Histological, immunohistochemical and mRNA gene expression responses in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded duodenal biopsies

Abstract

Background: There is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease. The gold standard for successful treatment is a healed small intestinal mucosa, and therefore, the outcome measures in proof-of-concept studies should be based on evaluation of small intestine biopsies. We here evaluated morphometric, immunohistochemical and messenger RNA (mRNA) expression changes in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded biopsies. Methods: Fifteen coeliac disease patients were challenged with 4 g of gluten per day for 10 weeks and 24 non-coeliac patients served as disease controls. A wide array of histological and immunohistochemical staining and mRNA-based gene expression tests (RT-qPCR and RNAseq) were carried out. Results: Digital quantitative villous height: Crypt depth ratio (VH: CrD) measurements revealed significant duodenal mucosal deterioration in all coeliac disease patients on gluten challenge. In contrast, the Marsh-Oberhuber class worsened in only 80% of coeliac patients. Measuring the intraepithelial CD3+ T-lymphocyte and lamina propria CD138+ plasma cell densities simultaneously proved to be a meaningful new measure of inflammation. Stainings for γδT cells and IgA deposits, where previously frozen samples have been needed, were successful in PAXgene fixed paraffin-embedded samples. Messenger RNA extraction from the same paraffin-embedded biopsy block was successful and allowed large-scale qRT-PCR and RNAseq analyses for gene expression. Molecular morphometry, using the mRNA expression ratio of villous epithelium-specific gene APOA4 to crypt proliferation gene Ki67, showed a similar significant distinction between paired baseline and post-gluten challenge biopsies as quantitative histomorphometry. Conclusion: Rigorous digitally measured histologic and molecular markers suitable for gluten challenge studies can be obtained from a single paraffin-embedded biopsy specimen. Molecular morphometry seems to be a promising new tool that can be used in situations where assessing duodenal mucosal health is of paramount importance. In addition, the diagnostically valuable IgA deposits were now stained in paraffin-embedded specimens making them more accessible in routine clinics.