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P-152 YI Detection and Measurement of Changes in mRNA Colonic Expression of Inflammatory Genes in Biopsies from Patients with Ulcerative Colitis

BACKGROUND: The ability to measure mRNA expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis is critical for proof of concept and mechanism of action studies. Little published data exist on how many biopsies per patient and how many patients need to be studied to provide accurate, precise, and reproducible results. We performed a cross sectional study in patients with ulcerative colitis with both inactive and active disease to systematically validate methods for detecting and measuring transcriptional changes in inflammatory genes in colonic biopsies from regions where inflammation is continuous and homogenous. METHODS: A total of 12 colonic biopsies were obtained from patients with ulcerative colitis during colonoscopy. Three biopsies were obtained from each of the: 1) ascending; 2) transverse; 3) descending and 4) recto-sigmoid colon. Using quantitative PCR, mRNA expression was assessed for inflammatory genes that have been associated with ulcerative colitis: Interleukin (IL)-1&bgr;, IL-6, IL-8, tumor necrosis factor-&agr; (TNF), IP-10 (interferon gamma-induced protein), C-X-C motif chemokine 13(CXCL13), matrix metalloproteinase-3 (MMP-3), and S100-A8 (calcium binding protein). The Mayo Score and endoscopic subscore of the Mayo score for each colonic segment were recorded for each participant. A linear mixed model utilizing repeat measures was used to calculate the power to detect a reduction in gene expression corresponding to concentrations in active as compared to inactive ulcerative colitis. RESULTS: Colonic biopsies were obtained from 13 patients with ulcerative colitis: 6 patients with inactive disease and 7 patients with active disease. Biopsies yielded sufficient RNA sufficient to measure approximately 260 inflammatory markers. Mayo Endoscopic subscores for each colonic segment correlated with inflammatory gene expression. The mean coefficient of variation for 3 colonic biopsies in a colonic segment ranged from 26% to 83% depending on which inflammatory gene was measured. Obtaining 2 rectal biopsies for TNF-&agr; reduced sampling error to acceptable levels with a standard error of less than 25%. Overall variability was assessed to calculate the power of sampling in similar cohorts to detect changes in inflammatory gene expression corresponding to active versus inactive ulcerative colitis. Depending on the specific inflammatory marker, 3 to 6 biopsies from 6 patients would obtain a power of 80% with alpha of 0.05. CONCLUSIONS: Early phase proof of concept and mechanism of action studies that measure mRNA expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis are feasible. Three to 6 biopsies obtained from 6 patients provide sufficient power for such studies. Table. No title available.