Exabis Library

Biologics are being used increasingly in the treatment of Inflammatory Bowel Disease. However, up to 40% of patients do not respond to biologics. Therefore, methods to predict response are imperative. We aimed to identify novel genes and pathways predictive of anti-TNF response in patients with Ulcerative Colitis (UC) undergoing electronic chromoendoscopy and probe confocal laser endomicroscopy (pCLE). We further evaluated the ex-vivo binding of fluorescent labelled biologics as markers of response

26 UC patients starting anti-TNF therapy as standard of care were recruited. Pre-treatment colonoscopy, with electronic chromoendoscopy and pCLE (Cellvizio, Mauna Kea) by injecting intravenous fluorescein (2.5-5mls), was performed to assess disease activity. Targeted biopsies were taken for fluorescein isothiocyanate (FITC)-labelled infliximab staining and RNA extraction and gene expression analysis. Ex vivo labelling was evaluated by an automated analysis: after a first pre-processing step to remove biases, the labelled regions were identified using statistical multi-level thresholding, and evaluated as area and intensity. To assess response, the same endoscopic procedure was repeated at week 12-14 after anti-TNF. cDNA libraries were prepared using QIAseq UPX 3’Transcriptome reagents and sequenced. Normalised gene expressions were obtained through the CLC Genomics Workbench. Differentially expressed genes (DEGs) (FDR-corrected P-value<0.05) were determined using the Limma package and PLS-DA modelling performed to calculate their importance (VIP score). Functionally related genes were identified and classified using DAVID tools. Strongest indicators of response were predicted by Random Forest area under the curve (AUC) analysis in this cohort and a similar validation cohort

At baseline increased binding of the labelled biologic was associated with a higher likelihood of response to treatment  (AUROC81%, accuracy77%, PPV100%, NPV63%). 342 DEGs (75 up-regulated, 267 down-regulated) distinguished responders from non-responders, 76 fell within enriched pathways. Pathways related to inflammation, chemotaxis, TGF-beta signalling, extracellular matrix and carbohydrate metabolism were reduced and cell-cell adhesion increased in responders pre-treatment. Among the 37 genes with VIP>1, CRIP2, CXCL6,EMILIN1,GADD45B, LAMA4 and MAPKAPK2 were upregulated in non-responders pre-treatment and were good predictors of response (AUROC>0.7) in this cohort and validation cohort

A higher mucosal binding of the biologics before treatment was observed in anti-TNF responders. Responsive UC patients have a less inflamed and fibrotic state pre-treatment. Chemotactic pathways, involving CXCL6 may be novel targets to treat non-responders

Interventions targeting key inflammatory mechanisms have expanded the therapeutic repertoire for ulcerative colitis (UC). However, exact molecular mechanisms associated with clinical response remain elusive. We conducted a multiplex-immunohistochemistry (IHC) study to monitor immune cell composition in biopsies from UC patients under 2 approved therapies targeting TNF (infliximab) and integrin (vedolizumab), and a phase IIa clinical trial with the selective IL-6 transsignalling inhibitor olamkicept, to identify spatiotemporal changes of mucosal immune cell compartments in relation to each mechanism of action.

Sigmoid biopsies from UC patients exposed to infliximab, vedolizumab or olamkicept (26 patients in total) at baseline and week 2, 6 and 14 after therapy induction were subjected to multiplex IHC for CD3, CD15, CD20, CD68, pSTAT3, and pan-cytokeratin (OPAL/Vectra Polaris, Akoya). Quantitative and spatial immune cell patterns captured by advanced image analysis (inForm, Akoya; R package PhenoptrReports) were analysed for differences between baseline and subsequent time points using Dunnett’s multiple comparisons test   (GraphPad Prism 8.4.3). Accepted significance levels: *p<0.05,**p<0.01, ***p<0.001.

Targeted therapies resulted in overall decrease of immune cell infiltrates (range 1.15-1.22 fold; p=0.017-0.007), irrespective of drug or specified endpoint (remission at week 14) (Fig. 1). Significant drug-specific changes of spatial immune cell distributions were discernible (Fig. 2). Anti-TNF treatment was mainly associated with decrease in CD3⁺ T cells (p=0.005) localized in the submucosa close to epithelium or in tertiary lymphoid organs. In contrast, integrin targeting resulted in fewer CD15⁺ neutrophils, mainly in the submucosal compartment (p=0.046). Olamkicept treatment resulted in unique depletion of pSTAT3⁺ cells in patients achieving remission at week 14 (not observed in remission after infliximab or vedolizumab). To decipher drug-independent features of clinical remission we assessed distance metrics between immune and intestinal epithelial cells in remission and non-remission patients and observed increase (endpoint compared to baseline) of the average distance to the nearest CD20 or CD15 cell in the remission (p=0.0054, p=0.0004) but not in the non-remission group (n.s.).

Our study strongly suggests that multiplexed spatiotemporally resolved immune cell phenotyping may provide novel insights into the dynamic shifts of immune cell compartments in UC patients undergoing targeted therapy. We propose that such highly resolved digital maps of immune cells could lead to novel tools for therapeutic stratification of IBD patients.