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

Abstract

Background The ability to measure the expression of pro-inflammatory cytokines from intestinal biopsies in patients with Crohn’s disease in an accurate and reproducible way is critical for proof-of-concept and mechanism-of-action trials; however, the number of biopsies from a segment of the ileum or colon required to yield reproducible results has not been rigorously evaluated. We examined intestinal biopsies from patients with Crohn’s disease to validate methods for detecting changes in inflammatory gene expression. Methods To evaluate the reproducibility of gene expression measurements, intestinal biopsies were obtained from designated segmentsfrom6 healthy controls, 6 patients with active Crohn’s disease, and 6 patients with inactive Crohn’s disease. Disease activity was based on the simple endoscopic score for Crohn’s disease (SES-CD). Expression of 7 pro-inflammatory genes was measured from each biopsy using quantitative PCR. Using a linear mixed effects model, the power to detect transcriptional changes corresponding to active and inactive Crohn’s disease was calculated. Results Total SES-CD score corresponds with expression of most inflammatory biomarkers. For most genes, 2 – 5biopsies are needed to reduce sampling error to <25% for most genes. To measure changes in mRNA expression corresponding to active versus inactive Crohn’s disease, one to two intestinal biopsies from 3 patients before and after treatment are needed to yield power of at least 80%. Conclusion Measuring pro-inflammatory gene expression from mucosal biopsies from patients with Crohn’s disease is practicable and provides objective biomarkers that can be utilized in proof-of-concept and mechanism-of-action trials to assess response to therapy.

Evaluation of optimal biopsy location for assessment of histological activity, transcriptomic and immunohistochemical analyses in patients with active Crohn’s disease

“…Analysis of proinflammatory gene expression may become a powerful objective tool in clinical practice and trials of patients with Crohn's disease. In addition to our work, this notion is supported by Boland et al's work, who confirmed that measuring proinflammatory gene expression from mucosal biopsies is a feasible and reproducible method to measure site‐specific inflammation in Crohn's disease, and that expression of some inflammatory genes correlates with endoscopic disease activity . Decreased mucosal expression of proinflammatory genes has been observed with successful treatment with infliximab and adalimumab, and normalisation of proinflammatory gene expression has been shown to predict long‐term remission with successful treatment with TNF antagonists .…”

“…Decreased mucosal expression of proinflammatory genes has been observed with successful treatment with infliximab and adalimumab, and normalisation of proinflammatory gene expression has been shown to predict long‐term remission with successful treatment with TNF antagonists . Boland et al have also suggested that it may be possible to analyse gene expression in biopsies sampled from the distal colon as a surrogate proxy for more proximal disease activity, potentially avoiding complete colonoscopy, especially in patients with Crohn's colitis and rectal inflammation …”

“…First strand complementary DNA was synthesised from mRNA using RevertAid reverse transcriptase (Fermentas; Thermo Fisher Scientific), according to the manufacturer's instructions. The expression of five genes previously shown to be significantly up‐regulated in colonic and/or ileal mucosa of Crohn's disease patients in the setting of acute inflammation and known to be implicated in the pathogenesis of Crohn's disease was analysed, including interleukin (IL)‐6, IL‐8, and IL‐23 (p19 and p40 subunits), platelet endothelial cell adhesion molecule (PECAM)‐1 (or CD31) and S100A9, a subunit of calprotectin . Expression levels of mRNA for the genes of interest as well as reference genes (β‐actin and cyclophilin) were determined in duplicate by quantitative real‐time reverse transcriptase polymerase chain reaction using SensiFAST SYBR ® No‐ROX Kit (GC‐Biotech, Alphen a/d Rijn, The Netherlands) according to the manufacturer's instructions on a LightCycler instrument (Roche, Alphen a/d Rijn, The Netherlands) and gene‐specific primers (See Table S1 for primers used).…”

“…However, to our knowledge no data from empiric studies are available to justify this guidance. Similarly, no standards exist to guide sampling for gene expression or immunohistochemical analyses in Crohn's disease. Accordingly, we conducted a prospective study in patients with Crohn's disease‐related ileocolonic ulcers to assess histological disease activity, messenger ribonucleic acid (mRNA) expression levels of proinflammatory genes and number of lamina propria inflammatory cells according to biopsy location relative to an ulcer edge.…”

Doença de Crohn: aspectos do tratamento na atualidade

“…As doenças inflamatórias intestinais (DII), como a doença de Crohn (DC), são condições inflamatórias crônicas [1][2][3][4] e recidivantes do intestino de etiologia desconhecida, [1][2][3] associando-se a uma morbidade significativa. 4 A DC ocorre principalmente em adultos, porém, 7% a 20% dos casos são diagnosticados na infância, sendo frequentemente descrita como mais grave e agressiva nas crianças do que nos adultos.…”