Using the Pediatric RISK and PROTECT Inception Cohorts to Define Important Clinical and Biologic Phenotypes Ted Denson, MD Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine

Disclosures & Objectives Grant support: NIH & CCFA Describe aims & composition of RISK & PROTECT cohort studies Share early analyses from RISK cohort Summarize future goals for using cohorts to refine patient clinical and biologic phenotypes

PRO-KIIDS Enrolling sites Thomas D. Walters, SickKids, Toronto, Canada Subra Kugathasan, Emory-Children’s Center, Atlanta, GA CCHMC - GI Lee Denson Yael Haberman CCHMC Bioinformatics core Bruce J Aronow Phillip Dexheimer Curtis Huttenhower Timothy L Tickle Ramnik J Xavier Dirk Gevers

Current IBD Clinical Phenotyping Suspect the diagnosis –History, exam, lab tests Determine disease location –Upper endoscopy, colonoscopy, CT/MR enterography Classify as CD, UC, or IBD-U –Montreal or Paris classification for disease location, extent, severity, and complications

Importance of IBD Clinical Phenotypes Louis et al Best Pract & Res Clin Gastro 2011

Crohn’s Disease Progresses on “Conventional Therapy” in Children: Vernier-Massouille et al. Gastroenterology 2008;135:1106 Inflammatory Stricturing Penetrating 34% at 5 yrs

Combined Genetic:Paneth Cell Phenotype Stappenbeck et al Gastro 2013

High Risk CD Patient for Complications: CD1 NOD2 gene mutation Ileal location High titer anti-microbial serologies

Siegel et al IBD 2011 CD1: High Risk Anti-TNF Responsive Validate this model in a prospective inception cohort

Clinical use of Gene Expression Panels to Improve Diagnostic or Prognostic Accuracy Several gene expression diagnostics for oncology Afirma Thyroid Cancer test 56,540 thyroid cancer cases per year Indeterminate pathology in 30% Expression of 142 genes in thyroid biopsy 49 site validation in 3789 patients: 92% accuracy Prevent 25,000 thyroid resections per year Charge: $4200, covered by Medicare and third party Alexander et al NEJM 2012

Paris Classification Genotype Environmental Exposures Serology Microbial Community/Gene Expression 1112 children with CD at diagnosis between Follow-up to 2017 Define patients with complication / surgery CCFA Sponsored Pediatric Clinical Research Network: PRO-KIIDS RISK Study

AIMS OF RISK Develop and validate a composite risk score for complicating CD based upon age, cytokine and microbial antigen sero-reactivity, and early anti-TNF therapy Compare the effectiveness of early versus late introduction of anti-TNF upon rates of complicating behavior Profile host gene expression & the microbial community structure and function in tissue and stool samples Identify and validate intestinal gene expression and microbial community profiles associated with complicated behavior which will improve the accuracy of a model based upon clinical, genetic, and serologic factors

RISK Cohort Original Specific Aims Funded in 2009Progress to date in a: Recruit 1100 children (age at diagnosis < 16 years) with newly diagnosed inflammatory CD using standardized diagnostic criteria. Recruited 1112 CD. Additionally, we have recruited 160 UC, 129 IBDU, and 393 non-IBD as disease / healthy controls. 1b: Collect clinical and demographic information from well-characterized newly diagnosed CD patients. Collected comprehensive data at enrollment and every 6 months on every subject with the diagnosis of CD, UC, and IBDU. 1c: Collect serum samples at diagnosis and annually for both current serological immune markers including ANCA, ASCA, OMPC, I2 and CBir1 and newly identified markers including GM-CSF auto-antibodies. Serum was collected and all the stated antibodies were measured at diagnosis for all CD, UC, IBDU, and non-IBD control. Serum has been collected annually on CD, UC, and IBDU. 1d: Collect and genotype samples for known candidate genes and newly discovered variants which may influence the stricturing/penetrating outcome, and/or CD susceptibility. Immunochip genotyping (over 200,000 SNPs) has been completed in all CD, UC, and IBDU and non-IBD control subjects. Preliminary analysis shows that we have found novel early onset susceptibility variants. 1e: Collect mucosal biopsies in a subset inception cohort stored for subsequent aggregative gene expression analysis to define minimal genomic signatures which will distinguish CD patients who experience complicated disease behavior from those who do not. We collected over 5300 individual biopsies from 950 subjects. DNA and RNA extraction has been completed for all biopsies, with sufficient quality and quantity to support 16S microbial community profiling and RNASeq gene expression analysis in > 95% of samples. 1f: Establish a Centralized Data Coordinating Center based at Toronto Sick Kids utilizing a Clinipace web-based Data Management Platform and a Biospecimen Procurement Center and Repository at Emory University. A DCC and Bio-repository were established and have functioned very well to support the RISK study. Details regarding quality control processes for both data and sample management are provided in the Research Plan and the Appendix.

RISK Enrollment Birth & Environmental History

Comparative Effectiveness of Early IM vs anti-TNF for Week 52 SFR in RISK 552 with complete data and 1 yr f/u Anti-TNFα n = 68 No early immunotherapy n = 236 Early IM only n=248 Propensity Score Matching Anti-TNFα n = 68 No early immunotherapy n = 68 IM only n = 68 Hyams et al Gastro 2013

TreatmentYes (n=136)No (n=68) Early anti-TNFα only (n=68) 58 (85%)10 (15%) Early IM only (n=68) 41 (60%)27 (40%) No early immunotherapy (n=68) 37 (54%)31 (46%) CS-free, Surgery free (p=0.0003) 12 Month Outcomes For The Three Early Therapy Approaches: No difference between early IM and no early IM Hyams et al Gastro 2013

What Does This Mean? In clinically similar populations of children with Crohn’s disease, early (<3 mon) therapy with anti-TNFα was superior to early IM or no early immunotherapy despite later addition of those agents: PCDAI remission, CRP, growth There was no particular clinical or laboratory characteristic that helped predict response or non- response to an initial therapeutic decision It doesn’t mean that everyone should get anti-TNFα therapy, rather that we need to better define further characteristics of patients, such as genetics, serology, microbiome. Confirmatory studies will be required. Hyams et al Gastro 2013

Using Next Gen Sequencing to Classify the Intestinal Microbiome and Genome at Diagnosis Processed ~ 5300 intestinal biopsies from 950 CD, UC, IBDU patients and non- IBD controls DNA yield: 10,500 (8,468,12,670) ng RNA yield: 11,490 (9,351, 13,640) ng RNA quality sufficient for PCR or RNASeq in >95% Microbiome: 1000 ng DNA RNASeq: 1000 ng RNA Completed ileal & rectal RNASeq for 317 CD, 79 UC, 9 IBDU & 52 non-IBD control Completed ileal, rectal, and fecal 16S- microbial DNA profile for 477 CD & 221 non-IBD control

Ileal Gene Expression Panel Associated with Complicated Behavior in High Risk Ser2+ Patients

Between: Genes from the APOA1 module (APOA1, CXCL9) Genes from DUOX2 module (DUOXA2, MUC4, LCT) Clinical phenotype (Ctl, UC, CD) endoscopic severity (ileal deep ulcers) Clinical severity (PCDAI) Multivariate Analysis by Linear Models (MaAsLin) ileal microbial community Controlling for: age, gender, body mass index (BMI), and NOD2, FUT2, and ATG16L1 risk allele carriage. & Haberman et al 2013

Host:Microbe Profiles Present at Diagnosis May Define IBD Biologic Phenotypes Haberman et al 2013 UC1 UC2 CD1 CD2 CD2 greater clinical severity at diagnosis Lower rate of SFR with conventional therapy Two-fold higher anti-TNF exposure

Treatment Response Phenotype: “5-ASA Responsive UC” Disease Severity Moderate Mild Severe Enteral Nutrition Budesonide Antibiotics Aminosalicylates Methotrexate Thiopurines Prednisone Surgery Biologics CD UC

1U01 DK

AIMS OF PROTECT Define rates of week 52 steroid-free remission with 5- ASA as sole maintenance therapy in an inception cohort of 430 pediatric UC patients receiving standardized induction therapy with 5-ASA/corticosteroids Test clinical and biologic predictors of week 52 SFR Define the host rectal global pattern and of gene expression and rectal and fecal microbial community structure and function Identify mucosal host and microbial biologic pathways associated with initial treatment response and week 52 SFR

PROTECT Recruitment

Future IBD Phenotyping RISK & PROTECT cohorts: 2000 CD & UC patients Prospective clinical course & treatment responses Genetics & serology in all Tissue gene expression and microbial community profile in fifty percent Optimizing technique to isolate RNA & DNA from clinical path specimens in order to perform gene expression and microbial community analysis on all patients Key to maintain accurate long-term follow-up to link these host and microbial profiles to clinical outcomes Utilize to define combined clinical/biologic/treatment response phenotypes