 15-PGDH compared with an ND group at day 1 after cessation of the DSS treatment. An HFD also elevated the serum concentration of TNF-α and the nuclear translocation of nuclear factor-kappa B. Regardless of whether they were given an ND or an HFD, colon cancer had higher expression of COX-2 and PGE2 but lower expression of 15-PGDH compared to normal tissues. In addition, expression of unphosphated (active) β-catenin was undetectable in normal tissues, however, its accumulation was significantly enhanced in the cytoplasmic and nuclear of colon carcinomas. Conclusions We concluded that high fat dietary strongly facilitated inflammation, thereby resulting in promoting AOM/DSS-induced colon carcinogenesis.
477 Effect of the Probiotic VSL#3 on the Transition From Chronic Inflammation to Dysplasia in a Model of Colitis-Associated Colon Cancer Caroline B. Appleyard, Idamary Passalacqua, Angel A. Isidro, Myrella L. Cruz, Raymond A. Isidro, Victor A. Rodriguez Rapale, Claudio De Simone Patients with long-standing colitis have a significantly increased risk of developing colorectal cancer. It is not yet clear how the presence of chronic inflammation results in neoplastic transformation and progression, but there is much evidence to support an important role for the microflora. Probiotics, through modulation of both immune and barrier mechanisms, may alter this process. Aim: To investigate the protective efficacy of the probiotic VSL#3 in preventing the transition from chronic inflammation to dysplasia in a novel model of colitis-associated colorectal cancer. Methods: Chronic colitis was induced in male Sprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for three days. To induce colitis-associated dysplasia and cancer the animals then received TNBS (iv) twice a week for ten weeks. One group of rats received the probiotic in drinking water (VSL#3; ~5 billion cfu /100 gram body weight) from one week prior to initial colitis induction until the time of sacrifice (n=22-23/group). After sacrifice the colons were removed and analyzed for total macroscopic damage, microscopic damage and presence of dysplasia or cancer. Cell proliferation was assessed by immunohistochemical staining. Results: Those animals receiving the probiotic had significantly less macroscopic (p<0.05) and microscopic damage (p<0.05) than the vehicle treated controls in all areas of the colon. Pathological analysis revealed that 29.4% of the vehicle treated animals developed carcinoma, which was confined to the proximal region. None of the probiotic treated animals developed carcinoma. Although there was no significant difference in the amount of dysplasia found in any region of the colon between the groups, treatment with the probiotic caused a shift to a less severe diagnosis with no high-grade dysplasia found in either the proximal or mid colon. Animals receiving the probiotic had more non-specific inflammation in any region of the colon with less diagnosis of IBD compared to vehicle treated. Treatment with the probiotic reduced crypt cell proliferation by 22% as assessed by measurement of BrdU incorporation in comparison with the vehicle treated group. Fecal alkaline phosphatase activity in the probiotic treated animals was half that found in those treated with vehicle. Conclusion: Our results suggest that treatment with the probiotic VSL#3 can attenuate the development of colonic damage and possibly delay the transition to dysplasia and cancer, offering its potential therapeutic use in patients with long-standing colitis.
480 Colorectal Cancer (CRC), Adenoma and IBD Detection in Blood Cells by Array PCR - Role of Circulating Free DNA as Mononuclear Cell Activator Béla Molnár, Kinga Tóth, Sándor Spisák, Orsolya Galamb, Ottó Jász, Alexandra Kalmar, Ferenc Sipos, Pál Miheller, Zsolt Tulassay Background: Whole genomic(WG) gene expression analysis of peripheral blood(PBL) and colon migrating cell populations could contribute to the identification of diagnostic mRNA markers of colorectal diseases. Aims: Validation and verification of WG blood based biomarkers on an independent test set were performed by RT-PCR. Identification of circulating free DNA (cFDNA) as activator molecule of the mononuclear cell fraction causing their local colon alteration specific mRNA expression changes was aimed. Materials and methods: Total RNA was isolated from PBL samples of 16 healthy(HE), 19 CRC, 12 adenoma(AD) and 11 IBD patients. After globin reduction, WG expression profile analysis was done. For classification of samples Prediction analysis of microarrays(PAM) was used. Array results were confirmed by sequence specific array RT-PCR on independent sample set (40 HE, 40 CRC, 40 AD, 40 IBD) in PBL and in selected cases in mononuclear (MNC) and polymorphonuclear cell compartments(PMNC). A linear logistic regression based model was built for the classification of random test set samples. In parallel cfDNA was isolated from 12 IBD patients using from 4 ml blood, each. The isolated cfDNA was added to Ficoll separated MNC fraction of HE subjects and the IBD related mRNA expression pattern analysis was performed. A CRC related DNA segment was artificially synthesized and injected into the tail vein of healthy mice(3). The analysis of cell type changes in colonic stroma and lymphoid follicles(LF) was performed using IHC for CD34, and CD133. Results: CRC could be classified from all of the other groups by 85% sensitivity(SE) and 86% specificity(SP) depending on the levels of 34 genes including septin 5, CD36, nuclear coactivator 1. ADs were detected by 95% SE and 91% SP in relation to HE on the expression of 21 genes including EGFR and MMP8. IBD and HE could be discriminated using 13 genes by 94% SE and 93% SP. Array RT-PCR confirmation yielded a highly reproducible gene set that worked on an independent set of patients (CRC SE:82%, AD SE:85%, IBD SE: 91% at 95% SP) and was present in MNC cells. IBD related cfDNA increased the expression of IBD related genes in MNCs of healthy subjects. Injection of CRC related cfDNA fragments into the tail of healthy mice, increased the volume of LF in the colon and the number of CD34 and CD133 cells, locally in the colon. Discussion: PBL based detection of local colorectal diseases can be performed by array RT-PCR after WG whole blood mRNA expression analysis with high sensitivity and specificity. One of the local alteration reporting molecules can be the cfDNA that can cause local stem cell infiltration in a mouse model.
478 Intestinal Microbiota: New Investigation Field in Colorectal Cancer (CRC) Iradj Sobhani, Jean-Pierre Furet, Francoise Roudot-Thoraval, Gérard Corthier Overweight is a risk factor of CRC. Commensal intestinal microbiota is changed in obese individuals while Bacteroides fragilis, is shown to favor colon cancer growth in animals (Nat Med, Oct 2009). The aim was to analyze dysbiosis, in average and higher risk individuals for CRC. Patients & Methods. After informed consent was obtained, stool samples were collected in 223 consecutive individuals 1-2 wks prior to colonoscopy. Stool bacteria DNAs were extracted after homogenization and mechanic destruction and quality of samples was verified. Bacteria DNA samples from 6 individuals with normal colonoscopy and 6 with invasive carcinoma were submitted to complete DNA pyrosequencing procedure by targeting V3-V4 region of 16S rDNA. Second, quantitative PCR (qPCR) measurements in separate sets, targeted specific sequences for all bacteria, and for those species with trends to difference between normal and tumoral subjects as revealed by full pyrosequencing. Appropriate statistical tests were used with P<0.05 as significant. Results. Stool DNAs of 119 normal subjects (N) were compared to those of 104 with colon or rectal tumours (Advanced adenomas-AD, n=44; invasive carcinoma, CA, n=60). These groups were balanced for all characteristics except for age (57 versus 67 yrs; p=0.01). Pyrosequencing results of two determinations per sample were reproducible and showed (mean of two values) 8 to 12% of no hits sequences according to 200 nucleotides minimal sequence reads and 98% matching rate. Significant and trends of differences regarding dominant and subdominant bacteria families were found. Consequently quantitative and qualitative comparisons were performed: qPCR expressed as mean±SD of log10 (bactéries/g stool) between N, AD and CA showed no difference (P=0.16) in All bacteria (11.88 ± 0.35, 11.97 ± 0.44 and 11.80 ± 0.56, respectively); among dominant or sub dominant bacteria families, those of Bacteroidetes were higher (All bacteria-Bacteriodetes; P=0 .009) in CA (1.04±0.55) than in AD (1.43±0.75) or N (1.40±0.83). ROC curve showed AUC of 0.656 [0.579 - 0.734] with a threshold of 1.2 that corresponded to 75 % sensitivity and 51.5 % specificity to identify cancers. Qualitative
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AGA Abstracts
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determined the composition of adherent bacteria in distal colons using PCR and terminal restriction fragment length polymorphism (TRFLP) of 16S rRNA genes. Terminal restriction fragment (TRF) peak height- and statistical analyses were performed using PRIMER VI and STATA respectively. Results: SPF Il10-/- mice developed colitis (ave. score = 2.6) with an average of 1.7 colorectal tumors/mouse compared to WT mice, which showed no signs of colitis and developed only 0.2 tumors/mouse. To further study the relationship between inflammation and colorectal cancer, we mono-associated Il10-/- germ-free mice with E. faecalis, a known inducer of colitis. Surprisingly, although E. faecalis-monoassociated germ free Il10-/- mice developed severe colitis (ave. score = 3.4), tumor development was strongly diminished (0.05 tumors/mouse) suggesting that inflammation alone is not sufficient to drive tumorigenesis. Principal component analysis showed that adherent bacterial communities present in mice with colorectal tumors cluster independently of those in non-tumor bearing mice (R = 0.189; P = 0.027). Strikingly, bacterial profiles in mice harboring invasive carcinomas cluster independently from those observed in mice with non-invasive tumors. We identified 8 TRFs each representing a bacterial community that showed significant variance between tumor- and non-tumor-bearing mice (P = 0.002). Conclusions: These findings indicate that bacterial composition strongly influences the development of CAC in Il10-/- mice and suggest that chronic inflammation is not the sole component leading to CAC development. Further investigation of the composition and influence of therapeutic modulation of the microbiota in patients with IBD and a high risk for CAC are warranted.