Background Despite the proven efficacy of biological treatments in Crohn’s disease (CD), many patients fail to respond or lose response over time. Therefore, predictive biomarkers for treatment efficacy would be of extreme value. Previous epigenome-wide association studies associated differential DNA methylation with CD-specific phenotypes, suggesting a potential use in classification and prediction of response to treatment. Methods We prospectively collected and measured longitudinal peripheral blood DNA methylation profiles of 184 adult CD patients prior to (T1) and after a median of 28 weeks (T2) following biological treatment with Adalimumab (ADA), Vedolizumab (VEDO) or Ustekinumab (USTE) in a discovery (n=88) and independently collected internal validation cohort (n=96) using the Illumina EPIC BeadChip array. Response (R) was defined as the combination of endoscopic response (≥50% reduction in SES-CD score) and steroid-free clinical response (≥3 point drop in HBI or HBI ≤4 AND no systemic steroids) and/or biochemical response (≥50% reduction in C-reactive protein (CRP) and fecal calprotectin or a CRP ≤5 g/mL and fecal calprotectin ≤250 µg/g). Biomarker identification and classification analyses were performed using stability selection gradient boosting on samples taken at T1 whereas samples taken at T2 and intraclass correlation (ICC) data were used to assess long-term stability of our identified CpG loci1. Results A total of 58 ADA-patients (NR=29, NNR=29), 64 VEDO-patients (NR=36, NNR=28) and 62 USTE-patients (NR=30, NNR=32) were included. Prior to treatment, at T1, we identified distinct panels of 100 ADA-, 22 VEDO- and 68 USTE-associated CpG loci that, in combination, predict clinical- and endoscopic response with high accuracy (AUC ADA=0.73, VEDO=0.89 and USTE=0.94) upon validation. Notably, for these CpG loci, methylation levels did not significantly differ between T1 and T2, implicating stability during both induction and maintenance treatment, irrespective of inflammatory status and therapeutic intervention. In addition, the majority of these CpG loci (>60%) demonstrated long-term hyper stability (ICC-values ≥0.90). Furthermore, genes annotated to the CpGs of interest suggest drug specific involvement in TNF-signaling, endothelial cell-cell adhesion, integrin dependent T-cell homing, the innate immune system and Th17/Treg differentiation, corroborating to the mode of action of each drug. Conclusion Here, we report on 3 validated panels of highly stable, epigenetic biomarkers that predict clinical and endoscopic response in CD patients treated with ADA, VEDO or USTE. Additional external- and clinical validation as part of EPIC-CD and the OMICROHN clinical trial are currently ongoing.
Background Abdominal pain is a common occurrence for approximately 39% of patients with inflammatory bowel diseases in remission (quiescent IBD; qIBD). There is increasing evidence for a contributing role of the gastrointestinal fungal community (i.e., mycobiome) in relation to intestinal inflammation and irritable bowel syndrome (IBS). As such, abundance of Candida spp. and sub-species variation of Candida albicans were previously associated with IBD severity. In the current study, we aim to investigate mycobiome of patients with qIBD and qIBD with abdominal pain (qIBD–AP). Methods Patients with qIBD (defined as faecal calprotectin (FCP) ≤250 μg/g) with (n=91) or without (n=58) abdominal pain provided faecal samples. Abdominal pain was scored based on either the Irritable Bowel Syndrome Symptom Severity Scale (IBS–SSS) or Gastrointestinal Symptom Rating Score (GSRS) questionnaire. Faecal fungal communities were determined based on Internal Transcribed Spacer 1 (ITS1) sequencing. Cultivable yeasts from faecal samples were identified using Matrix Assisted Laser Deionization Time–of–Flight Mass Spectrometry. C. albicans strains (n=137 from 29 patients) were genotyped by ITS Sanger sequencing analysis and typing of seven microsatellite loci. Release of virulence-related enzymes (proteinase, phospholipase, lipase, esterase) by C. albicans was assessed through determination of precipitation zones on solid agar mediums containing enzyme-specific substrates. Results Descriptive mycobiota analysis revealed limited differences between compositions of qIBD and qIBD–AP patients, but a discriminative signature for abdominal pain was extracted using machine-based learning models. While ITS Sanger sequencing of faeces-derived C. albicans was insufficient to elucidate genetic variability, analysis of microsatellite loci revealed extensive variability and clustering into six clone clusters and a likely distinction between qIBD and qIBD-AP patients (Fig. 1). Phospholipase enzymatic activity of C. albicans strains correlated with GSRS–Abdominal Pain sub-score, as did proteinase activity with severity of abdominal pain according to the IBS–SSS (Fig. 2). Additionally, lipase activity inversely correlated with faecal calprotectin (Fig. 2). Conclusion The faecal gut mycobiome is associated with self–reported abdominal pain in patients with IBD in remission. This notion is based on both compositional and culture-dependent methods and specified clones of C. albicans may selectively contribute to abdominal pain for qIBD patients. This study opens further possibilities to investigate the role of faecal gut fungi in light of abdominal pain for qIBD-AP patients.
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