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Background Inflammatory bowel diseases (IBDs) involve an aberrant host response to intestinal microbiota causing mucosal inflammation and gastrointestinal symptoms. Patient-reported outcomes (PROs) are increasingly important in clinical care and research. Our aim was to examine associations between PROs and fecal microbiota in patients 0 to 22 years of age with IBD. Methods A longitudinal, prospective, single-center study tested for associations between microbial community composition via shotgun metagenomics and PROs including stool frequency and rectal bleeding in ulcerative colitis (UC) and abdominal pain and stool frequency in Crohn’s disease (CD). Mucosal inflammation was assessed with fecal calprotectin. A negative binomial mixed-effects model including clinical characteristics and fecal calprotectin tested for differentially abundant species and metabolic pathways by PROs. Results In 70 CD patients with 244 stool samples, abdominal pain correlated with increased relative abundance of Haemophilus and reduced Clostridium spp. There were no differences relative to calprotectin level. In 23 UC patients with 76 samples, both rectal bleeding and increased stool frequency correlated with increased Klebsiella and reduced Bacteroides spp. Conversely, UC patients with lower calprotectin had reduced Klebsiella. Both UC and CD patients with active symptoms exhibited less longitudinal microbial community stability. No differences in metabolic pathways were observed in CD. Increased sulfoglycolysis and ornithine biosynthesis correlated with symptomatic UC. Conclusions Microbial community composition correlated with PROs in both CD and UC. Metabolic pathways differed relative to PROs in UC, but not CD. Data suggest that microbiota may contribute to patient symptoms in IBD, in addition to effects of mucosal inflammation.

Lay Summary Active cytomegalovirus (CMV) infection complicates management of inflammatory bowel disease, but the relationship of resolved CMV infection to Crohn’s disease (CD) behavior or localization is unknown. This article reports a striking risk (9-fold) of Crohn’s disease localization to the colon with prior CMV infection. It also reports imputed mucosal cellular composition, HLA class 1, and KIR gene variants that delimit prior observations regarding HLA and KIR associations with Crohn’s disease risk and behavior.

Background Develop a clinical and biological predictive model for colectomy risk in children newly diagnosed with ulcerative colitis (UC). Methods This was a multicenter inception cohort study of children (ages 4-17 years) newly diagnosed with UC treated with standardized initial regimens of mesalamine or corticosteroids (CS) depending upon initial disease severity. Therapy escalation to immunomodulators or infliximab was based on predetermined criteria. Patients were phenotyped by clinical activity per the Pediatric Ulcerative Colitis Activity Index (PUCAI), disease extent, endoscopic/histologic severity, and laboratory markers. In addition, RNA sequencing defined pretreatment rectal gene expression and high density DNA genotyping by the Affymetrix UK Biobank Axiom Array. Coprimary outcomes were colectomy over 3 years and time to colectomy. Generalized linear models, Cox proportional hazards multivariate regression modeling, and Kaplan-Meier plots were used. Results Four hundred twenty-eight patients (mean age 13 years) started initial theapy with mesalamine (n = 136), oral CS (n = 144), or intravenous CS (n = 148). Twenty-five (6%) underwent colectomy at ≤1 year, 33 (9%) at ≤2 years, and 35 (13%) at ≤3 years. Further, 32/35 patients who had colectomy failed infliximab. An initial PUCAI ≥ 65 was highly associated with colectomy (P = 0.0001). A logistic regression model predicting colectomy using the PUCAI, hemoglobin, and erythrocyte sedimentation rate had a receiver operating characteristic area under the curve of 0.78 (95% confidence interval [0.73, 0.84]). Addition of a pretreatment rectal gene expression panel reflecting activation of the innate immune system and response to external stimuli and bacteria to the clinical model improved the receiver operating characteristic area under the curve to 0.87 (95% confidence interval [0.82, 0.91]). Conclusions A small group of children newly diagnosed with severe UC still require colectomy despite current therapies. Our gene signature observations suggest additional targets for management of those patients not responding to current medical therapies.

BACKGROUND The majority of children with ulcerative colitis present with extensive colitis at diagnosis, but the response to therapy is heterogenous. Identifying the optimal window for biologic treatment and risk stratification of patients remains an unmet need. AIM To develop a pathology based histomic model to predict corticosteroid free clinical remission (CSF) with mesalamine alone at one year. METHODS 292 hematoxylin and eosin diagnostic treatment naïve rectal mucosal biopsies from the multi-center PROTECT study were digitized. Whole slide images (WSIs) underwent two-step pre-processing a)stain normalization and b)informative patch selection (size 512x512). We trained machine learning (ML) models using 250 histomic features (texture, color, histogram and nuclei features) with 5-fold cross-validation, for patch-level classification. Feature importance was determined by the Gini index. We re-trained the classifier using the top features. Slide-level prediction was defined by threshold voting. Performance metrics at the patch and WSI level was evaluated. A total of 161 patients underwent high-throughput RNA sequencing to define rectal gene expression. We undertook unsupervised weighted gene co-expression network analysis (WGCNA) to discover networks of co-expressed genes with shared biologic functions correlated with histomic features, histological traits, and outcomes. RESULTS 187571 informative patches from 292 patients (Male:55%; Age:12.7y (IQR:11-15); CSF remission:41%) were trained on 23 ML classifiers. The best model trained on 250 features at the patch-level was random forest (RF). At a remission ratio threshold of 0.48, WSI area under the receiver operator curve (AUROC) was 0.90 (95%CI:0.70, 1.00), accuracy 90.4%, precision 90.9% and recall 84.7%. 18 top features were identified and trained, and the corresponding WSI AUROC was 0.87 (95%CI:0.72, 1.00), accuracy 90.1%, precision 89.4%, and recall 83.9% (Fig. 1). We re-trained the 18 features on an independent real-world dataset of 131 UC patients and the model WSI AUROC was 0.85 (95%CI:0.74, 1.00) and an accuracy of 88.5%. Of the 13 modules identified by WGCNA analysis, six modules significantly correlated with clinical and/or histomic features (Fig. 2). Two of the gene co-expression modules were negatively associated with baseline clinical, endoscopic, and histologic measures of severity, and positively associated with nuclei features (Otsu area, perimeter, and equivalent diameter) and the outcome measure of CSF remission. Intersection of genes with adult single cell RNA seq data demonstrated enrichment for enterocytes (SLC26A3) and extracellular matrix (IHH) CONCLUSION We developed a predictive model for UC disease course using histomic features from standard of care pre-treatment pathology images. Characterization of the underlying molecular basis of the histomic features is ongoing.

Background Reports on the feasibility and effectiveness of translating proactive, antitumor necrosis factor (TNF) therapeutic drug monitoring (TDM) for inflammatory bowel disease into practice-wide quality improvement (QI) are lacking. We aimed to determine whether a TDM QI program improved outcomes at a large academic pediatric gastroenterology practice. Methods We instituted local anti-TNF TDM practice guidelines to proactively monitor and optimize drug levels (goal >5 μg/mL). We conducted a retrospective single-center cohort analysis of patient outcomes before (pre-TDM) and after (post-TDM) guideline institution and assessed the independent effect by multivariable regression. Primary outcome was sustained clinical remission (SCR22-52), defined as physician global assessment (PGA) of inactive from 22 to 52 weeks and off corticosteroids at 52 weeks. Results We identified 108 pre-TDM and 206 post-TDM patients. The SCR22-52 was achieved in 42% of pre-TDM and 59% of post-TDM patients (risk difference, 17.6%; 95% CI, 5.4–29%; P = 0.004). The post-TDM group had an increased adjusted odds of achieving SCR22-52 (odds ratio, 2.03; 95% CI, 1.27–3.26; P = 0.003). The adjusted risk of developing high titer antidrug antibodies (ADAs) was lower in the post-TDM group (hazard ratio, 0.18; 95% CI, 0.09–0.35; P < 0.001). Although the risk of anti-TNF cessation for any reason was not significantly different, there was a lower adjusted risk of cessation related to any detectable ADA in the post-TDM group (hazard ratio, 0.45; 95% CI, 0.26–0.77; P = 0.003). Conclusions A practice-wide proactive anti-TNF TDM QI program improved key clinical outcomes at our institution, including sustained clinical remission, incidence of high titer ADA, and anti-TNF cessation related to ADA.

Background Viral infections have been suggested as possible triggers for the onset of ulcerative colitis (UC). Methods We employed VirCapSeq-Vert, a high-throughput sequencing virus capture platform, to examine the stool virome of children with newly diagnosed moderate to severe UC. We surveyed fecal samples collected at presentation, after symptom remission, and from a control group diagnosed with irritable bowel syndrome. Results Seventy subjects with UC (mean age 13 years, 45 had moderate symptoms, 25 had severe, 69 of 70 had a Mayo endoscopy subscore 2/3) were studied. We detected a wide range of animal viruses that were taxonomically classified into 12 viral families. A virus was present in 50% of fecal samples collected at presentation, 41% of samples collected after remission, and 40% of samples in our control group. The most frequently identified viruses were diet-based gyroviruses. The UC cohort had a significantly higher prevalence of anelloviruses compared with the control cohort. However, we did not identify a single virus that can be implicated in the onset of UC and did not find an association between UC disease severity and viral presence. Conclusion Presence of virus in stool was not associated with the onset of pediatric UC., We used VirCapSeq-VERT, a high-throughput sequencing virus capture platform, to examine the stool virome of children with newly diagnosed moderate to severe ulcerative colitis. We did not identify a link between a viral infection and the onset of ulcerative colitis.

Precision medicine is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, novel technologies, and pragmatic clinical research. The Challenges in IBD Research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the precision medicine section is focused on highlighting the main gap areas that must be addressed to get closer to treatments tailored to the biological and clinical characteristics of each patient, which is the aim of precision medicine. The main gaps were identified in: 1) understanding and predicting the natural history of IBD: disease susceptibility, activity, and behavior; 2) predicting disease course and treatment response; and 3) optimizing current and developing new molecular technologies. Suggested approaches to bridge these gaps include prospective longitudinal cohort studies to identify and validate precision biomarkers for prognostication of disease course, and prediction and monitoring of treatment response. To achieve this, harmonization across studies is key as well as development of standardized methods and infrastructure. The implementation of state-of-the-art molecular technologies, systems biology and machine learning approaches for multi-omics and clinical data integration and analysis will be also fundamental. Finally, randomized biomarker-stratified trials will be critical to evaluate the clinical utility of validated signatures and biomarkers in improving patient outcomes and cost-effective care.

Background The neutrophil fecal biomarkers, calprotectin (FCP) and lactoferrin (LCT), and peripheral blood neutrophil CD64 surface receptor (nCD64) are biomarkers for mucosal inflammation in inflammatory bowel disease (IBD). Although FCP has been evaluated as a biomarker for mucosal healing, cut points for LCT and nCD64 are less known. We aimed to identify the cut points for LCT and nCD64 that were associated with FCP remission, with a secondary aim to evaluate the relationship between biochemical outcomes and infliximab (IFX) trough concentrations. Methods We analyzed FCP, LCT, and nCD64 before and after IFX induction in a pediatric Crohn’s disease (CD) cohort study. Week-14 FCP biomarker remission was defined as FCP 17.5 improvement. Predictive outcomes were calculated by receiver operating characteristics (ROCs). Results Among 56 CD patients, ROC analysis identified an infusion 4 LCT 9.4 µg/mL (AUROC, 0.799, P = 0.002) and >11.5 µg/mL (AUROC, 0.835, P = 0.003) were associated with a FCP 5 µg/mL had a median FCP improvement (dose 1 to dose 4) of 90% compared with a median of 35% with levels Conclusions This study establishes cut points in neutrophil stool and blood biomarkers for both biochemical remission and therapeutic trough levels following induction therapy. Further studies that evaluate pharmacodynamic biomarker targets for endoscopic and histologic healing are warranted.

Author(s): Denson, Lee A; Jurickova, Ingrid; Karns, Rebekah; Shaw, Kelly A; Cutler, David J; Okou, David; Valencia, C Alexander; Dodd, Anne; Mondal, Kajari; Aronow, Bruce J; Haberman, Yael; Linn, Aaron; Price, Adam; Bezold, Ramona; Lake, Kathleen; Jackson, Kimberly; Walters, Thomas D; Griffiths, Anne; Baldassano, Robert N; Noe, Joshua D; Hyams, Jeffrey S; Crandall, Wallace V; Kirschner, Barbara S; Heyman, Melvin B; Snapper, Scott; Guthery, Stephen L; Dubinsky, Marla C; Leleiko, Neal S; Otley, Anthony R; Xavier, Ramnik J; Stevens, Christine; Daly, Mark J; Zwick, Michael E; Kugathasan, Subra | Abstract: BACKGROUND:Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS:Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS:We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P l 0.0001). CONCLUSIONS:Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

Background Inflammatory bowel disease (IBD) mainly consists of Crohn's disease (CD) and ulcerative colitis (UC). About 10%-15% of patients with IBD cannot be firmly diagnosed with CD or UC; hence, they are initially diagnosed as inflammatory bowel disease unclassified (IBD-U). Having a firm diagnosis is clearly preferred to guide treatment choices, and better understanding of the nature of IBD-U is required. Methods We performed an analysis of a subset of pediatric subjects from an inception IBD cohort of patients initially enrolled in a prospective multicenter study (the RISK study). Initial diagnosis and 2-year follow-up data from the subjects diagnosed with IBD-U were analyzed. An expert panel verified final diagnosis using predefined criteria as a guide. Serological and disease-relevant ileal and rectal tissue gene expression profiles were investigated. The use and the time to initiate anti-TNFα treatment was analyzed among the outcome groups. Results A total of 1411 subjects were enrolled with initial diagnosis of IBD, and among them, 136 subjects were initially diagnosed as IBD-U at enrollment. And 26% were reclassified as UC and 14% as CD within 2 years of diagnosis, while 60% remained as IBD-U. Of those who were reclassified, there was a 2:1 ratio, UC (n = 35) to CD (n = 19). The molecular and serological features of IBD-U at the end of follow-up were very similar to UC and very different from CD. There was less likelihood of receiving anti-TNFα agents if the diagnosis was IBD-U compared with CD (P < 0.0001). Conclusions In our cohort, 60% of the IBD-U subjects remained as unclassified at 2 years; of those subsequently classified, a higher percentage followed a course more similar to UC. Most of the IBD-U subjects at diagnosis had serological and molecular signatures that are very similar to UC. Although the atypical presentations made the clinician to make an interim diagnosis of IBD-U, results of the molecular and serological factors performed at the time of diagnosis suggests that they were very similar to UC. However, long-term studies are needed to better understand the natural history and molecular characterization of pediatric onset IBD-U. 10.1093/ibd/izy136_video1Video 1.Video 1. Watch now at https://academic.oup.com/ibd/article-lookup/doi/10.1093/ibd/izy136izy136.video15791389938001.

INTRODUCTION Use of vedolizumab in pediatric inflammatory bowel disease (IBD) often requires an interval intensification from standard dosing due to distinct pharmacokinetics (PK) in children. Adult population PK models found inclusion of albumin, weight, anti-TNF naivety and antibodies-to-vedolizumab (ATV) as covariates of drug clearance (CL) improved model accuracy. We aimed to build a population-PK model for children and identify novel covariates of drug CL to better account for pediatric-specific variability in vedolizumab PK. METHODS This was a prospective multicenter cohort study for pediatric IBD patients starting vedolizumab. Vedolizumab dosing regimens were managed by the treating clinician. Stool, serum and plasma were collected at standardized intervals for robust PK sampling and biomarker analysis. Population PK analysis were conducted with nonlinear mixed effects modeling (NONMEM 7.5). A 1-compartment model with proportional error model was created based on the PK data. In the final model, novel covariates were identified using a stepwise approach. A decrease in the objective function value (OFV) >3.84 was considered significant (P RESULTS Twenty-one patients had a total of 277 infusions with 274 measured vedolizumab peak and trough concentrations. Among these, 57% were male, 76% had Crohn’s disease, and 81% had prior anti-TNF exposure. The median (IQR) age was 16 years (13-18), and median weight was 52kg (41-71). Baseline biomarkers are described in Table 1. Baseline CL was inversely correlated with albumin (R=-0.54, P= 0.012), hemoglobin (R=-0.45, P= 0.048), and platelets (R=-0.5, P= 0.026). There was a positive correlation of CL with CRP (R=0.8, P< 0.001), neutrophil CD64 (nCD64, R=0.68, P= 0.001), fecal calprotectin (FCP, R=0.63, P= 0.009), and BSA (R=0.59, P= 0.007). The final model estimated CL as 0.168 L/d (relative standard error (RSE), 13%) and volume (V) distribution as 3.97L (8%). The interindividual variability (IIV) of the final model was 62.2% (25%) of CL and 37% (8%) of V. Covariates that significantly decreased the OFV were incorporated into the final model including weight for V and albumin, ESR, and nCD64 for CL. Prior anti-TNF, immunomodulators (MTX, n=3) or ATV (n=1) did not impact CL. Baseline CL CONCLUSIONS We describe a novel pediatric PK model for vedolizumab that identified novel covariates, including ESR and nCD64, that may guide model-informed precision dosing. Larger cohorts should establish whether additional compartments and pediatric-specific covariates can further decrease the remaining unexplained variability.

BACKGROUND Human gastrointestinal (GI) organoid technologies have transformed GI disease research. By recapitulating the essential steps that occur during embryonic organ development, we could generate in vitro human colonic organoids (HCOs) (Munera et al, Cell Stem Cell. 2017) and human intestinal organoids (HIOs) (Spence et al, Nature. 2011) from iPSCs. Interestingly, HCOs contain both epithelial and surrounding mesenchymal derivatives, including myofibroblasts and tissue-resident immune cells. Our preliminary data demonstrate that HCOs co-develop CD163 positive macrophages derived from the hemogenic endothelium that develops within the colonic mesenchyme. Conversely, HIOs do not spontaneously develop tissue-resident immune cells. OBJECTIVE To incorporate tissue-resident immune cells into HIOs and use this new model to interrogate diseases such as IBD and Crohn’s Disease. RESULTS HIOs did not form endothelial tubes and immune cell floaters as seen in HCOs under the microscope 20 days after the start of differentiation. Moreover, HIO supernatants yielded few to no CFUs in Methocult assays compared to HCOs. Therefore, we tried two approaches to generate a novel HIO model system containing resident macrophages. First, we tried to transfer mesenchymal cells innately present in HCOs. We dissociate mesenchyme from HCOs and recombine them with HIO epithelium around day 20 from the start of differentiation (Figure 1A). We could successfully dissociate HCO-mesenchymal components from their epithelium and recombined them with HIO epithelium, but we could not detect CD163 positive macrophages in recombined HIOs. Second, we tried to transfer the supernatant of HCOs to HIOs around day 20 from the start of differentiation (Figure 1B). We confirmed the presence of CD163+ macrophages in day 35 HIOs using immunostaining and RNA sequencing (Figure 2A, B). We also confirmed the expression of cytokines such as IL10, IL12, and TGFβ via qPCR. Furthermore, we wanted to investigate whether these tissue resident macrophages were maintained after further maturation. As previously described, HIO transplantation into the mouse kidney capsule for 12 weeks allows for tissue maturation enabling crypt/villus formation and smooth muscle differentiation (Figure 2C) (Watson et al, Nat. Med. 2014). After recombined HIOs were transplanted, human CD163+ macrophages displayed expansion and were not displaced by bone marrow derived mouse macrophages (expressing F4/80). In the future, we also plan to differentiate monocytes directly from iPSCs and inject the monocytes into HIOs to see if they resident in the tissue as macrophages (Figure 1C). CONCLUSION We generated HIOs containing tissue-resident macrophages by transferring supernatant of HCOs. We expect to accelerate our understanding of the mechanisms of IBD such as CD because of the development of HIOs technology containing of immune cells.