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P364. Resected fibrostenotic intestine from Crohn's disease patients express significant TNF in proliferative fibroblasts and in extracellular matrix

R. Hundal1, S. Gui2, R. Panaccione1, M. Iacucci1, G. Kaplan1, J. Heatherington1, S. Liu2, H. Becker1, A. Ueno1, S. Ghosh1, 1University of Calgary, Division of Gastroenterology & IBD Clinic, Calgary, Canada, 2University of Calgary, Department of Pathology and Laboratory Medicine, Calgary, Canada

Background

We have recently reported that patients with B2 or stricturing Crohn's disease prior to commencement of biological therapy, are at increased risk of intestinal resection, despite anti-TNF (tumour necrosis factor) therapy [1]. We investigated TNF expression in the fibroblasts and extracellular matrix in the resected fibrostenotic intestine. We hypothesized that TNF expression in fibrostenotic intestinal compartments contributes to decreased efficacy of therapy including anti-TNF treatment in this subset of patients.

Methods

Utilizing a prospective case series design, patients with fibrostenotic Crohn's disease requiring surgical resection were consecutively identified. Representative tissue sections of the fibrostenotic region of resected diseased bowel were taken. Immunostains for TNF and Actin were performed on 4 µm thick formalin-fixed paraffin-embedded sections subjected to heat-induced antigen retrieval and then incubated with TNF or Actin mouse monoclonal antibody. These specimens were further assessed microscopically to characterize histologic changes and to assign phenotype as part of the Montreal Classification for Crohn's disease.

Results

Eleven specimens from patients with Crohn's disease (7 male, 4 female; mean age 34.5) are included. All patients were confirmed to have B2 or fibrostenotic phenotype from their resected specimens (10 terminal ileum and 1 colon). 7 of 11 patients had previously been treated with anti-TNF therapy. All specimens revealed significant submucosal expansion with marked fibrosis (fibroblast proliferation and collagen deposition). In addition, submucosal adipocyte and smooth muscle proliferation, muscularis mucosae dissection and thickening as well as muscularis propria hypertrophy were present. On immunohistochemistry, the areas of fibrosis demonstrated diffuse deposition of TNF within the cytoplasm of fibroblasts and in the extracellular matrix in all specimens. The TNF-positive fibroblasts included but were not limited to myofibroblasts (with immunopositivity for actin). No or minimal TNF deposition was seen within the muscular or adipose tissues.

Conclusion

We have confirmed the presence of diffuse and significant TNF expression in the fibrostenotic bowel wall of Crohn's disease patients. Fibrostenotic disease is also associated with significant TNF expression by fibroblasts with the presence of TNF in the fibroblastic cytoplasm and in the extracellular matrix. Anti-TNF therapy may be less effective in fibrostenotic disease as considerable fibroblast and extracellular matrix TNF expression is entrapped in the fibrotic segment and inaccessible to anti-TNF antibodies.

1. Moran GW et. al. Clin Gastroenterol Hepatol. 2013 Aug 24. pii: S1542-3565(13)01228–7.