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Treating beyond endoscopic remission, aiming for histological remission, has shown to reduce relapse and hospitalization rates in patients with ulcerative colitis (UC). However, very little is known on how histological remission associates with patient reported outcomes (PROMs).

PROMs (Simple clinical colitis activity index [SCCAI], IBD disk and Visual Analogue Scales [VAS]) were prospectively collected through a digital questionnaire in all patients with UC undergoing colonoscopy between July 21^st 2020-Jan 21^st 2021. Mayo endoscopic sub score and UCEIS were determined, as well as the Nancy histologic index (NHI) of the most affected area. Endoscopic remission was defined as Mayo endoscopic sub score 0 and UCEIS 0; histologic remission as NHI 0, absence of active inflammation as NHI ≤ 1. PRO2 remission was defined as stool frequency ≤ 1 (absolute stool frequency ≤ 3 OR 1-2 stools more than usual) and rectal bleeding score of 0.

Fifty-six paired assessments were collected in 48 unique patients (Table 1), with a histologic, endoscopic and PRO-2 remission rate of 23.2%, 28.6% and 38.2% respectively. Patients with histologic remission or absence of histologic inflammation had a significantly lower overall IBD disability (p=0.007, p=0.003) and disease activity score (p=0.003, p<0.001), as compared to patients without. In line, NHI correlated with the overall IBD disk (r=0.40, p=0.002) and SCCAI score (r=0.50, p<0.001). Many individual components of both scores (abdominal pain, arthralgia, impact on education and work/interpersonal interactions/sexual function, regulation of defecation, blood loss, general wellbeing, joint pain, numbers of stools during night/day, urgency) differed significantly between patients with and without histologic remission. VAS scores assessing general wellbeing (r=0.33, p=0.01), impact on daily activities (r=0.41, p=0.002), UC-related symptoms (r=0.42, p=0.001) and worries (r=0.40, p=0.002) correlated with histology.  Quartile analysis of the overall IBD disk and SCCAI scores confirmed the highest likelihood for histologic remission in patients with the lowest scores (Q1-Q2 vs Q3-Q4 39.3% vs 7.1%, p=0.01; 40.0% vs 9.7%, p=0.01) (Figure 1). Nevertheless, the overall accuracy of the IBD disk (0.75) or SCCAI score (0.76) for histologic remission is lower (p<0.05) than the accuracy of the Mayo endoscopic (0.90) or UCEIS (0.90) score.

Table 1 : Baseline features

In patients with UC, PROMs for disability and clinical disease activity reflect histologic disease activity and should therefore be further explored in (trial) endpoint discussions. However, they cannot fully replace endoscopic and histologic findings, and should be considered complementary.

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.

In Crohn’s disease (CD), biologics can induce mucosal healing and stable remission. After reaching this target, treatment de-escalation could be considered but the risk of relapse needs to be estimated. Current biomarkers used to predict relapse (C-reactive protein: CRP, faecal calprotectin) offer a limited prognostic capacity. Furthermore, they only monitor inflammation while we recently highlighted various and distinct pathological processes associated with the risk of short-term (<6 months) and mid/long-term (>6 months) relapse in CD patients stopping infliximab. Herein, the aim of our study was to further characterise this distinction.

Serum abundance of 92 proteins were measured by proximity extension assay (immune response panel, Olink)at baseline of the STORI cohort (infliximab diScon-Tinuation in CrOhn’s disease patients in stable Remission on combined therapy with Immunosuppressors, n=102). Association of markers with the risk of relapse was determined by univariable Cox model in stratified (relapse <6 months or >6 months) and non-stratified datasets. Study of protein characteristics and enrichment analyses were performed to find biological patterns differentiating short-term from mid/long-term relapsers. To evaluate the predictive capacity of markers, we combined them systematically by pairs (‘AND’ or ‘OR’ logical operators) and used log-rank statistics with false discovery rate (FDR) correction (Benjamini-Hochberg).

The risk of mid/long-term relapse was associated with a decreased circulating level of anti-inflammatory effectors while the risk of short-term relapse was associated with an increased circulating level of pro-inflammatory effectors (Fig. 1A, 1B).

The risk associated with the downstream signalling of cytokine and pattern recognition receptors showed an opposite pattern in the short-term versus mid/long-term relapsers (Fig. 1D, 1E).

The risk of short-term relapse was characterised by a perturbed circulating level of proteins inducing tolerance and immunity in antigen presenting cells (Fig. 2A, 2B).

The risk of mid/long-term relapse was characterised by an increased circulating level of proteins promoting lymphocyte tolerance (Fig. 2D, 2E) and a decreased circulating level of cellular junction proteins (Fig. 3).

In CD patients stopping infliximab, blood proteins linked to immunoregulation or cellular junctions support the distinct profiles of short-term and mid/long-term relapsers. These proteins showed a capacity to predict the relapse.