Identification of immune cell markers associated with ulcerative colitis histological disease activity in colonic biopsies.

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.

Effect of storage time on peripheral blood mononuclear cell isolation from blood collected in vacutainer CPT™ tubes.

BACKGROUND: Clinical trials of novel therapies for the treatment of ulcerative colitis (UC) may benefit from immune cell profiling, however implementation of this methodology is limited in the multicenter trial setting by necessity of timely (within 6 to 8 h) isolation and processing of peripheral blood mononuclear cells (PBMC) from whole blood samples. Becton Dickinson Vacutainer CPT™ Cell Preparation Tubes (CPT™) limit required processing prior to shipping to a central lab to an initial centrifugation step within 24 h of sample collection. As shipping may delay final processing beyond 24 h, we analyzed cell viability and T cell composition in whole blood stored in CPT™ to determine if their use may accommodate processing delays typical for multicenter clinical trials. METHODS: Whole blood samples from 3 patients with UC were collected in CPT™ (15 tubes/patient) and PBMC were processed at various timepoints (24-96 h). Cell viability and T cell composition (26 types) were evaluated by flow cytometry. Variability between technical and biological replicates was evaluated in the context of cell-type abundance, delayed processing time, and data normalization. RESULTS: Total cell viability was <50% when processing was delayed to 48 h after collection and was further reduced at later processing timepoints. The effect of delayed processing on cell abundance varied widely across cell types, with CD4+, CD8+, naïve effector CD8+, and Tcm CD4 + T cells displaying the least variability in abundance with delayed processing. Normalization of cell counts to cell types other than total T cells corrected for the effect of delayed processing for several cell types, particularly Th17. CONCLUSIONS: Based on these data, processing of PBMC in CPT™ should ideally be performed within 48 h. Delayed processing of PBMC in CPT™ may be considered for cell types that are robust to these conditions. Normalization of cell abundance to different parental cell-types may reduce variability in quantitation and should be used in conjunction with the expected effect size to meet the experimental goals of a multicenter clinical trial.

Existing Bowel Preparation Quality Scales Are Reliable in the Setting of Centralized Endoscopy Reading.

BACKGROUND: Development of bowel preparation products has been based upon colon cleansing rating by a local endoscopist. It is unclear how bowel preparation scales perform when centrally evaluated. AIMS: To evaluate the reliability of bowel preparation quality scales when assessed by central readers. METHODS: Four central readers evaluated 52 videos in triplicate, 2 weeks apart, during the entire endoscopic procedure (insertion/withdrawal of the colonoscope) and exclusively on colonoscope withdrawal using the Boston Bowel Preparation Scale (BBPS), Chicago Bowel Preparation scale, Harefield Cleansing Scale, Ottawa Bowel Preparation Quality Scale (OBPQS), Aronchick score, a visual analogue scale, and additional items proposed in a modified Research and Development/University of California Los Angeles appropriateness process. Reliability was assessed with intraclass correlation coefficients. RESULTS: Intraclass correlation coefficients (95% confidence interval) for inter-rater reliability of the quality scales ranged from 0.51 to 0.65 (consistent with moderate to substantial inter-rater reliability) during the entire procedure. Corresponding intraclass correlation coefficients for intra-rater reliability ranged from 0.69 to 0.77 (consistent with substantial intra-rater reliability). Reliability was highest in the right colon and lowest in the left colon. No differences were observed in reliability when assessed for the procedure overall (insertion/withdrawal) relative to assessment on withdrawal alone. CONCLUSION: All five bowel preparation quality scales had moderate to substantial inter-rater reliability. Panelists considered the Aronchick score too simplistic for clinical trials and recognized that assessment of residual fluid in the Ottawa Bowel Preparation Quality Scale was not amenable to central assessment.

Development and Validation of a Digital Analysis Method to Quantify CD3-immunostained T Lymphocytes in Whole Slide Images of Crohn's Disease Biopsies.

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.

Responsiveness of Magnetic Resonance Enterography Indices for Evaluation of Luminal Disease Activity in Crohn's Disease.

BACKGROUND & AIMS: Magnetic resonance enterography (MRE) is having an increasing role in Crohn's disease; however, fully validated indices are needed. We evaluated the responsiveness of 4 MRE indices in luminal Crohn's disease. METHODS: Paired MRE images (pretreatment and post-treatment at weeks 12 or 14) from 41 patients were scored by 3 blinded radiologists. Disease activity was scored for 4 MRE indices (magnetic resonance index of activity [MaRIA], simplified MaRIA, London index, and London extended index) and a 100-mm visual analog scale (VAS) of overall disease activity. The criterion for change was an improvement by at least one half of an SD in the VAS after treatment. Responsiveness was evaluated using the standardized effect size (SES). Longitudinal validity was evaluated using correlations between changes in MRE index scores and disease activity measures including endoscopy and the VAS. RESULTS: The SES was 1.17 (95% CI, 0.56-1.77) for the simplified MaRIA, 0.98 (95% CI, 0.42-1.55) for the MaRIA, 0.95 (95% CI, 0.38-1.51) for the London extended index, and 0.85 (95% CI, 0.31-1.39) for the London index. The simplified MaRIA was significantly more responsive than the London index (ΔSES, 0.32; 95% CI, 0.05-0.58) but not the MaRIA (ΔSES, 0.18; 95% CI, -0.01 to 0.38) or the London extended index (ΔSES, 0.22; 95% CI, -0.05 to 0.50). Correlations with endoscopy (simplified MaRIA: r = 0.72) were not different from correlations with the VAS (London extended index: r = 0.70). CONCLUSIONS: Evaluated MRE indices showed moderate-to-large responsiveness and are suitable for use in clinical trials. The simplified MaRIA may be preferred because of its responsiveness and nonreliance on gadolinium administration.

Evaluating the optimum number of biopsies to assess histological inflammation in ulcerative colitis: a retrospective cohort study.

BACKGROUND: The optimal ulcerative colitis biopsy protocol is unclear. AIM: To evaluate the number of biopsies required to accurately assess microscopic disease activity in ulcerative colitis METHODS: Biopsies 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. RESULTS: Forty-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). CONCLUSIONS: A 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.

Response to Placebo, Measured by Endoscopic Evaluation of Crohn's Disease Activity, in a Pooled Analysis of Data From 5 Randomized Controlled Induction Trials.

BACKGROUND & AIMS: Endoscopy is used to measure activity of Crohn's disease (CD) and determine eligibility and outcomes of participants in randomized controlled trials of therapeutic agents. We aimed to estimate the rate of response to placebo in trials, based on endoscopic evaluation of CD activity, and identify factors that affect this response. METHODS: We collected patient-level data from randomized, double-blind, placebo-controlled trials of therapeutic agents for CD that included centrally-read endoscopic assessments with validated scoring indices. We analyzed data from induction trials of eldelumab, filgotinib, risankizumab, and ustekinumab (from 188 patients given placebo). The primary outcome was the rate of response to placebo, based on endoscopic assessment of CD activity (>50% reduction in the simple endoscopic score for CD). Rate of remission, based on endoscopic score, was a secondary outcome. Overall rates of response to placebo were calculated using the inverse variance-weighted average method and presented with 95% CIs. We performed a multi-variable meta-regression analysis to identify determinants of response to placebo, assessed endoscopically, using patient-level data from the filgotinib and ustekinumab trials. RESULTS: The pooled rate of response among patients given placebo was 16.2% (95% CI, 10.5%-22.0%) and the rate of remission in this group was 5.2% (95% CI, 1.7%-8.8%). Prior exposure to tumor necrosis factor antagonists (odds ratio, 0.31; 95% CI, 0.10-0.93; P = .036) and increased concentration of C-reactive protein at baseline (odds ratio, 0.93; 95% CI, 0.87-0.98; P = .014 per 10 mg/L increase) were independently associated with lower rates of response to placebo. CONCLUSIONS: Rates of response and remission to placebo, determined by centrally-read endoscopy, in induction trials of therapies for CD are low. These estimates are important for sample size calculations for randomized placebo-controlled trials that use the Simple Endoscopic Score for CD as an endpoint. They also provide a benchmark to interpret findings from non-placebo controlled, prospective, randomized, unblinded trials.

Development and Validation of a Magnetic Resonance Index for Assessing Fistulas in Patients With Crohn's Disease.

BACKGROUND & AIMS: There is no validated magnetic resonance imaging (MRI) index for assessment of perianal fistulas in patients with Crohn's disease (CD). We developed and internally validated a new instrument. METHODS: We used paired baseline and week-24 MRI scans from 160 participants in a randomized placebo-controlled trial of stem cell therapy for patients with perianal fistulizing CD. Four radiologists scored disease activity using index items identified during previous studies and exploratory items. Reliability was assessed using intraclass correlation coefficients. We developed an index using backward elimination linear regression analysis, in which potential independent variables were items having intraclass correlation coefficients of at least 0.4 and the dependent variable was perianal fistulizing disease activity, measured on a 100-mm visual analogue scale. The final model was internally validated using the .632 bootstrap method to correct model optimism and quantify calibration accuracy. We evaluated responsiveness of the index by assessing longitudinal validity and estimating standardized effect sizes. RESULTS: We developed the magnetic resonance novel index for fistula imaging in CD (MAGNIFI-CD) using 6 items. The optimism-corrected R2 of the model was 0.71, which was comparable to R2 for the original sample (0.74). The calibration slope for the model was 0.98. Compared with the original and modified versions of the Van Assche Index, the MAGNIFI-CD had improved operating characteristics. Estimates of intraclass correlation coefficients for MAGNIFI-CD, the modified Van Assche Index, and Van Assche Index were 0.85 (95% confidence interval [CI], 0.77-0.90), 0.81 (95% CI, 0.74-0.86), and 0.81 (95% CI, 0.71-0.86) for intra-rater reliability, and 0.74 (95% CI, 0.63-0.80), 0.67 (95% CI, 0.55-0.75) and 0.68 (95% CI, 0.56-0.77) for inter-rater reliability. Corresponding standardized effect size estimates were 1.02 (95% CI, 0.65-1.39), 0.84 (95% CI, 0.48-1.21), and 0.68 (95% CI, 0.33-1.03). CONCLUSIONS: We developed an index called the MAGNIFI-CD, which is based on 6 items. It assesses MRI data and determines perianal fistulizing CD activity with improved operating characteristics compared to previous indices. This index may be used as an outcome measure in clinical trials comparing treatment effects in patients with perianal fistulizing CD. Although the performance of the MAGNIFI-CD indicates its stability and reasonable external validity, external validation is needed.

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

BACKGROUND: The appropriate location for biopsy procurement relative to an ulcer in active Crohn's disease is unknown. AIM: To explore the relationship between biopsy location, histological disease activity, proinflammatory gene expression and the presence of inflammatory cells. METHODS: Fifty-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. RESULTS: Histological 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. CONCLUSIONS: Biopsies 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.

Evaluation of the effect of storage condition on cell extraction and flow cytometric analysis from intestinal biopsies.

BACKGROUND: Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. METHODS: Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. RESULTS: Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. CONCLUSION: Storage of intestinal tissue biopsies at -20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.

Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study.

OBJECTIVES: Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. RESULTS: Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. CONCLUSIONS: Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.