Jesse S Siffledeen, Travis Murdoch, Carolyn Slupsky 1, Haili Wang, Richard N Fedorak University of Alberta, Edmonton, Alberta, Canada Center of Excellence For Gastrointestinal Inflammation and Immunity Research UC Davis, California, USA 1 Metabolomics In IBD: Development of Novel Diagnostic Tests for IBD, Polyps and Cancer

Background 25,000 Genes 7500 Enzymes 4000 chemicals Metabolomics Proteomics Genomics Metabolomics The characterization of small molecular weight compounds in biofluids or tissues. The urinary metablolomic fingerprint reflects the somatic (minor) and luminal bacterial (major) constituency

1.Altered luminal microflora play a key pathogenic role in IBD and colonic dysplasia… 2.Changes in microflora have been shown to markedly alter small molecule metabolites… Analysis of urinary metabolite profile (metabolomics) could differentiate individuals with different luminal bacterial profiles Background Hypothesis

1.Find urinary metabolomic profiles that differentiated patients with IBD from those without IBD and …. 2.…. those with colonic polyps from those without colonic polyps 3.Develop a commercial biomarker for IBD/colonic polyps/cancer and a predictor of response to therapy Objective

Patients with IBD provided a spot urine sample and completed a demographics questionnaire, HBI (CD; n=30) and modified Mayo Score (UC; n=30) Patients with IBS (n=30) were controls Metabolite concentrations were derived from analysis of 600Hz NMR spectra and sorted by partial least squares discriminate analysis Methods

Metabolomics Workflow Biofluids or Extracts NMR or MS Chemical Analysis pp m Data Analysis

Urine Metabolomics NMR spectra analysis defines 69 metabolites (Chenomx®NMRSuite v7.0)

Urine Metabolomics N= 69 metabolites in order of contribution to separation of group A from group B Concentration Partial least square discriminate analysis definesvery important metabolites and their concentrations most contributing metabolites least contributing metabolites

Results – IBD vs Control Principle component analysis uses 69 metabolites to separate IBD vs Control Sensitivity = 78% Specificity = 89%

Results – Active CD vs Active UC Separation of patients with active CD vs active UC

Change in Urinary Metabolites MetaboliteCrohns Disease Ulcerative Colits Levoglucosan* Sucrose Hippurate* Fucose* Creatine TMAO* DMA* * of bacterial origin

N=1075 >50 yr screening Colonoscopy + biopsy (gold standard) FOBT (FGT&FIT) Urine metabolomics Detection of: Polyps N=116 colon cancer Surgery + biopsy Urine metabolomics Detection of: Colon cancer Urine metabolomics from 77 CRC & 236 polyp & 422 normal patients analyzed to develop ColoDx TM and PolypDx TM = open label diagnostic cohort Urine metabolomics from 39 CRC & 119 polyp & 211 normal patients blinded for ColoDx TM and PolypDx TM validation = blinded validation cohort ColoDx & PolypDx Clinical Trial 87 Excluded samples due to: Incomplete colonoscopy Other diagnoses Corrupted urine sample

ColoDx & PolypDx Clinical Trial Principal component analysis uses 69 metabolites to separate each group NORMALPOLYP

ColoDx & PolypDx Clinical Trial After polypectomy the urine metabolomic fingerprint reverts back to a normal state POLYP Post Polypectomy

Dx TM a screening test for colon cancer & polyps ThresholdsSensitivitySpecificityAUC ColoDx 197%90% 0.99 ColoDx 287%96% ColoDx 376%99% ColoDx TM detects colon cancer PolypDx TM detects adenomatous polyps Finding adenomatous polyps is the real need ! ColoDx & PolypDx Clinical Trial MetabolitesSensitivitySpecificityAUC PolypDx 181%50% 0.73 PolypDx 271%61% PolypDx 366%65%

TestSensitivitySpecificity PolypDx TM 7161 Hemoccult II (guaiac) 199 Hemoccult ICT (immune) 697 Magstrema HemSp/HT (immune) 1595 Finding adenomatous polyps is the real need ! PolypDx Clinical Trial PolypDx TM sensitivity to detect adenomatous polyps compared to current tests

TestSensitivitySpecificity PolypDx TM 2395 Hemoccult II (guaiac) 199 Hemoccult ICT (immune) 697 Magstrema HemSp/HT (immune) 1595 PolypDx TM sensitivity to detect adenomatous polyps compared to current tests – specificity held constant Finding adenomatous polyps is the real need ! PolypDx Clinical Trial

Engage subject yearly PolypDx ® screening test in commercial development Colonoscopy Follow-up/Surveillance positive negative The PolypDx Opportunity