- Email: [email protected]
S1186 The Diagnostic Accuracy of Faecal Calprotectin in Inflammatory Bowel Disease (IBD): A Systematic Review
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against several standard measures for assessing IBD disease activity. Methods: Participants enrolled in the Manitoba IBD Cohort Study, a population based longitudinal cohort (n= 356) were assessed periodically by survey, clinical interview, and blood sample. The MI is based on patient report of symptom persistence for the previous 6 mos, using a 6-level response format: active (a) constantly (daily symptoms) (b) often (symptoms most days) (c) sometimes (symptoms some days e.g.,1-2 days/wk) (d) occasionally (symptoms 1-2 days/ mo) (e) rarely (symptoms few days past 6 mos) and (f) inactive (absence of symptoms). Disease was defined as active if respondents answered any of a-d and inactive if answered e or f. Physical symptoms (GI and systemic), clinical disease indices (HB, PT), IBD medication use, daily productivity loss, and psychological functioning (e.g. health anxiety, distress) were all assessed. Biological markers included hemoglobin and CRP. Sensitivity analyses and discriminant function analyses were used to validate the MI, using data from baseline (i.e., month 0), 12 and 24 months points. Results: The MI had good sensitivity when compared to HB, PT, and IBDQ scores (range 0.84 to 0.90). It correlated well with all these measures (0.39 to 0.62, p<0.01) and individual symptom measures (e.g., abdominal, joint, diarrhea, bowel movement urgency; 0.27 to 0.62, p < 0.01), despite the different assessment time frames. In separate discriminant analyses for CD and UC, common discriminating variables included clinical indices (HB for CD, PT for UC), IBDQ subscales of bowel and systemic symptoms, and symptom ratings (e.g., abdominal and joint pain, tiredness, diarrhea). Conclusions: A single-item patient-defined disease activity measure, the Manitoba Index, showed a high degree of sensitivity for classifying individuals with active disease compared to existing disease activity measures, and strong convergent validity with expected proxy measures of disease. These relationships were consistent across a two-year period. Thus, the MI shows promise as a valid brief tool for measuring disease activity.
AGA Abstracts
Serum Protein Signatures Determined By Mass Spectrometry (SELDI-ToF) Accurately Distinguishes Crohn's Disease (CD) from Ulcerative Colitis (UC) Venkataraman Subramanian, Devika Subramanian, Richard C. Pollok Background: Accurate diagnosis of UC or CD is essential to guide patient management. Current tests are invasive and carry risks. A meta analysis suggested that diagnosis of CD by ASCA (Anti-Saccharomyces cerevisiae antibodies) has a sensitivity of 54.6%. pANCA positivity has a sensitivity of 55.3% in the diagnosis of UC. Aim: We aimed to detect novel serum biomarkers by protein fingerprinting using surface-enhanced laser desorption ionization time-of-flight (SELDI-ToF) mass spectrometry to differentiate CD from UC. Methods: Serum was collected prospectively from 62 patients with UC and 48 with CD and stored at -80 0C. Samples were applied to CM10 protein chip arrays and time-of- flight spectra were generated using a PBS-II mass spectrometer (Ciphergen, Freemont, CA, USA). The mass spectrometry data was extracted and preprocessed prior to analysis. The spectra were analyzed using support vector machine (SVM) analysis utilizing recursive feature elimination (RFE) techniques with radial basis function and 5-fold cross validation. We then developed a boolean pattern classifier on the thresholded peaks (based on median value) detected by SVM. The identity of the selected peaks was confirmed using immunodepletion methods. ASCA and ANCA were determined by ELISA and indirect immunofluorescence respectively. Results:12 key peaks were identified using SVM with RFE (RBF kernel width of 0.00075 and C of 100) in a five-fold cross validation setting. These peaks were then thresholded to being above or below the median value. We clustered the data using kmeans with two clusters. Using 8 of these thresholded peaks two well separated clusters were identified. A boolean pattern classifier was developed on the 12 thresholded peaks. The specificity and sensitivity of this boolean classifier was 96% for the first cluster and 94% for the second cluster. ASCA had a sensitivity of 70.8% in detecting CD and overall accuracy of 80.9%. p ANCA had a sensitivity of 64.5% for detecting UC. The peaks identified as important had m/z values of 2.7, 4.1, 4.2, 5.6, 6.4, 6.8, 7.8, 7.9, 8.6, 8.7, 9.3, 1.6 kDa. These were confirmed to be fragments of inter alpha trypsin inhibitor 4 (2.7 and 4.2), Apolipoprotein C1 and probably its SPA adduct (6.4 and 6.8), Platelet activated factor 4 variants (7.8 and 7.9). Conclusions: Using protein signatures of patients with UC and CD we have developed a classification model which is accurate, sensitive and more informative than currently available serological tests. There seem to be distinct proteome based clusters of CD and UC patients. The clinical, pathogenic and prognostic significance of these clusters needs to be further characterized
S1185 Faecal Calprotectin's Utility in the Prediction of Inflammatory Bowel Disease (IBD) Relapses Javier P. Gisbert, Fernando Bermejo, Jl Perez-Calle, Carlos Taxonera, Isabel Vera, Yago Gonzalez-Lama, Alicia Algaba, P. Lopez-Serrano, N. López-Palacios, Marta Calvo, Adrian G. McNicholl, Ja Carneros, M. Velasco, José Maté-Jiménez BACKGROUND: IBD activity flares are usually unpredictable. If we had an available and reliable marker to estimate the risk of relapse, we could prescribe early medication to prevent or treat the relapse. Inflammation is a continuous process, so a biological marker able to estimate the intensity of inflammation might provide us a pre-symptomatic quantitative measure of the risk of imminent clinical relapse AIM: To determine faecal calprotectin's utility in the prediction of IBD relapses. METHODS: Prospective multicenter study including Crohn's Disease (CD) and Ulcerative Colitis (UC) patients who had been in clinical remission (based on Truelove and CDAI scores) during the preceding 6 months. Disease and treatment data collection and calprotectin's basal levels measurements were done during screening. Follow up period was 12 months in those patients showing no relapse and until activity flare in relapsed patients. RESULTS: Seventy-seven patients, 44 with CD and 35 with UC, have completed the study so far. Treatments: oral 5-ASA (62%), topical 5-ASA (7%), AZA/ MP (39%), and infliximab (4%). 25% of the patients had suffered a flare the year preceding the study. Seventeen patients (22%) relapsed during follow up (after an average of 20 weeks). Average levels of faecal calprotectin were 179±157 µg/g (ranging from 9 to 851 µg/g). Calprotectin concentrations in those patients who suffered a relapse were higher than in those who maintained remission (255±143 vs. 157±154 µg/g; p<0.05). This difference was only demonstrated in CD patients (317 vs. 162 µg/g; p<0.05) but not in UC patients (200 vs. 151 µg/g). The percentage of IBD patients relapsing was significantly different between those having calprotectin concentrations <100 µg/g and those with higher concentrations (10% vs 30% relapses respectively, p<0.05). These differences were even clearer in CD patients (6% for calprotectin <100 µg/g and 28% for >100 µg/g). Faecal calprotectin's (>100 µg/g) sensitivity and specificity to predict relapses in CD were 82% and 47% (positive and negative predictive values of 30% and 90% respectively). The area under the ROC curve to predict IBD relapse using calprotectin determination was 0.72, and considering only CD data was 0.81%. The best global cut-off point was 166 µg/g (sensitivity 82%; specificity 67%), but in CD patients it was 170 µg/g (sensitivity 88%; specificity 68%). Considering only the prediction of relapse during the first 6 months of follow up, results were better (area under the ROC curve in CD was 0.91) CONCLUSIONS: High calprotectin concentrations in faeces are associated with a higher risk of clinical relapse in IBD patients.
S1183 A Combination of Serum Adiponectin, Pepsinogen I and Anti-Saccharomyces Cerevisiae Antibody (ASCA) Levels Can Accurately Distinguish Patients with Crohn's Disease Involving the Small Bowel from Colonic Crohn's Disease Venkataraman Subramanian, Geetha Balarajah, Richard C. Pollok Background: Serum Pepsinogen I (PG I) level reliably correlates with the number of chief cells in the gastric corpus mucosa. Gastric heterotopia of the small intestine in Crohn's disease (CD) has been well described and PG I & II noted on immunohistochemisty in the metaplastic pyloric and oxyntic glands in the ileum. Adiponectin levels are known to be elevated in patients with CD with mesenteric adipose tissue hypertrophy and not in patients with UC. ASCA has been associated with more proximal involvement rather than colonic CD. Diagnosis of small intestinal involvement in patients with CD has major implications on patient management. Aim: The aim of this pilot study was to use serum PG I , serum adiponectin and IgA and IgG ASCA levels to develop an algorithm that distinguishes patients with colonic CD from those with CD involving the small intestine. Methods: Serum was prospectively collected and stored at -80 0C from patients with histologically proven CD. PG I levels (Biohit plc, Finland) and Adiponectin levels (B-bridge International, USA), IgA and IgG ASCA (Aesku Lab, Germany) were determined in duplicate by ELISA. Patients were grouped into those with small intestinal involvement and those with colonic CD. Differences between groups were tested using Mann-Whitney U test of significance. Results: Mean PG I levels in the small bowel CD group (n=18) was 97.89 ± 33.07 µg/l. Mean PG I levels in the colonic CD group (n=14) was 83.25 ± 29.74 µg/l (p=0.08). Mean Adiponectin levels in the small bowel CD group was 3.89 ± 1.87 ng/ml and in the colonic CD group was 2.65 ± 1.50 ng/ml (p=0.02). Area under the Receiver operating curve for Adiponectin was 0.74 and for Serum PG I was 0.68. A cut off value of more than 1.58 ng/ml for serum Adiponectin and more than 78 µg/l for serum PG I with either a positive IGA or IgG ASCA had a sensitivity of 88.9 %, specificity of 85.7 % and overall accuracy of 87.5% in detecting small bowel involvement in CD. Only 2 patients each with colonic and small intestinal CD were misclassified. Conclusion: Adiponectin levels are significantly higher in CD patients with small bowel disease. This could be because patients with colonic CD alone are less likely to have mesenteric fat hypertrophy. Serum PG1 levels showed a trend towards significance among patients with small intestinal CD. This could reflect both gastric heterotropia in the small intestine as well as inflammation due to CD in the stomach. Further studies are required to confirm these findings. A combination of Adiponectin , PG I levels and ASCA serology could help in non-invasive diagnosis of small bowel involvement in patients with CD.
S1186 The Diagnostic Accuracy of Faecal Calprotectin in Inflammatory Bowel Disease (IBD): A Systematic Review Javier P. Gisbert, Adrian G. McNicholl BACKGROUND: The presence of calprotectin in faeces is directly proportional to neutrophil migration towards the intestinal tract. The potential strength of faecal calprotectin assessment is that it is a direct measure of mucosal inflammatory activity, and levels in the stool seem to be unaffected by a variety of non-intestinal conditions, which may result in an elevation of the systemic inflammatory markers. AIM: To review the diagnostic accuracy of faecal calprotectin determination 1) to distinguish organic from functional disorders in symptomatic patients, and 2) for the diagnosis of IBD. METHODS: Bibliographical searches were performed in MEDLINE up to 2007 looking for the following words (all fields): (“inflammatory bowel disease” OR “Crohn's disease” OR “ulcerative colitis”) AND (calprotectin OR faecal marker). The mean sensitivity and specificity of faecal determination was calculated and expressed as weighted mean (and corresponding 95% confidence interval; 95% CI) to make due allowance for the number of patients included in each study. Subanalyses were performed depending on the IBD type: ulcerative colitis (UC) vs. Crohn's disease (CD). RESULTS: 1) Thirteen studies, including a total of 2475 patients, measured faecal calprotectin concentrations aiming to distinguish organic from functional intestinal disease in symptomatic patients. Mean sensitivities and specificities of 83% (95% CI, 81-84%) and 84% (95% CI, 82-85%), respectively, were calculated. 2) Fourteen studies, including a total of 754 patients, evaluated the diagnostic precision of faecal calprotectin for the diagnosis of IBD. In most of these studies, symptomatic patients with IBD or irritable bowel syndrome, in addition to controls,
S1184 The Manitoba Index: Psychometric Evidence for a New and Simple Indicator of IBD Activity Ian Clara, Lisa M. Lix, John Walker, Lesley A. Graff, Norine Miller, Linda Rogala, Patricia Rawsthorne, Charles N. Bernstein Many indicators of disease activity for IBD can be time consuming or difficult to administer, as they require invasive procedures (endoscopy), clinical evaluation (CDAI), or completion of multiple-item scales (IBDQ). Simpler indices such as the Harvey-Bradshaw (HB) and Powell-Tuck (PT) provide only a brief cross-sectional account of disease activity at the time of a clinic visit and do not adequately characterize disease activity over an extended period. A single-item indicator of disease activity, the Manitoba Index (MI) is proposed, and compared
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items). Originally developed in a Canadian cohort, it has since been translated and used in both English and non-English speaking countries. The general validity and reliability of the complete IBDQ has been repeatedly confirmed. However, the proposed 4 domain structure has not been successfully reproduced in multiple adaptations, including in the UK, Norway, Sweden, Greece, and Spain. This has raised concern over the validity of the 4 domain structure of the IBDQ. Aims: 1) To determine whether the IBDQ four domain structure can be supported by factor analysis using a large dataset; 2) to compare the domain structure of the IBDQ for ulcerative colitis and Crohn's disease; 3) if the 4 domain structure is not supported, to propose an alternate domain structure. Methods: Using baseline IBDQ data from the ACT and ACCENT trials (725 patients with UC and 876 patients with Crohn's), factor analysis was performed using principal component analysis with varimax rotation to identify domains. Cluster analysis of the IBDQ data was performed. Dendrograms were used to design a new domain structure. Results: Factor analysis identified 5 domains with four outlier questions (stool frequency, consistency, blood, and sleep) in Crohn's, and six domains in UC. In Crohn's, the emotional, social and systemic domains were largely intact, but the bowel domain broke into a rectal/emotional consequences domain and an abdominal discomfort domain. In UC, the emotional domain was largely intact, the social and systemic domains were merged, a rectal/emotional consequences domain was separate from an abdominal discomfort domain, a stool frequency/consistency/bleeding domain was found, and a gas/bloating/weight domain was found. We designed and tested a 5 domain (10 item) Brief IBDQ for Crohn's and a 6 domain (12 item) version for UC, which had the predicted domain structures, and correlated highly (0.94) with the complete IBDQ. Conclusions: The original 4 domain structure of the IBDQ is not valid. The four domain subscores of the IBDQ should not be used. The IBDQ in both Crohn's and UC is better described with a 5 or 6 domain structure; however, the domain content for UC and Crohn's are not identical. A 10-item Brief IBDQ maintains an identical five domain structure in UC and Crohn's, and is highly correlated with the original IBDQ. A sixth domain can be added (2 items) for a 6 domain UC version.
S1187 Colonic Pre-Inflammation in Quiescent Ulcerative Colitis Is Due to the Presence of Lamina Propria Residing Cells Jacob T. Bjerrum, Jørgen Olsen, Ole Haagen Nielsen Background: Genome-wide gene expression profiles of mucosal colonic biopsies have been employed in the pursuit of a more consistent diagnostic tool to differentiate between Crohn's disease (CD), ulcerative colitis (UC) and controls. With this approach a refinement of the diagnostics has been established and the existence of a pre-inflammatory state of UC suggested. However, as colonic biopsies hold several different cell types (e.g. colonocytes, monocytes, macrophages, and fibroblasts) it is uncertain in which cells the essential information about the pre-inflammatory state is contained. Aim: The aim was to use genome-wide gene expression profiling of mucosal colonic biopsies and isolated colonocytes from UC patients and controls in order to identify the cell types responsible for definition of the pre-inflammatory state. Methods: Five adjacent mucosal colonic biopsies were attained endoscopically from the left sided colon in active UC (n=8), quiescent UC (n=9), and in healthy controls (n=10). The colonocytes were subsequently isolated from four pooled biopsies, which mean that each of the 27 included patients contributed with two samples (colonic biopsy and colonocytes). After extraction of total RNA, genome-wide gene expression data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA) containing transcripts for approximately 38.500 human genetic sequences. Data analysis was carried out by principal component analysis (PCA) and partial least squares regression (PLSR) using the Simpca 11 software (Umetrics, Umeå, Sweden). Results: Both PCA and PLSR classification of the biopsy samples yielded a 100% separation between active UC, quiescent UC and control samples without any samples being misclassified. Oppositely, when isolated colonocytes were analysed the quiescent UC samples (n=9) were misclassified as either controls (7/9) or as active UC (2/9). The PCA and PLSR models consequently suggest that the cells residing outside the epithelium, i.e. lamina propria residing cells, define the pre-inflammatory state. Moreover, histological analysis of adjacent samples was performed in all patients and confirmed the diagnosis, hence, confirming the absence of inflammation in quiescent UC samples. Conclusion: This study demonstrates the presence of a pre-inflammatory state in quiescent UC colonic mucosa. The pre-inflammatory state reflects an altered gene expression profile of lamina propria residing cells, whereas the gene expression profile of mucosal colonocytes in quiescent UC and controls are similar. This knowledge is of outmost importance in the quest for a more consistent diagnostic tool.
S1190 The Differential Expression of MicroRNAs in Patients with Ulcerative Colitis Feng Wu, Michelle N. Zikusoka, Themistocles Dassopoulos, Theodore M. Bayless, Steven R. Brant, Shukti Chakravarti, John H. Kwon Background: Multiple global gene expression studies demonstrate that inflammatory bowel disease (IBD) is associated with an increased mRNA expression of genes involved in inflammation and fibrosis. MicroRNAs, a group of small, non-coding RNAs encoded in the human genome, are increasingly recognized as major negative regulators of gene expression in many processes, including development, cancer and inflammation. However, the expression and role of microRNAs in specific inflammatory diseases has not been investigated. Aim: To determine whether microRNAs are differentially expressed in the chronic IBD, ulcerative colitis (UC). Methods: Endoscopic pinch biopsies were obtained from the sigmoid colon of patients with active UC (15), inactive UC (15) and healthy patients undergoing screening colonoscopies (15). Additional biopsies were collected from twelve patients with microscopic colitis, infectious colitis and irritable bowel syndrome (IBS). MicroRNA microarrays were performed on small RNA fractions isolated from each biopsy. Differentially expressed microRNAs were validated by quantitative RT-PCR. Results: Our microarray data demonstrate that four microRNAs (miR-192, miR-320, miR-375 and miR-422b) are significantly decreased in sigmoid colon biopsy tissues of patients with active UC as compared to normal healthy control subjects. Twenty-two microRNAs (miR-15, miR-16, miR-20a, miR-21, miR-23a, miR-23b, miR-24, miR-26a, miR-27b, miR-29a, miR-106, miR-126, miR-150, miR-155, miR-195, miR-199a, miR-203, miR-221, let-7e, let-7f, let-7g, let-7i) are significantly increased in UC tissues, as compared to normal healthy control subjects. The pattern of expression of these microRNAs in patients with active UC is distinct from patients with inactive UC, microscopic colitis, infectious colitis and IBS. Conclusions: MicroRNAs are differentially expressed in active UC. These findings expand the possible roles of microRNAs by demonstrating the differential expression of microRNAs in a chronic inflammatory disease. Further studies may result in the development of microRNA-based therapies and diagnostic tests for IBD.
S1188 Protein Microarray Experiments for Profiling of the Autoimmune Response in Inflammatory Bowel Disease; Identification of PHLA1 Nathalie Vermeulen, Severine Vermeire, Georges Michiels, Marie Joossens, Paul J. Rutgeerts, Xavier Bossuyt Objective: Circulating antibodies are common in patients with inflammatory bowel disease (IBD) and directed against bacterial and/or self-antigens. The aim of the project was to characterize the autoantibody profile in patients with IBD and identify novel autoantigens. Methods: Sera from 10 patients with ulcerative colitis (UC), 15 with Crohn's disease (CD) and 5 healthy subjects (HC) were profiled using human protein microarrays (ProtoArray, Invitrogen). After scanning, fluorescence signals were quantified. Cut off value for positivity was calculated using M-statistics and p-values <0.05 were considered statistically significant. For validation, standard 1D-gelectrophoresis and western blotting was performed with sera from UC patients, CD patients, non-IBD gastrointestinal and healthy controls. Results: The protein microarray experiments revealed 75 proteins to which patients with IBD had a significantly higher reactivity than the healthy controls and 88 proteins to which the healthy subjects had a higher reactivity than the IBD patients. One of the identified autoantigens in IBD was pleckstrin homology-like domain, family A, member 1 (Phla1). Phla1 is described as a pro-apoptotic protein involved in cell death mechanisms in different cell types. The protein microarray revealed antibodies against Phla1 in 5 of 10 (50.0%) UC patients and in 9 of 15 (60.0%) CD patients, compared to 0 of 5 healthy subjects. In the validation experiment on a second cohort of patients and controls, 27 of 63 (42.8%) UC patients and 33 of 66 (50.0%) CD patients had anti-Phla1 antibodies, in contrast to 19 of 66 (28.7%) healthy and 22 of 66 (33.3%) non-IBD gastrointestinal controls. (p<0.05 ; IBD versus controls) Conclusion: Our data confirm the protein microarray as a novel technology to identify serological antibodies. Using this technology, we showed that Phla1 is an autoantigen in IBD. Independent cohorts are needed to confirm these first results.
S1191 Use of Methodological Standards in IBD Diagnosis Research Thomas A. Ullman, Arvind J. Trindade, Jianlin Xie, Yevgenia Pashinsky Introduction: The introduction of new diagnostic tests in IBD remains an unmet need due to the expensive and invasive nature of usual evaluations in suspected IBD. Doubt about the utility of new diagnostic tests exists despite publication of seemingly promising results. Previous investigations have noted a lack of adherence to methodological standards in general diagnostic test research. Aim: To determine the frequency with which accepted methodological standards are met in IBD diagnostic test research studies published in high impact GI journals. Methods: A Medline search of 3 prominent clinical GI journals (Gastroenterology, Gut, and The American Journal of Gastroenterology) was performed using ‘Crohn's,' ‘colitis,' and ‘IBD' as keywords. Abstracts were then reviewed and studies of diagnosis were included. Studies limited to prognosis, activity, and evaluation of therapeutics were excluded. Qualifying articles were then evaluated for their description of the following previously published standards: 1) specification of spectrum of patients; 2) report of test performance in clinical subgroups; 3) avoidance of verification bias; 4) avoidance of review bias; 5) reporting of precision of test accuracy with confidence intervals surrounding estimates; 6) reporting of indeterminate results; 7) specification of test reproducibility. Results: 29 articles met the stated inclusion criteria and were reviewed by 2 independent reviewers. No studies met all 7 or even 6 of the 7 criteria. 83% met 4 or fewer standards. The distribution of how many studies met fewer than 6 standards is described in Table 1. Table 2 reports the percentage of studies meeting each individual standard. Conclusions: Published reports of new diagnostic tests in IBD fall below a number of accepted standards for evaluation of their scientific accuracy. Improved adherence to methodological standards might allow for more widespread acceptance of diagnostic tests following their publication. Table 1: Number of Standards Met
S1189 Renovating the Domain Structure of the Inflammatory Bowel Disease Questionnaire Dahlia Awais, Peter D. Higgins Background: The Inflammatory Bowel Disease Questionnaire (IBDQ) is a widely used measure of disease-specific health-related quality of life. It includes 32 items designed to be divided into 4 domains: systemic (5 items), bowel (10 items), emotional (12 items), and social (5
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were included. Mean sensitivities and specificities of 80% (95% CI, 77-82%) and 76% (95% CI, 72-79%), respectively, were calculated for the diagnosis of IBD (taken together UC and CD). When a subanalysis was performed depending on the IBD type, a slightly higher accuracy was calculated for CD [sensitivity 83% (95% CI, 80-87%), specificity 85% (95% CI, 81-89%)] than for UC [sensitivity 72% (95% CI, 66-79%), specificity 74% (95% CI, 6780%)]. CONCLUSION: Faecal calprotectin has a good diagnostic precision for distinguishing organic from functional intestinal disease in symptomatic patients, and also for separating IBD from non-IBD diagnoses, providing better results than those previously reported for the classically recommended systemic inflammatory markers such as erythrocyte sedimentation rate, C-reactive protein, anti-neutrophil cytoplasmic antibodies, and anti-Saccharomyces cerevisiae antibodies.
against several standard measures for assessing IBD disease activity. Methods: Participants enrolled in the Manitoba IBD Cohort Study, a population based longitudinal cohort (n= 356) were assessed periodically by survey, clinical interview, and blood sample. The MI is based on patient report of symptom persistence for the previous 6 mos, using a 6-level response format: active (a) constantly (daily symptoms) (b) often (symptoms most days) (c) sometimes (symptoms some days e.g.,1-2 days/wk) (d) occasionally (symptoms 1-2 days/ mo) (e) rarely (symptoms few days past 6 mos) and (f) inactive (absence of symptoms). Disease was defined as active if respondents answered any of a-d and inactive if answered e or f. Physical symptoms (GI and systemic), clinical disease indices (HB, PT), IBD medication use, daily productivity loss, and psychological functioning (e.g. health anxiety, distress) were all assessed. Biological markers included hemoglobin and CRP. Sensitivity analyses and discriminant function analyses were used to validate the MI, using data from baseline (i.e., month 0), 12 and 24 months points. Results: The MI had good sensitivity when compared to HB, PT, and IBDQ scores (range 0.84 to 0.90). It correlated well with all these measures (0.39 to 0.62, p<0.01) and individual symptom measures (e.g., abdominal, joint, diarrhea, bowel movement urgency; 0.27 to 0.62, p < 0.01), despite the different assessment time frames. In separate discriminant analyses for CD and UC, common discriminating variables included clinical indices (HB for CD, PT for UC), IBDQ subscales of bowel and systemic symptoms, and symptom ratings (e.g., abdominal and joint pain, tiredness, diarrhea). Conclusions: A single-item patient-defined disease activity measure, the Manitoba Index, showed a high degree of sensitivity for classifying individuals with active disease compared to existing disease activity measures, and strong convergent validity with expected proxy measures of disease. These relationships were consistent across a two-year period. Thus, the MI shows promise as a valid brief tool for measuring disease activity.
AGA Abstracts
Serum Protein Signatures Determined By Mass Spectrometry (SELDI-ToF) Accurately Distinguishes Crohn's Disease (CD) from Ulcerative Colitis (UC) Venkataraman Subramanian, Devika Subramanian, Richard C. Pollok Background: Accurate diagnosis of UC or CD is essential to guide patient management. Current tests are invasive and carry risks. A meta analysis suggested that diagnosis of CD by ASCA (Anti-Saccharomyces cerevisiae antibodies) has a sensitivity of 54.6%. pANCA positivity has a sensitivity of 55.3% in the diagnosis of UC. Aim: We aimed to detect novel serum biomarkers by protein fingerprinting using surface-enhanced laser desorption ionization time-of-flight (SELDI-ToF) mass spectrometry to differentiate CD from UC. Methods: Serum was collected prospectively from 62 patients with UC and 48 with CD and stored at -80 0C. Samples were applied to CM10 protein chip arrays and time-of- flight spectra were generated using a PBS-II mass spectrometer (Ciphergen, Freemont, CA, USA). The mass spectrometry data was extracted and preprocessed prior to analysis. The spectra were analyzed using support vector machine (SVM) analysis utilizing recursive feature elimination (RFE) techniques with radial basis function and 5-fold cross validation. We then developed a boolean pattern classifier on the thresholded peaks (based on median value) detected by SVM. The identity of the selected peaks was confirmed using immunodepletion methods. ASCA and ANCA were determined by ELISA and indirect immunofluorescence respectively. Results:12 key peaks were identified using SVM with RFE (RBF kernel width of 0.00075 and C of 100) in a five-fold cross validation setting. These peaks were then thresholded to being above or below the median value. We clustered the data using kmeans with two clusters. Using 8 of these thresholded peaks two well separated clusters were identified. A boolean pattern classifier was developed on the 12 thresholded peaks. The specificity and sensitivity of this boolean classifier was 96% for the first cluster and 94% for the second cluster. ASCA had a sensitivity of 70.8% in detecting CD and overall accuracy of 80.9%. p ANCA had a sensitivity of 64.5% for detecting UC. The peaks identified as important had m/z values of 2.7, 4.1, 4.2, 5.6, 6.4, 6.8, 7.8, 7.9, 8.6, 8.7, 9.3, 1.6 kDa. These were confirmed to be fragments of inter alpha trypsin inhibitor 4 (2.7 and 4.2), Apolipoprotein C1 and probably its SPA adduct (6.4 and 6.8), Platelet activated factor 4 variants (7.8 and 7.9). Conclusions: Using protein signatures of patients with UC and CD we have developed a classification model which is accurate, sensitive and more informative than currently available serological tests. There seem to be distinct proteome based clusters of CD and UC patients. The clinical, pathogenic and prognostic significance of these clusters needs to be further characterized
S1185 Faecal Calprotectin's Utility in the Prediction of Inflammatory Bowel Disease (IBD) Relapses Javier P. Gisbert, Fernando Bermejo, Jl Perez-Calle, Carlos Taxonera, Isabel Vera, Yago Gonzalez-Lama, Alicia Algaba, P. Lopez-Serrano, N. López-Palacios, Marta Calvo, Adrian G. McNicholl, Ja Carneros, M. Velasco, José Maté-Jiménez BACKGROUND: IBD activity flares are usually unpredictable. If we had an available and reliable marker to estimate the risk of relapse, we could prescribe early medication to prevent or treat the relapse. Inflammation is a continuous process, so a biological marker able to estimate the intensity of inflammation might provide us a pre-symptomatic quantitative measure of the risk of imminent clinical relapse AIM: To determine faecal calprotectin's utility in the prediction of IBD relapses. METHODS: Prospective multicenter study including Crohn's Disease (CD) and Ulcerative Colitis (UC) patients who had been in clinical remission (based on Truelove and CDAI scores) during the preceding 6 months. Disease and treatment data collection and calprotectin's basal levels measurements were done during screening. Follow up period was 12 months in those patients showing no relapse and until activity flare in relapsed patients. RESULTS: Seventy-seven patients, 44 with CD and 35 with UC, have completed the study so far. Treatments: oral 5-ASA (62%), topical 5-ASA (7%), AZA/ MP (39%), and infliximab (4%). 25% of the patients had suffered a flare the year preceding the study. Seventeen patients (22%) relapsed during follow up (after an average of 20 weeks). Average levels of faecal calprotectin were 179±157 µg/g (ranging from 9 to 851 µg/g). Calprotectin concentrations in those patients who suffered a relapse were higher than in those who maintained remission (255±143 vs. 157±154 µg/g; p<0.05). This difference was only demonstrated in CD patients (317 vs. 162 µg/g; p<0.05) but not in UC patients (200 vs. 151 µg/g). The percentage of IBD patients relapsing was significantly different between those having calprotectin concentrations <100 µg/g and those with higher concentrations (10% vs 30% relapses respectively, p<0.05). These differences were even clearer in CD patients (6% for calprotectin <100 µg/g and 28% for >100 µg/g). Faecal calprotectin's (>100 µg/g) sensitivity and specificity to predict relapses in CD were 82% and 47% (positive and negative predictive values of 30% and 90% respectively). The area under the ROC curve to predict IBD relapse using calprotectin determination was 0.72, and considering only CD data was 0.81%. The best global cut-off point was 166 µg/g (sensitivity 82%; specificity 67%), but in CD patients it was 170 µg/g (sensitivity 88%; specificity 68%). Considering only the prediction of relapse during the first 6 months of follow up, results were better (area under the ROC curve in CD was 0.91) CONCLUSIONS: High calprotectin concentrations in faeces are associated with a higher risk of clinical relapse in IBD patients.
S1183 A Combination of Serum Adiponectin, Pepsinogen I and Anti-Saccharomyces Cerevisiae Antibody (ASCA) Levels Can Accurately Distinguish Patients with Crohn's Disease Involving the Small Bowel from Colonic Crohn's Disease Venkataraman Subramanian, Geetha Balarajah, Richard C. Pollok Background: Serum Pepsinogen I (PG I) level reliably correlates with the number of chief cells in the gastric corpus mucosa. Gastric heterotopia of the small intestine in Crohn's disease (CD) has been well described and PG I & II noted on immunohistochemisty in the metaplastic pyloric and oxyntic glands in the ileum. Adiponectin levels are known to be elevated in patients with CD with mesenteric adipose tissue hypertrophy and not in patients with UC. ASCA has been associated with more proximal involvement rather than colonic CD. Diagnosis of small intestinal involvement in patients with CD has major implications on patient management. Aim: The aim of this pilot study was to use serum PG I , serum adiponectin and IgA and IgG ASCA levels to develop an algorithm that distinguishes patients with colonic CD from those with CD involving the small intestine. Methods: Serum was prospectively collected and stored at -80 0C from patients with histologically proven CD. PG I levels (Biohit plc, Finland) and Adiponectin levels (B-bridge International, USA), IgA and IgG ASCA (Aesku Lab, Germany) were determined in duplicate by ELISA. Patients were grouped into those with small intestinal involvement and those with colonic CD. Differences between groups were tested using Mann-Whitney U test of significance. Results: Mean PG I levels in the small bowel CD group (n=18) was 97.89 ± 33.07 µg/l. Mean PG I levels in the colonic CD group (n=14) was 83.25 ± 29.74 µg/l (p=0.08). Mean Adiponectin levels in the small bowel CD group was 3.89 ± 1.87 ng/ml and in the colonic CD group was 2.65 ± 1.50 ng/ml (p=0.02). Area under the Receiver operating curve for Adiponectin was 0.74 and for Serum PG I was 0.68. A cut off value of more than 1.58 ng/ml for serum Adiponectin and more than 78 µg/l for serum PG I with either a positive IGA or IgG ASCA had a sensitivity of 88.9 %, specificity of 85.7 % and overall accuracy of 87.5% in detecting small bowel involvement in CD. Only 2 patients each with colonic and small intestinal CD were misclassified. Conclusion: Adiponectin levels are significantly higher in CD patients with small bowel disease. This could be because patients with colonic CD alone are less likely to have mesenteric fat hypertrophy. Serum PG1 levels showed a trend towards significance among patients with small intestinal CD. This could reflect both gastric heterotropia in the small intestine as well as inflammation due to CD in the stomach. Further studies are required to confirm these findings. A combination of Adiponectin , PG I levels and ASCA serology could help in non-invasive diagnosis of small bowel involvement in patients with CD.
S1186 The Diagnostic Accuracy of Faecal Calprotectin in Inflammatory Bowel Disease (IBD): A Systematic Review Javier P. Gisbert, Adrian G. McNicholl BACKGROUND: The presence of calprotectin in faeces is directly proportional to neutrophil migration towards the intestinal tract. The potential strength of faecal calprotectin assessment is that it is a direct measure of mucosal inflammatory activity, and levels in the stool seem to be unaffected by a variety of non-intestinal conditions, which may result in an elevation of the systemic inflammatory markers. AIM: To review the diagnostic accuracy of faecal calprotectin determination 1) to distinguish organic from functional disorders in symptomatic patients, and 2) for the diagnosis of IBD. METHODS: Bibliographical searches were performed in MEDLINE up to 2007 looking for the following words (all fields): (“inflammatory bowel disease” OR “Crohn's disease” OR “ulcerative colitis”) AND (calprotectin OR faecal marker). The mean sensitivity and specificity of faecal determination was calculated and expressed as weighted mean (and corresponding 95% confidence interval; 95% CI) to make due allowance for the number of patients included in each study. Subanalyses were performed depending on the IBD type: ulcerative colitis (UC) vs. Crohn's disease (CD). RESULTS: 1) Thirteen studies, including a total of 2475 patients, measured faecal calprotectin concentrations aiming to distinguish organic from functional intestinal disease in symptomatic patients. Mean sensitivities and specificities of 83% (95% CI, 81-84%) and 84% (95% CI, 82-85%), respectively, were calculated. 2) Fourteen studies, including a total of 754 patients, evaluated the diagnostic precision of faecal calprotectin for the diagnosis of IBD. In most of these studies, symptomatic patients with IBD or irritable bowel syndrome, in addition to controls,
S1184 The Manitoba Index: Psychometric Evidence for a New and Simple Indicator of IBD Activity Ian Clara, Lisa M. Lix, John Walker, Lesley A. Graff, Norine Miller, Linda Rogala, Patricia Rawsthorne, Charles N. Bernstein Many indicators of disease activity for IBD can be time consuming or difficult to administer, as they require invasive procedures (endoscopy), clinical evaluation (CDAI), or completion of multiple-item scales (IBDQ). Simpler indices such as the Harvey-Bradshaw (HB) and Powell-Tuck (PT) provide only a brief cross-sectional account of disease activity at the time of a clinic visit and do not adequately characterize disease activity over an extended period. A single-item indicator of disease activity, the Manitoba Index (MI) is proposed, and compared
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items). Originally developed in a Canadian cohort, it has since been translated and used in both English and non-English speaking countries. The general validity and reliability of the complete IBDQ has been repeatedly confirmed. However, the proposed 4 domain structure has not been successfully reproduced in multiple adaptations, including in the UK, Norway, Sweden, Greece, and Spain. This has raised concern over the validity of the 4 domain structure of the IBDQ. Aims: 1) To determine whether the IBDQ four domain structure can be supported by factor analysis using a large dataset; 2) to compare the domain structure of the IBDQ for ulcerative colitis and Crohn's disease; 3) if the 4 domain structure is not supported, to propose an alternate domain structure. Methods: Using baseline IBDQ data from the ACT and ACCENT trials (725 patients with UC and 876 patients with Crohn's), factor analysis was performed using principal component analysis with varimax rotation to identify domains. Cluster analysis of the IBDQ data was performed. Dendrograms were used to design a new domain structure. Results: Factor analysis identified 5 domains with four outlier questions (stool frequency, consistency, blood, and sleep) in Crohn's, and six domains in UC. In Crohn's, the emotional, social and systemic domains were largely intact, but the bowel domain broke into a rectal/emotional consequences domain and an abdominal discomfort domain. In UC, the emotional domain was largely intact, the social and systemic domains were merged, a rectal/emotional consequences domain was separate from an abdominal discomfort domain, a stool frequency/consistency/bleeding domain was found, and a gas/bloating/weight domain was found. We designed and tested a 5 domain (10 item) Brief IBDQ for Crohn's and a 6 domain (12 item) version for UC, which had the predicted domain structures, and correlated highly (0.94) with the complete IBDQ. Conclusions: The original 4 domain structure of the IBDQ is not valid. The four domain subscores of the IBDQ should not be used. The IBDQ in both Crohn's and UC is better described with a 5 or 6 domain structure; however, the domain content for UC and Crohn's are not identical. A 10-item Brief IBDQ maintains an identical five domain structure in UC and Crohn's, and is highly correlated with the original IBDQ. A sixth domain can be added (2 items) for a 6 domain UC version.
S1187 Colonic Pre-Inflammation in Quiescent Ulcerative Colitis Is Due to the Presence of Lamina Propria Residing Cells Jacob T. Bjerrum, Jørgen Olsen, Ole Haagen Nielsen Background: Genome-wide gene expression profiles of mucosal colonic biopsies have been employed in the pursuit of a more consistent diagnostic tool to differentiate between Crohn's disease (CD), ulcerative colitis (UC) and controls. With this approach a refinement of the diagnostics has been established and the existence of a pre-inflammatory state of UC suggested. However, as colonic biopsies hold several different cell types (e.g. colonocytes, monocytes, macrophages, and fibroblasts) it is uncertain in which cells the essential information about the pre-inflammatory state is contained. Aim: The aim was to use genome-wide gene expression profiling of mucosal colonic biopsies and isolated colonocytes from UC patients and controls in order to identify the cell types responsible for definition of the pre-inflammatory state. Methods: Five adjacent mucosal colonic biopsies were attained endoscopically from the left sided colon in active UC (n=8), quiescent UC (n=9), and in healthy controls (n=10). The colonocytes were subsequently isolated from four pooled biopsies, which mean that each of the 27 included patients contributed with two samples (colonic biopsy and colonocytes). After extraction of total RNA, genome-wide gene expression data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA) containing transcripts for approximately 38.500 human genetic sequences. Data analysis was carried out by principal component analysis (PCA) and partial least squares regression (PLSR) using the Simpca 11 software (Umetrics, Umeå, Sweden). Results: Both PCA and PLSR classification of the biopsy samples yielded a 100% separation between active UC, quiescent UC and control samples without any samples being misclassified. Oppositely, when isolated colonocytes were analysed the quiescent UC samples (n=9) were misclassified as either controls (7/9) or as active UC (2/9). The PCA and PLSR models consequently suggest that the cells residing outside the epithelium, i.e. lamina propria residing cells, define the pre-inflammatory state. Moreover, histological analysis of adjacent samples was performed in all patients and confirmed the diagnosis, hence, confirming the absence of inflammation in quiescent UC samples. Conclusion: This study demonstrates the presence of a pre-inflammatory state in quiescent UC colonic mucosa. The pre-inflammatory state reflects an altered gene expression profile of lamina propria residing cells, whereas the gene expression profile of mucosal colonocytes in quiescent UC and controls are similar. This knowledge is of outmost importance in the quest for a more consistent diagnostic tool.
S1190 The Differential Expression of MicroRNAs in Patients with Ulcerative Colitis Feng Wu, Michelle N. Zikusoka, Themistocles Dassopoulos, Theodore M. Bayless, Steven R. Brant, Shukti Chakravarti, John H. Kwon Background: Multiple global gene expression studies demonstrate that inflammatory bowel disease (IBD) is associated with an increased mRNA expression of genes involved in inflammation and fibrosis. MicroRNAs, a group of small, non-coding RNAs encoded in the human genome, are increasingly recognized as major negative regulators of gene expression in many processes, including development, cancer and inflammation. However, the expression and role of microRNAs in specific inflammatory diseases has not been investigated. Aim: To determine whether microRNAs are differentially expressed in the chronic IBD, ulcerative colitis (UC). Methods: Endoscopic pinch biopsies were obtained from the sigmoid colon of patients with active UC (15), inactive UC (15) and healthy patients undergoing screening colonoscopies (15). Additional biopsies were collected from twelve patients with microscopic colitis, infectious colitis and irritable bowel syndrome (IBS). MicroRNA microarrays were performed on small RNA fractions isolated from each biopsy. Differentially expressed microRNAs were validated by quantitative RT-PCR. Results: Our microarray data demonstrate that four microRNAs (miR-192, miR-320, miR-375 and miR-422b) are significantly decreased in sigmoid colon biopsy tissues of patients with active UC as compared to normal healthy control subjects. Twenty-two microRNAs (miR-15, miR-16, miR-20a, miR-21, miR-23a, miR-23b, miR-24, miR-26a, miR-27b, miR-29a, miR-106, miR-126, miR-150, miR-155, miR-195, miR-199a, miR-203, miR-221, let-7e, let-7f, let-7g, let-7i) are significantly increased in UC tissues, as compared to normal healthy control subjects. The pattern of expression of these microRNAs in patients with active UC is distinct from patients with inactive UC, microscopic colitis, infectious colitis and IBS. Conclusions: MicroRNAs are differentially expressed in active UC. These findings expand the possible roles of microRNAs by demonstrating the differential expression of microRNAs in a chronic inflammatory disease. Further studies may result in the development of microRNA-based therapies and diagnostic tests for IBD.
S1188 Protein Microarray Experiments for Profiling of the Autoimmune Response in Inflammatory Bowel Disease; Identification of PHLA1 Nathalie Vermeulen, Severine Vermeire, Georges Michiels, Marie Joossens, Paul J. Rutgeerts, Xavier Bossuyt Objective: Circulating antibodies are common in patients with inflammatory bowel disease (IBD) and directed against bacterial and/or self-antigens. The aim of the project was to characterize the autoantibody profile in patients with IBD and identify novel autoantigens. Methods: Sera from 10 patients with ulcerative colitis (UC), 15 with Crohn's disease (CD) and 5 healthy subjects (HC) were profiled using human protein microarrays (ProtoArray, Invitrogen). After scanning, fluorescence signals were quantified. Cut off value for positivity was calculated using M-statistics and p-values <0.05 were considered statistically significant. For validation, standard 1D-gelectrophoresis and western blotting was performed with sera from UC patients, CD patients, non-IBD gastrointestinal and healthy controls. Results: The protein microarray experiments revealed 75 proteins to which patients with IBD had a significantly higher reactivity than the healthy controls and 88 proteins to which the healthy subjects had a higher reactivity than the IBD patients. One of the identified autoantigens in IBD was pleckstrin homology-like domain, family A, member 1 (Phla1). Phla1 is described as a pro-apoptotic protein involved in cell death mechanisms in different cell types. The protein microarray revealed antibodies against Phla1 in 5 of 10 (50.0%) UC patients and in 9 of 15 (60.0%) CD patients, compared to 0 of 5 healthy subjects. In the validation experiment on a second cohort of patients and controls, 27 of 63 (42.8%) UC patients and 33 of 66 (50.0%) CD patients had anti-Phla1 antibodies, in contrast to 19 of 66 (28.7%) healthy and 22 of 66 (33.3%) non-IBD gastrointestinal controls. (p<0.05 ; IBD versus controls) Conclusion: Our data confirm the protein microarray as a novel technology to identify serological antibodies. Using this technology, we showed that Phla1 is an autoantigen in IBD. Independent cohorts are needed to confirm these first results.
S1191 Use of Methodological Standards in IBD Diagnosis Research Thomas A. Ullman, Arvind J. Trindade, Jianlin Xie, Yevgenia Pashinsky Introduction: The introduction of new diagnostic tests in IBD remains an unmet need due to the expensive and invasive nature of usual evaluations in suspected IBD. Doubt about the utility of new diagnostic tests exists despite publication of seemingly promising results. Previous investigations have noted a lack of adherence to methodological standards in general diagnostic test research. Aim: To determine the frequency with which accepted methodological standards are met in IBD diagnostic test research studies published in high impact GI journals. Methods: A Medline search of 3 prominent clinical GI journals (Gastroenterology, Gut, and The American Journal of Gastroenterology) was performed using ‘Crohn's,' ‘colitis,' and ‘IBD' as keywords. Abstracts were then reviewed and studies of diagnosis were included. Studies limited to prognosis, activity, and evaluation of therapeutics were excluded. Qualifying articles were then evaluated for their description of the following previously published standards: 1) specification of spectrum of patients; 2) report of test performance in clinical subgroups; 3) avoidance of verification bias; 4) avoidance of review bias; 5) reporting of precision of test accuracy with confidence intervals surrounding estimates; 6) reporting of indeterminate results; 7) specification of test reproducibility. Results: 29 articles met the stated inclusion criteria and were reviewed by 2 independent reviewers. No studies met all 7 or even 6 of the 7 criteria. 83% met 4 or fewer standards. The distribution of how many studies met fewer than 6 standards is described in Table 1. Table 2 reports the percentage of studies meeting each individual standard. Conclusions: Published reports of new diagnostic tests in IBD fall below a number of accepted standards for evaluation of their scientific accuracy. Improved adherence to methodological standards might allow for more widespread acceptance of diagnostic tests following their publication. Table 1: Number of Standards Met
S1189 Renovating the Domain Structure of the Inflammatory Bowel Disease Questionnaire Dahlia Awais, Peter D. Higgins Background: The Inflammatory Bowel Disease Questionnaire (IBDQ) is a widely used measure of disease-specific health-related quality of life. It includes 32 items designed to be divided into 4 domains: systemic (5 items), bowel (10 items), emotional (12 items), and social (5
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were included. Mean sensitivities and specificities of 80% (95% CI, 77-82%) and 76% (95% CI, 72-79%), respectively, were calculated for the diagnosis of IBD (taken together UC and CD). When a subanalysis was performed depending on the IBD type, a slightly higher accuracy was calculated for CD [sensitivity 83% (95% CI, 80-87%), specificity 85% (95% CI, 81-89%)] than for UC [sensitivity 72% (95% CI, 66-79%), specificity 74% (95% CI, 6780%)]. CONCLUSION: Faecal calprotectin has a good diagnostic precision for distinguishing organic from functional intestinal disease in symptomatic patients, and also for separating IBD from non-IBD diagnoses, providing better results than those previously reported for the classically recommended systemic inflammatory markers such as erythrocyte sedimentation rate, C-reactive protein, anti-neutrophil cytoplasmic antibodies, and anti-Saccharomyces cerevisiae antibodies.