Short summary

In the NORDTREAT cohort study, a Nordic prospective multicentre study, we aim to identify novel molecular biomarkers of diagnostic value by assessing the diagnostic test accuracy, as well as the prognostic utility when used as prognostic markers in the long-term (cohort study).

Rationale

Inflammatory bowel disease (IBD), primarily ulcerative colitis (UC) and Crohn's disease (CD), is a chronic disease entity affecting individuals of all ages, including children, which severely impacts the lives of the patients and their families as well as society. Individuals with IBD may have to live with relapsing symptoms, such as diarrhea, abdominal pain, and fatigue. Further, a substantial proportion of patients develop serious complications such as bowel obstruction, fistula, and disease manifestations from other organs than the gut (e.g. skin, eyes, joints) and some develop complicating liver disease and eventually colorectal cancer. The consequences are that many patients suffer hospitalizations, recurring sick-leave, life-long medication, and surgical interventions. As IBD has become increasingly common in Western populations, with a prevalence of up to 1.0% in some Nordic countries, it is posing an increasing burden on the society in the form of sick-leave and use of health care resources. Hence, there is a clear need to improve the outcome from IBD. IBD is a heterogeneous disease entity with substantial differences between patients. Personalized Medicine may help provide strategies for better treatment of IBD patients. Currently, one of the main unmet needs is the glaring lack of robust biomarkers for individual disease characterization. This lack leads to delayed diagnosis, worse outcomes, increased mortality and an amplified disease burden. Personalized medicine uses a person's individual genetic and other characteristics (e.g. molecular profile, phenotype, imaging) to arrive at "the right diagnosis, the right treatment, at the right dose, to the right patient, at the right time". Ultimately, personalized medicine represents a shift from reactive to proactive and preventive care (ICPerMed.eu). Diagnosis of IBD is difficult. The European guideline on diagnosis of IBD states: "A single reference standard for the diagnosis of Crohn's disease [CD] or ulcerative colitis [UC] does not exist. At a minimum, the diagnosis of CD or UC is based on a combination of clinical, biochemical, stool, endoscopic, and histological investigations. When CD is suspected, it may be necessary to visualize [radiologically] the small intestine. Infectious colitis, including Clostridium difficile, should be excluded." Early diagnosis is crucial as it helps avoid the development of irreversible organ damage. Therefore, there is an emerging focus on the development of simple, non-invasive, and cheap biomarkers which can support clinical decision-making in IBD. Several potential biomarkers for diagnostic purpose have been described. Fecal calprotectin (FC) is widely used is clinical practice. Calprotectin (an heterodimer made of Myeloid Related Protein 8 and 14 (MRP8 and MRP14)), a product of activated neutrophils, macrophages, epithelial cells and monocytes, responds to Microbe-Associated Molecular Pattern (MAMP) and Pattern Recognition Receptor (PRR) being involved in the early steps of the immune response. Various studies have found FC to have a high sensitivity and specificity in diagnosing IBD among patients suspected for IBD. In a review including 670 cases the pooled sensitivity and specificity of FC to diagnose IBD with colonic disease was found to more than 90% although the performance may be lower for small bowel disease. It has been found that FC better reflects UC than CD endoscopic activity. Serum calprotectin (SC) may offer advantages for diagnostic purposes in clinical practice. First of all, as it is measured in blood it may reflect systemic inflammation, and thus have the potential to add information to the FC, i.e. it may show its value in patients with an upper gastrointestinal disease location. Other advantages include that SC can be used where FC is not feasible and may be more easily included in a fast-track pipeline for response on the day of visit. Only few studies have been performed investigating the potential of SC in IBD. Kalla et al investigated new patients with IBD (N=83) and a control group of healthy controls and patients with other disease (N=73) in a tertiary hospital setting. SC was higher among the IBD patients than among the controls and was built into a diagnostic score based on a multivariate model (SC>852ng/ml). In another study focusing on CD, Meuwis et al, found SC correlated with disease activity (CDAI) but not with endoscopic disease activity (CDEIS). Other potential new biomarkers include NETs, found in UC, serum biomarkers of degraded collagens and other products. The generation of new and better biomarkers for clinical use requires prospective clinical cohorts with rigorously collected information. The clinical circumstances should reflect the situation that the biomarkers are going to be used for, including the clinical setting (primary care, hospital etc.), the individuals that the test are going to be applied to, and the reference standard (the diagnostic procedures). This study protocol describes a Nordic, prospective, multicenter, clinical study with the aim of identifying markers that are associated with the diagnosis of IBD and prediction of clinical outcomes with various disease manifestations. Importantly, this study will evaluate the markers in a relevant clinical setting, i.e. among patients referred to the hospital for suspicion on IBD using the ECCO Criteria. Study Aims, Hypotheses, and Objectives: - Improve the accuracy to define the prognosis of IBD The main hypothesis of the study is that biochemical biomarkers can discriminate between individuals diagnosed with IBD and individuals without IBD (non-IBD) as well as distinguishing different disease progress pathways for individuals with a suspected IBD. The primary aim is to identify molecular markers (e.g. in the blood and stools) for discrimination between individuals diagnosed with IBD (incl. CD, UC, and IBD-U), and those without (non-IBD). Among the individuals with suspected IBD at baseline, with various disease pathways, will be evaluated again using a 1-year cohort study. Diagnosis and clinical outcome will be evaluated at referral and after 1 year of observation. The secondary aims are, in addition to the molecular information, to investigate whether the inclusion of information on clinical and lifestyle factors as well as combination hereof (e.g. gene-environment interaction analyses) can improve the predictive potential of identifying IBD and distinguish the prognosis. In both the diagnostic and prognostic setting, probability estimates are commonly based on combining information from multiple predictors observed or measured from an individual. Information from a single biomarker predictor is often insufficient to provide reliable estimates of diagnostic or prognostic probabilities or risks. We will develop multivariable prediction models relating multiple predictors for a particular individual to the probability of or risk for the presence (diagnosis of IBD at baseline) or future occurrence (prognosis) of a particular outcome such as intestinal surgery within the first year. Predictors such as biomarkers are covariates, explored as prognostic factors -more statistically-these are independent variables. Objective for the cross-sectional study: To determine the sensitivity and specificity of biomarkers of interest to diagnose inflammatory bowel disease by using the ECCO Diagnostic criteria as the reference standard. Objective for the prospective longitudinal cohort study: To examine whether the biomarkers of interest predicts poor disease outcome defined as severe IBD within one year among people referred to a gastrointestinal department with suspicion of IBD. A composite outcome measure will be applied as our primary endpoint. The composite outcome of the proportion of patients who experience severe IBD within 52 weeks after inclusion is defined as: IBD-related surgery or IBD-related hospitalization (except planned procedures) or IBD-related death.

Description of the cohort

This is a prospective Nordic multi-center study on prognostic factors for the diagnosis and characterization of IBD among patients referred to the hospital on suspicion of IBD. All patients referred to the hospitals due to a suspicion of IBD will be invited to take part in the study. All participants will be treated according to standard clinical practice by the clinical departments. Thus, there is no intervention. In total, 300 participants will be included in the Nordic study. The participants will be followed up until week 52. The participants of the cohort will be classified as IBD and non-IBD based on the criteria defined below.

Data and biological material

Biological material will be obtained four times, at week-2/0, and 12, 26 and 52 after the diagnosis has been established. The visit, at week-2/0 is the baseline visit before the potential diagnosis. Study subjects where the diagnosis of IBD is not established (non-IBD) will only have biological material obtained at baseline in the project (that is visit -2/0) and will have a clinical interview after 52 weeks. Blood, fecal, urine and hair sampling will take place in relation to the subjects' visit at an outpatient clinic and do not require any additional visit. We aim to collect all biological material at the planned visits. However, if the collection is not possible, the participants will still be able to stay in the study. Blood samples At each study visit specific blood sampling, two dry tubes for serum (10 ml), two EDTA coated tubes (10 ml), one EDTA whole blood sample (4 ml) and two blood sample (3 ml) drawn in PAXGene tube (BD Bioscience) will be collected, a total of maximum 57ml. All blood samples will be stored at minus 80 degrees. Fecal samples Fecal samples will be collected at the patient's home, as described in the patient guidelines and handed in to the project team or send by mail. Fecal samples at inclusion and week 54 are preferably taken from the last stool before colonic cleansing begins, NOT after cleansing is started. If this is not possible, then collection will be performed 3 to 7 days after the colonoscopy. One sample will be collected at each time point and distributed in 3 different containers, 2 with stabilizing agents: RNA later and DNA stabilizer. Biopsies 14 mucosal biopsies for the biobank with video recording will be collected from all study subjects undergoing ileocolonoscopy at visit -2/0 and 52 weeks. However, this is not mandatory for participation in the study. Routine biopsies will be taken from all patients investigated for IBD according to international guidelines and include formalin fixated paraffin-embedded biopsies (FFPE). Urine and hair samples Spot urine and hair samples (10-20ug) will be collected at the hospital where they will be aliquoted and frozen according to SOPs. Urine and hair samples will be collected at visit -2/0 and 52 weeks.

Collaborating researchers and departments

1) Department of Medical Gastroenterology S, Odense University Hospital, Odense, Denmark

• Professor Jens Kjeldsen
• PhD fellow Anja Fejrskov

2) Molecular Diagnostics and Clinical Research Unit, Institute of Regional Health Research, University Hospital of Southern Denmark, Hospital Sønderjylland, Aabenraa, Denmark

• Andersen V

3) Section for Biostatistics and Evidence-Based Research, the Parker Institute, Bispebjerg and Frederiksberg Hospital, Copenhagen, Denmark

• Christensen R

4) Internal Medicine & Emergency Department Svendborg, Odense University Hospital, Svendborg, Denmark

• Aalykke C

5) Department of Internal Medicine, Örebro University Hospital, Region Örebro län, Örebro, Sweden

• Halfvarson J
• Rejler M
• Repsilber D
• Söderholm JD

6) Department of Gastroenterology, Oslo University Hospital, Oslo, Norway

• Høivik ML

7) Department of Internal Medicine-Gastroenterology, University Hospital of Southern Denmark, Lillebaelt Hospital, Vejle, Denmark

• Jørgensen MT

8) Gastrointestinal Diseases, Nordic Bioscience A/S, Herlev, Denmark

• Mortensen JH

9) Research Unit of Medical gastroenterology and hepatology, University Hospital of Southern Denmark, Hospital South West Jutland, Esbjerg, Denmark

• Jensen MD
• Nielsen LN