Computer-Aided Imaging Analysis of Probe-Based Confocal Laser Endomicroscopy With Molecular Labeling and Gene Expression Identifies Markers of Response to Biological Therapy in IBD Patients: The Endo-Omics Study

Abstract

Background: We aimed to predict response to biologics in inflammatory bowel disease (IBD) using computerized image analysis of probe confocal laser endomicroscopy (pCLE) in vivo and assess the binding of fluorescent-labeled biologics ex vivo. Additionally, we investigated genes predictive of anti-tumor necrosis factor (TNF) response.

Methods: Twenty-nine patients (15 with Crohn's disease [CD], 14 with ulcerative colitis [UC]) underwent colonoscopy with pCLE before and 12 to 14 weeks after starting anti-TNF or anti-integrin α4β7 therapy. Biopsies were taken for fluorescein isothiocyanate-labeled infliximab and vedolizumab staining and gene expression analysis. Computer-aided quantitative image analysis of pCLE was performed. Differentially expressed genes predictive of response were determined and validated in a public cohort.

Results: In vivo, vessel tortuosity, crypt morphology, and fluorescein leakage predicted response in UC (area under the receiver-operating characteristic curve [AUROC], 0.93; accuracy 85%, positive predictive value [PPV] 89%; negative predictive value [NPV] 75%) and CD (AUROC, 0.79; accuracy 80%; PPV 75%; NPV 83%) patients. Ex vivo, increased binding of labeled biologic at baseline predicted response in UC (UC) (AUROC, 83%; accuracy 77%; PPV 89%; NPV 50%) but not in Crohn's disease (AUROC 58%). A total of 325 differentially expressed genes distinguished responders from nonresponders, 86 of which fell within the most enriched pathways. A panel including ACTN1, CXCL6, LAMA4, EMILIN1, CRIP2, CXCL13, and MAPKAPK2 showed good prediction of anti-TNF response (AUROC >0.7).

Conclusions: Higher mucosal binding of the drug target is associated with response to therapy in UC. In vivo, mucosal and microvascular changes detected by pCLE are associated with response to biologics in inflammatory bowel disease. Anti-TNF-responsive UC patients have a less inflamed and fibrotic state pretreatment. Chemotactic pathways involving CXCL6 or CXCL13 may be novel targets for therapy in nonresponders.

Keywords: Crohn’s disease; RNA transcriptomics; artificial intelligence; biological agents; endoscopic molecular labeling; probe confocal laser endomicroscopy; ulcerative colitis.

Overview of the study methodology.

Figure 1.

Summary of probe confocal laser endomicroscopy findings in vivo and ex vivo.

Figure 2.

Immunohistochemistry for tumor necrosis factor (TNF) and integrin-β7 was performed on biopsies sampled before and after biologic treatment. Expression was scored on a scale of 0 to 3 in which 0 = none, 1 = low (<30%), 2 = medium (30%-60%), and 3 = high (>60%). A, Representative images of tissues with high and low expression. B, Summary of scores for tissues from patients classified as nonresponder (NR), partial responder (PR), and responder (R). i and ii indicate actual scores. iii and iv show reduction in score from pre- to posttreatment (pre score minus post score). Numbers indicate the number of patients represented where data points overlap.

Figure 3.

A, A diagrammatic representation of the direction of regulation of the differentially expressed genes (DEGs) when partial responders are considered as responders. B, A principal component analysis score plot performed on the 342 DEGs demonstrating clustering of the responders vs nonresponders. Dots represent patients and are colored according to the subject cohort. Ellipses represent 95% confidence.

Figure 4.

A selection of response predictive markers for anti-tumor necrosis factor (TNF) therapy. An area under the curve (AUC) analysis was performed on the enriched genes with variable importance in projection >1 that were also measured in the validation study. A, Heat map summaries of the 7 selected targets in the Crohn’s disease (CD)/ulcerative colitis (UC) and validation cohorts. B, Sensitivity vs specificity receiver-operating characteristic curves for combined use of CRIP2, CXCL6, EMILIN1, GADD45B, LAMA4, and MAPKAPK2 as predictors of TNF response in the optima cohort and validation cohorts. CI, confidence interval.

Figure 5.

Correlation between ex vivo probe confocal laser endomicroscopy findings and genes. Ex vivo data with mean value of the high threshold and low threshold on area intensity were used to link with selected genes. Three different samples, responder and nonresponder combined, responder, and nonresponder, were selected. Only significant Spearman correlation values are shown (P < .05). Red = positive correlation; blue = negative correlation. The ACTN1 expression value was 0 across all the samples for nonresponder and hence was not used. IBD, inflammatory bowel disease.