Validated gene expression biomarker analysis for biopsy‐based clinical trials in ulcerative colitis

Abstract

Summary Background Accurate and reproducible measurement of expression of pro-inflammatory cytokines in colonic biopsies from patients with ulcerative colitis (UC) is essential for proof-of-concept and mechanism-of-action studies. Few studies have rigorously established the number of biopsies required for accurate and reproducible biomarker measurements. Aim To validate methods for measuring changes in gene expression in colonic biopsy samples. Methods Twelve colonic biopsies were obtained from each of 6 healthy controls, 6 patients with inactive UC, and 7 patients with active UC. Mayo endoscopic scores were used as a clinical reference standard. Quantitative PCR was used to assess mRNA expression of eight known inflammatory genes. The power to detect a reduction in gene expression in active versus inactive UC was calculated using a linear mixed effect model. Results mRNA analysis of colonic biopsies is a sensitive and feasible approach for measuring inflammatory gene expression in colonic biopsies. Inflammatory biomarkers correlate with Mayo endoscopic subscores for each colonic region. For most genes, 3 rectal biopsies from 2–4 patients are required to detect changes in gene expression corresponding to active versus inactive UC to achieve a power of 80% with an alpha of 0.05. Conclusions Our data suggest that systematic measurement of inflammatory biomarkers at the mRNA level can be a valuable tool for hypothesis testing and assessment of clinical activity and response to therapy in ulcerative colitis.

Validation of Gene Expression Biomarker Analysis for Biopsy-based Clinical Trials in Crohnʼs Disease

“…In effect, our results effectively establish the number of intestinal biopsies required in order to provide sampling reliability comparable to a single biopsy. We previously showed 40 in UC that 2 – 5 intestinal biopsies are generally needed to reduce standard error to <25%, and we now extend this finding to CD.…”

“…40 Two to 4 rectal biopsies in UC, as compared to 2–5 intestinal or colonic biopsies in CD, were needed to reduce sampling error to <25%. In the study of patients with UC, the mixed model for power calculations used a fixed number of colonic biopsies to determine the number of patients required to obtain a power of 80%, and 3 rectal biopsies before and after treatment from 2 patients would yield power of at least 80% or greater.…”

“…In the study of patients with UC, the mixed model for power calculations used a fixed number of colonic biopsies to determine the number of patients required to obtain a power of 80%, and 3 rectal biopsies before and after treatment from 2 patients would yield power of at least 80% or greater. 40 When we applied this model using three intestinal or colonic biopsies before and after treatment to gene expression in CD, we found that 2 patients with CD were also needed to obtain a power of 80% or greater (Table S5). Together these two sets of data suggest that similar strategies may be applied to UC and CD to detect statistically significant differences between active and inactive disease.…”

“…A similar approach for proof-of-concept and mechanistic clinical trials using serial synovial biopsies has been successfully in rheumatoid arthritis, 41, 42 and an analogous biopsy-based study from patients with UC provided a framework for performing serial biopsy studies. 40 Further validity for our approach comes from the association between SES-CD and inflammatory gene expression, indicating that inflammatory biomarkers correlate with endoscopic disease activity. Ultimately, using the variability from the intestinal sampling, we determined the minimum number of patients and the number of biopsies prior to and following treatment needed to power a study to detect a reduction in gene expression corresponding to a transition from active to inactive disease.…”

Characterizing the inflammatory response in esophageal mucosal biopsies in children with eosinophilic esophagitis

“…Prior to our study, cytokine/chemokine expression in EoE had been mainly based on mRNA abundance in whole biopsy extracts and immunohistochemical staining for pre-defined targets. Although measuring inflammatory biomarkers at the mRNA level is reliable in making the diagnoses and monitoring disease activity, 17 knowing protein levels will add to the functional role immune cells have in EoE. We sought to approach biomarker mining in EoE from a different perspective by utilizing standard immunohistochemical and ex vivo techniques including flow cytometry and biopsy explant cultures.…”