Viegen, Tanja van

Aims Accurate determination of histological activity in ulcerative colitis (UC) is essential given its diagnostic and prognostic importance. Data on the relationship between histology and immune cell markers are limited. We aimed to evaluate the association between histological disease activity and immune cell marker concentration in colonic biopsies from patients with UC. Methods Sigmoid colon biopsies from 20 patients with UC were retrospectively assessed using the Robarts Histopathology Index (RHI). Targeted mass spectrometry determined the concentration of 18 immune cell markers (cluster of differentiation (CD) 4, CD8, CD19, CD20, CD40, CD56, CD68, CD103, forkhead box p3 (FOXP3), human leucocyte antigen, DR alpha chain (HLA-DRA), interleukin 10 (IL-10), IL-23 subunit alpha (IL-23A), IL-23 receptor (IL-23R), IL-2 receptor alpha chain (IL-2RA), Ki67, lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1) and PD ligand 1 (PD-L1)). The association between RHI score and immune cell marker concentration was quantified using Spearman’s rank correlation coefficient (ρ) and related 95% CIs. Results Fourteen of the 18 immune cell marker proteins were detected, with tissue concentration ranging from 0.003 to 11.53 fmol/µg. The overall RHI score was positively correlated with CD19, CD20, CD40, FOXP3, LAG-3, PD-1 and PD-L1 concentration (ρ=0.596–0.799) and negatively correlated with CD56 concentration (ρ=−0.460). There was no significant association between RHI score and CD4, CD8, CD68, CD103, HLA-DRA or Ki67 concentration. Conclusions This study provides insight into the correlation between immune cell marker expression and histological disease activity and the possible molecular and immunological determinants underlying microscopic disease activity in UC.

SummaryBackgroundThe optimal ulcerative colitis biopsy protocol is unclear.AimTo evaluate the number of biopsies required to accurately assess microscopic disease activity in ulcerative colitisMethodsBiopsies from patients with ≥4 rectosigmoid samples, and clinical and endoscopic data, were retrospectively obtained from a prospective biobank. Histology and endoscopic videos were read blindly. A 4‐biopsy Robarts Histopathology Index (RHI) reference score, consisting of the worst item‐level ratings from four biopsies, was compared to 1‐, 2‐ and 3‐biopsy estimates. Agreement was determined using bivariate errors‐in‐variable regression analysis (acceptance interval: ±8.25). Endoscopic activity and disease location subgroup analyses were also performed.ResultsForty‐six patients had ≥4 rectosigmoid biopsies available (N = 287). The 2‐biopsy (tolerance interval: −7.66, 4.79) and 3‐biopsy (tolerance interval: −4.86, 3.46) RHI scores demonstrated acceptable agreement with 4‐biopsy scores. One‐biopsy scores demonstrated unacceptable agreement (tolerance interval: −13.99, 7.78). Mean RHI scores using the 2‐, 3‐ and 4‐biopsy approaches were similar (6.1 ± 9.6 P = 0.36; 6.8 ± 10.5, P = 0.7; 7.5 ± 11.2), whereas the 1‐biopsy estimate was lower (4.4 ± 8.1, P = 0.06). Histological remission rates were identical for the 2‐, 3‐ and 4‐biopsy methods (65.2%, P = 1.0). Subgroup analysis demonstrated that three biopsies were required in patients with endoscopically active disease. Sampling additional colonic locations yielded lower histological remission rates compared to rectosigmoid sampling alone (33.3% vs 61.9%, P = 0.1).ConclusionsA minimum of two — conservatively, three — biopsies are required to reliably assess disease activity in a single colonic segment using the RHI. Further studies are needed of endoscopically active patients and sampling locations. These results have implications for biopsy strategies in clinical trials and practice.

The T-lymphocyte–mediated inflammation in Crohn's disease can be assessed by quantifying CD3-positive T-lymphocyte counts in colonic sections. We developed and validated a process to reliably quantify immunohistochemical marker-positive cells in a high-throughput setting using whole slide images (WSIs) of CD3-immunostained colonic and ileal tissue sections. In regions of interest (ROIs) and/or whole tissue sections of 40 WSIs from 36 patients with Crohn's disease, CD3-positive cells were quantified by an expert gastrointestinal pathologist (gold standard) and by image analysis algorithms developed with software from 3 independent vendors. Semiautomated quantification of CD3-positive cell counts estimated in 1 ROI per section were accurate when compared with manual analysis (Pearson correlation coefficient, 0.877 to 0.925). Biological variability was acceptable in digitally determined CD3-positive cell measures between 2 to 5 ROIs annotated on the same tissue section (coefficient of variation <25%). Results from computer-aided analysis of CD3-positive T lymphocytes in a whole tissue section and the average of results from 2 to 5 ROIs per tissue section lacked reliability (overestimation or underestimation and systematic bias), suggesting that absolute quantification of CD3-positive T lymphocytes in a whole tissue section may be more accurate. Semiautomated image analysis in WSIs demonstrated reproducible CD3-positive cell measures across 3 independent algorithms. A computer-aided digital image analysis method was developed and validated to quantify CD3-positive T lymphocytes in colonic and ileal biopsy sections from patients with Crohn's disease. Results support consideration of this digital analysis method for use in future Crohn's disease clinical studies.

SummaryBackgroundThe appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown.AimTo explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells.MethodsFifty‐one patients with Crohn's disease and ulcers >0.5 cm diameter in the colon and/or ileum were prospectively enrolled at three centres. Biopsies were obtained from 0 mm, 7 to 8 mm and 21 to 24 mm from the edge of the largest ulcer. Histological activity was blindly assessed with the Global Histological Disease Activity Score, the Robarts Histopathology and Nancy Histological indices. Messenger ribonucleic acid (mRNA) levels for interleukins‐6, ‐8 and ‐23 (p19 and p40 subunits), CD31 and S100A9 were measured using quantitative polymerase chain reaction. The number of CD3+, CD68+ and myeloperoxidase‐positive cells was quantified by immunohistochemistry. Data were analysed using mixed models with location and segment as fixed effects and patients as random effect to account for correlation among segments within a patient.ResultsHistological disease activity scores (P < 0.0001), proinflammatory gene expression levels (P < 0.005) and numbers of myeloperoxidase‐positive cells (P < 0.0001) were highest in biopsies from the ulcer edge in the colon and ileum, with decreasing gradients observed with distance from the edge (P < 0.05). No differences between colonic and ileal samples were detected for the parameters measured at any location.ConclusionsBiopsies from the ulcer edge in patients with Crohn's disease yielded the greatest histological disease activity and mRNA levels and had similar readouts in the colon and ileum. Research is needed to confirm this conclusion for other measures.