Ultra-high Magnification Endocytoscopy and Molecular Markers for Defining Endoscopic and Histologic Remission in Ulcerative Colitis-An Exploratory Study to Define Deep Remission

Abstract

Background: Endoscopic and histological remission are both important treatment goals in patients with ulcerative colitis (UC). We aimed to define cellular architecture, expression of molecular markers, and their correlation with endoscopic scores assessed by ultra-high magnification endocytoscopy (ECS) and histological scores.

Methods: Patients with UC (n = 29) were prospectively recruited. The correlation among ECS score (ECSS), Mayo endoscopic score (MES), and histological scores were determined. Area under curve were plotted to determine the best thresholds for ECSS that predicted histological remission by Robarts (RHI) and Nancy Histological Index (NHI).Soluble analytes relevant to inflammation were measured in serum and mucosal culture supernatants using ProcartaPlex Luminex assays and studied by partial least square discriminant analysis and logistic model. Mucosal RNA sequencing and bioinformatics analysis were performed to define differentially expressed genes/pathways.

Results: Endocytoscope scoring system correlated strongly with RHI (r = 0.89; 95% CI, 0.51-0.98) and NHI (r = 0.86; 95% CI, 0.42-0.98) but correlated poorly with MES (r = 0.28; 95% CI, 0.27-0.70). We identified soluble brain-derived neurotrophic factors (BDNF), macrophage inflammatory proteins (MIP-1 α) and soluble vascular cell adhesion molecule 1 (sVCAM-1) predicted histological remission. Mucosal biopsy cultures also identified sVCAM-1 associated with healed mucosa. RNA-seq analysis identified gene expressions shared between ECSS, RHI, or NHI defined healing. A number of gene expressions and pathways were identified including inflammation and metabolic and tumor suppressors that discriminated healed from nonhealed mucosa.

Conclusions: Endocytoscopy represents an interesting tool that may sit between endoscopy and histology-but closer to the latter-identifying gene expression markers and pathways that are also identified by histology.

Keywords: RNA-sequencing; endocytoscope; histological healing; mucosal healing; noninvasive markers.

FIGURE 1.

A–C, Endoscopic MH assessed by HD white light endoscopy and NBI with and without magnification showing elongated crypts as observed in endoscopic mucosal healing in UC. D–F, Ultra-high magnification endocytoscope after using dye chromoendoscopy with methylene blue 1% showed increase infiltration with cells between the crypts (arrow) and elongated distorted crypts (arrow) as per histological healing changes.

FIGURE 2.

ROC curves of serum BDNF and MIP-1 that, when combined together, were best at predicting histological healing (RHI ≤3 and NHI ≤1) with an AUROC of 0.82.

FIGURE 3.

Principal component analysis score plots represented on the differentially expressed transcriptome datasets. Each of the plots demonstrating clustering of patients according to the healed vs nonhealed categories. Each of the dots (red or green) represent samples (or patients) and are colored according to the subject cohort (healed vs nonhealed). Ellipses represent 95% confidence healed or nonhealed patients. Results are plotted according to the top 2 principal components scores: principal component 1 (PC1) and principal component 2 (PC2). PC1 and PC2 scores, with the percentage variation explained by the x and y axis. Four different definitions of the healed vs nonhealed defined as (A) RHI scores, (B) NHI, (C) ECCS scores, and (D) Mayo score.

FIGURE 4.

Overlap of upregulated genes in healed and nonhealed mucosa as defined by (A) ECSS and RHI scores, and (B) ECSS and NHI scores.

FIGURE 5.

Volcano plot representations of the differentially expressed genes in healed vs nonhealed mucosa as defined by (A) ECSS, (B) Mayo, (C) NHI, and (D) RHI scores.

FIGURE 6.

GO and KEGG pathway enrichment analysis of commonly upregulated genes in healed mucosa defined by ECSS and RHI scores (A and B) and ECSS and NHI scores (C and D). Pathways for which P < 0.05 are shaded red.