Introduction

Mucosal healing (MH) has become a treatment goal for inflammatory bowel disease (IBD), especially for ulcerative colitis. However, MH is less feasible to assess frequently in patients with Crohn’s disease (CD). In this study, we investigated Protein Fingerprint Assays (PFA) of collagen degradation and formation, and neutrophil and macrophage activity markers in the ImageKids study as non-invasive markers of MH.

Aims & Methods

This is a proteomics planned sub-study of the multi-center prospective ImageKids study which included serum samples from children with CD (n=178) undergoing MRE and ileocolonoscopy concurrently (ImageKids study: NCT01881490). Serum from healthy age and gender-matched controls were obtained from BioIVT (n=83). PFA panel for mucosal damage (C1M, C3M, C4M, C6M, ELP-3), Immune cell activity panel (CPa9-HNE, VICM, C4G), and tissue fibrosis panel (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6, PRO-C11, PRO-C22) were determined. MH was classified as having an SES-CD score of 0 and global radiologist assessment of inflammation in the MRE of 0, using a visual analogue scale (VAS) 0-100mm, and mucosal damage (MD) was defined as having SES-CD or MRE VAS>0. Spearman rho correlations, one-way ANOVA with Tukey correction, and receiver operator characteristics (ROC) curves were applied for statistical analysis.

Results

Some of the previous biomarkers demonstrated to correlate with the SES-CD score and ulcer size (C1M, C3M, C6M, PRO-C4, VICM, PRO-C1, PRO-C3). CPa9-HNE, ELP-3, and PRO-C22 also correlate with the SES-CD score (table 1). The combined SES-CD and MRE VAS score of MH correlated positively with CPa9-HNE (r=0.30, p<0.0001), VICM (r=0.39, p<0.0001), C1M (r=0.29, p=0.0001), C3M (r=0.22, p=0.0022), C6M (r=0.35 p<0.0001), PRO-C4 (r=0.2, p<0.0001), PRO-C22 (r=0.32, p<0.0001), and showed significantly lower serum levels in pCD patients with MH compared to patients with mild, moderate, or severe mucosal damage (p<0.001). PRO-C3 correlated negatively with the combined SES-CD and MRE VAS score (r=-0.17, p=0.0211). These PFA biomarkers were combined in a multivariate regression model, demonstrating increased accuracy to identify patients in MH healing compared to the individual biomarkers (p<0.001, MH vs. MD: AUC=0.82 [CI:0.69-0.95), MH+Mild MD vs. moderate+severe MD: AUC=0.77 [0.70-0.84], (figure 1).

Conclusion

PFA biomarkers of Neutrophil activity (CPa9-HNE), Macrophage activity (VICM), mucosal damage (C1M, C3M, C4M, C6M, ELP-3), and tissue fibrosis (PRO-C3, PRO-C22) are potential surrogate biomarkers for identifying pCD patients with mucosal healing and for monitoring of mucosal damage and stenosis.

Disclosure

Joachim H. Mortensen, Martin Pehrsson, Marta Alexdottir, Morten A. Karsdal are fulltime employee at Nordic Bioscience A/S. Joachim H. Mortensen and Morten A. Karsdal owns stocks in Nordic Bioscience A/S

Introduction

Crohn ‘s disease (CD) is characterized by chronic inflammation in the gut, where severe complications such as fibrotic strictures require surgical resection. Stricture development involves excessive extracellular matrix (ECM) deposition due to continuous activation of intestinal myofibroblasts, that further contribute to intestinal fibrogenesis. Emerging data suggests that stricture-related intestinal myofibroblasts overexpress a serine protease called fibroblast activation protein (FAP). Furthermore, type III collagen deposition is increased during fibrosis in all layers of the intestinal tract, and studies have shown elevated collagen degradation in serum from patients with CD. The present study aimed at evaluating the potential of FAP-cleaved type III collagen [C3F] as a serum marker for intestinal fibrosis in CD.

Aims & Methods

The cohort consisted of 49 patients with luminal CD and 62 patients with stenotic CD. Clinical assessment was conducted for all patients at baseline and 12 months post study initiation. For the luminal phenotype, endoscopy was performed at both time points. For the stenotic group, radiographical assessment was done prior to surgery and endoscopy was performed at 12 months. C3F was measured in serum at all timepoints for baseline, post-operative day (POD), 1, 3, 6 and 12 months. To compare C3F levels at baseline for luminal vs stenotic phenotype, Mann-Whitney U-test was conducted. For the stenotic group, mixed-effect analysis, with a Dunnett’s multiple comparison applying Bonferroni Correction, was conducted to compare C3F levels for baseline vs POD and Spearman’s rank correlation was applied.

Results

C3F baseline levels were significantly higher in patients with stenotic CD compared to luminal CD (Table 1). In the stenotic group, C3F was significantly increased in patients at POD compared to baseline (Table 1). In the luminal group, C3F was significantly elevated in patients at 12 months compared to baseline. Furthermore, C3F showed a positive correlation with C-reactive protein (CRP) (ρ=0.350, p=0.006) and a significant negative correlation with disease duration (ρ= -0.257, p=0.048) for patients with the stenotic CD phenotype (Table 1).


Table 1. C3F levels are elevated in the stenotic CD vs the luminal CD group, with increased levels at POD compared to baseline for the stenotic phenotype. Spearman’s rank correlation, depicting the relationship between C3F and routine blood demographics, as well as demographics. Significant findings (p<0.05) are highlighted in bold.

Conclusion

C3F was elevated in patients with stenotic CD, with even higher levels after the surgical resection. The data suggests that C3F reflects fibroblast activity during fibrostenosis and tissue remodeling following surgery. The positive correlation with CRP supports the association with inflammation leading to collagen deposition and fibrosis. Interestingly, C3F shows negative correlation with disease duration suggesting that C3F could reflect fibrosis at early stages of stenotic CD. These findings highlight the potential use of C3F as a novel biomarker for intestinal fibrosis in CD.

Disclosure

TTK, MSA, MP are employed at Nordic Bioscience. ACBJ, JHM and MAK are employees and stock owners at Nordic Bioscience.

Introduction

Inflammatory bowel disease (IBD) is characterized by chronic inflammation due to an uncontrolled immune response orchestrated through, i.e. neutrophils. The basement membrane acts as a front-line defense promoting tissue integrity, where the α-3 chain of type IV collagen is solely expressed on the outermost layer of the mucosa. Detection of subclinical ulceration is crucial since early-stage IBD presents a unique window of opportunity for intervention, as the disease might become a self-sustaining process once deep inflammation becomes established. We therefore, wanted to develop a biomarker reflecting early tissue injury, aiding in the implementation of prompt treatment.

Aims & Methods

A competitive enzyme-linked immunosorbent assay quantifying a human neutrophil elastase (HNE) degraded fragment of type IV collagen α-3 chain was developed (C4A3-HNE). Dilution- and spiking recovery, inter- and intra-variability, and interference were evaluated. Acceptance criteria for technical validation were a recovery of 100% ±25 and an inter-intra variability of <15%. Commercial serum samples were purchased from ProteoGenex. Biological relevance was validated in an observational study from Odense University Hospital (OUH) and healthy controls. Groups were compared using Kruskal Wallis (Dunn‘s corrected).

Results

The technical validation of the C4A3-HNE assay demonstrated acceptable results for recovery, variation, and interference. Biological relevance was assessed in commercial IBD samples and healthy donors. C4A3-HNE was elevated in both CD and UC compared to healthy (p=0.001 and <0.0001)(AUC [95% CI]: 0.820 [0.669-0.971]; 0.875 [0.775-1.000]). The results were validated in an independent cohort of IBD patients (both p<0.0001). For UC, analysis showed that compared to patients in endoscopic remission, C4A3-HNE is elevated in patients with mild endoscopic activity, decreasing dose-dependently for moderate and severe disease (T1). Fecal calprotectin and CRP levels were also lower in IBD patients with high levels of C4A3-HNE (T1). With a cut-off value of 54.1 ng/ml, C4A3-HNE could significantly discriminate between mild and severe endoscopic disease (T1).

Conclusion

Our study demonstrates the biological and possible clinical relevance of the novel C4A3-HNE biomarker. The biomarker is elevated in patients with IBD compared with healthy, and high levels may reflect early mucosal damage in patients with UC. Furthermore, inflammatory markers were lower in IBD patients with high levels of C4A3-HNE, pointing towards high levels being indicative of early disease. C4A3-HNE may provide early detection of mucosal damage or prediction of relapse; however, further studies are required to assess the clinical validity of this biomarker.

Disclosure

MA, MP, MK, and JM are employees of Nordic Bioscience A/S. MK owns stocks in Nordic Bioscience A/S.

Introduction

Data on the long-term benefit of transmural healing (TH) are scarce in children.

Aims & Methods

We investigated the outcomes of pediatric Crohn's disease (CD) patients with TH comparing them to those with mucosal healing (MH) and with “no healing”.
We conducted a prospective, single-center, observational study conducted at the Pediatric Gastroenterology Unit of Sapienza University of Rome. Children with an established diagnosis of CD and under biological therapy undergoing an MRE and an ileocolonoscopy within a 3-month interval were consecutively enrolled. TH was defined as the complete healing of both the mucosa at endoscopy and of the bowel layers at the MRE. In the presence of a normal colonoscopy but an active MRE children were deemed as MH. The group “no healing” (NH) included children with an active endoscopy, regardless of the MRE classification. At 6, 12, and 24 months number disease flares, CD-related hospitalization, CD-related surgery, need for step-up treatment, and occurrence of complications were collected.

Results

93 children were included (mean age 11,2±3,8). Twenty-three (25%) were classified as TH, 27 (29%) as MH and 43 (46%) as NH. The risk of any unfavorable outcome was lower in both TH and MH patients compared to NH (Log Rank p<0.0001 and p=0.002). Children with TH were at lower risk of hospitalization (p=0.02), and complications (p=0.007) compared to those with NH. No difference was found between MH and NH children for the same outcomes. Both TH and MH children were at lower risk of step-up treatment (p<0.0001 and p=0.0002, respectively) compared to NH. TH and MH patients were comparable for all the outcomes evaluated.

Conclusion

Achieving TH in pediatric CD is associated with better outcomes, although the additional benefit over MH alone does not appear significant. However, the persistence of inflammation is associated with worse outcomes in all patients.

Introduction

IIn Crohn’s disease, chronic inflammation drives fibroblast activation, excessive collagen deposition, and creeping fat expansion, all contributing to fibrostenotic strictures. We examined serum markers related to collagen formation, macrophage and neutrophil activation, and NETosis, exploring their associations with stricturing Crohn’s disease in a treatment-naïve IBD inception cohort.

Aims & Methods

142 Serum samples from CD patients were analysed (IBSEN III population matched baseline and 1-year follow-up). The Montreal classification (luminal, fibrostenotic strictures, fistula) was applied to assess disease complications at the time of serum sampling, e.g. baseline biomarkers measurements were paired with baseline Montreal B assessment, and one-year followup biomarkers measurements were paired with one-year followup Montreal B assessment. The biomarkers C4M, C6M, C7M, PRO-C3, PRO-C4, PRO-C5, PRO-C6, PRO-C11, PRO-C16, PRO-C22, VICM, CTX-III, CPa9-HNE were measured in the serum samples at baseline (total n=142; luminal n=114; fibrostenotic n=23, fistula n=5) and 1-year follow-up (total n=142; luminal n=111; fibrostenotic n=24, fistula n=7). One-way-ANOVA with LSD correction was applied for statistical analysis.

Results

The PRO-C6 (p<0.001) and CPa9-HNE (p<0.05) biomarkers were significantly elevated in CD patients with fibrostenotic strictures compared to luminal CD patients at baseline, and PRO-C6 was continually elevated in serum after 1 year of follow-up in patients with fibrostenotic strictures (p<0.01). At baseline, C7M was demonstrated to be elevated in fistulizing CD patients compared to luminal and fibrostenotic CD patients (P<0.01). PRO-C5 (p<0.001) and PRO-C16 (p<0.01) were significantly elevated at 1-year follow-up in the CD patients with fibrostenotic strictures compared with luminal CD patients. None of the remaining biomarkers were associated with stenotic or fistulizing CD.

Conclusion

These encouraging biomarker data from the IBSEN III, especially PRO-C6, seem to be a promising biomarker for intestinal fibrosis, as PRO-C6 was demonstrated to be elevated at baseline and after 1 year follow-up. CPa9-HNE levels were highest in the fibrostenotic group, suggesting neutrophil activity and NETosis involvement in intestinal fibrogenesis. Elevated levels of C7M were associated with fistula in CD patients, suggesting that C7M is reflective of transmural inflammation. The biomarkers PRO-C5 and PRO-C16 are also relevant biomarkers of established intestinal fibrosis, as these were elevated after 1 year of follow-up. Collectively, these biomarker data highlight the importance of quantifying collagen protein fragments in serum as tools for assessing intestinal fibrosis in Crohn’s disease.

Disclosure

Full time employee at Nordic Bioscience A/S

Introduction

Infiltration of neutrophils into the intestinal tissue is a crucial component of the inflammatory response in Crohn‘s disease (CD). CPa9-HNE, a calprotectin fragment released by human neutrophil elastase during NETosis, has been shown to strongly correlate with endoscopic disease activity in inflammatory bowel disease (IBD) and serves as an indicator for pharmacodynamic response. Furthermore, emerging data suggests that neutrophil extracellular traps (NETs) significantly contribute to the pathogenesis of fibrosis.

Aims & Methods

For the first time, we investigated the association of CPa9-HNE with histological grading of both inflammation and fibrosis. The cohort included 62 patients with stenotic Crohn‘s disease. Inclusion criterion was the presence of stenotic disease requiring bowel resection, as confirmed by radiographic assessment. Upon surgery, the resected specimens were collected and histologically graded according to transmural fibrosis burden, and inflammation (D'Haens score). Serum was drawn at baseline (before resection). CPa9-HNE was measured, reflecting a human neutrophil elastase-derived fragment of calprotectin. Spearman’s rank correlation was applied.

Results

CPa9-HNE showed an overall positive correlation to the D'Haens score (ρ=0.279, p<0.005). Significant positive correlation was observed between CPa9-HNE and the presence of neutrophils, both in the epithelium and the lamina propria (ρ=0.309, ρ=0.349, p<0.005 both), as well as with epithelial damage (ρ=0.262, p<0.005). We also observed a positive correlation between CPa9-HNE and transmural fibrosis burden, specifically fibrosis in the submucosa (ρ=0.294, p<0.005)

Conclusion

This study showed that CPa9-HNE, a biomarker of neutrophil activity, correlated with the presence of neutrophils in the intestinal tissue, as well as epithelial damage. Interestingly, CPa9-HNE also showed association to submucosal fibrosis, emphasizing the importance of considering neutrophil activity when targeting the fibro-inflammatory axis. These findings highlight the potential use of CPa9-HNE as a complementary tool for histological grading of inflammation and fibrosis burden, and could be used to further explore the role of neutrophil extracellular traps in intestinal fibrosis.

Disclosure

MSA, TKA, MP, MAK, ACBJ and JHM are employees of Nordic Bioscience. MAK, ACBJ and JHM are shareholders of Nordic Bioscience.