Discovery of proteic biomar k ers for C rohn Disease and U lcerative Colitis by SELDI-TOF-MS

1. M-A. Meuwis1, D de Seny1, M Fillet1, E Louis2, J Belaiche2, P Geurts3,L Wehenkel3, M Malaise 4 , M-P. Merville1. CD vsUC CD vsUC IBDvsHC, IC IBDvsHC, IC CD vsUC, IC, HC UCvsCD, IC, HC CD vsUC, IC, HC UCvsCD, IC, HC Discovery of proteic biomarkers for Crohn Disease and Ulcerative Colitis by SELDI-TOF-MS 1Laboratory of Medical Chemistry and Human Genetics, CTCM, CHU Sart-Tilman, 4000 Liege 2Stochastic Methods, Sart Tilman 4000 LG. 3 Hepato-Gastroenterology. CHU Sart Tilman Lg. 4 Clinical Sciences- Rhumatology, CHU, Sart Tilman, Lg. CBIG Université de Liège. 1 INTRODUCTION 3 Technic: SELDI -TOF-MS Crohn Disease ( CD) and Ulcerative Colitis (UC) both generally known as Inflammatory Bowel Disease (IBD) are chronic autoimmune inflammatory pathologies affecting the gastro intestinal tract. Their ethiopathogenesis has not been fully elucidated and involve a complex interplay among genetic, environmental, pathogenic and immune factors. The still growing knowledge in the ethiology of these disorders gave rise to new promising treatments. Nevertheless, the success of those drugs are cases dependent: CD or UC. Therefore, accurate and early diagnosis is a real important step in circumventing these pathologies. Today, clinical diagnosis are made on many biological data such as CRP, ASCA, ANCA determinations, according to severity of symptoms ( recorded in Harvey-Bradshow test) or with invasive techniques as gastro-endoscopies. No simple, rapid, unique and efficient technique is able to discriminate IBD from other inflammatory diseases (as infectious colitis) or among IBD itself: CD versus UC. Here, we present a strategy of sera protein profiling on SELDI-TOF ( Surface Enhanced Laser Desorption-Ionization, Time of Flight Mass Spectrometry ) and the downstream statistical analysis. Methods :Protein profiling by SELDI-TOF-MS (Surface Enhanced Lazer Desorption Ionisation -Time Of Flight - Mass Spectrometry) Different types of surfaces interactions : Ion exchange, hydrophobe, normal phase, IMAC, (affinity antibody capture…) Sera sample loadded on chip in specific conditions ( pH, salinity, detergent…) Washing steps discarding unbonded material Addition of energy absorbing molecule, EAM To discover powerful biomarkers able to discriminate sera of patients suffering from CROHN disease :CD, from patients UC (Ulcero Hemoragic Recto Colitis) or sera from IBD patients (CD and UC) from other inflammatory diseases (IC)and healthy controls (HC). 2 AIMS Trace View « reading » of chip: IBD PROFILES Laser HC Detector TOF-MS Gel View • Comparaisons of profiles :on « ion exchange » surfaces : CM10 – Q10 • CROHN (C) - 30 samples • UHRC (UC) - 30 samples • healthy controls (HC) - 30 samples • non IBD inflammatory controls (IC) - 30 samples • 120 samples, in quadruplicate, on 2 different surfaces = 960 spectra !!!!!!!!!!!!!!! • Many comparisons : CDvsUC • CD vs (HC, IC, UC) • UCvs (HC, IC,UC) • IBD (UC, CD) vs ( IC, HC) 4 STRATEGY : IBD «  pValue » calculation : by  Biomarker Wizards Ciphergen Models of Classification based on calculations of Decision Trees (unique or multiple) by different statistical approches :(Geurts et al., 2004) Note : Calculations are made on 2 sets of data :integrated peaks and « raw data » = every m/z increments of the spectra. 5 STATISTICS : Probability of missclassification - good if <10 -3 statistical analysis Sensitivity (true positives) and Specificity ( true negatives) of the models Multiple decision trees : by boosting and extra tree arethe best methods and are selfs validated RESULTS : 6 Specificity and sensitivity of the proposed models by « boosting » : Classification of m/z values used to build this model with « boosting » and associated « pValue » illustrating their power of discrimination : comparison :Q10CM10 specificity sensitivityspecificity sensitivity CD vs UC80.83 81.67% 85.00% 90.83% CD vs (HC, IC, UC) 95.83% 70.83% 92.76% 77.50% UCvs (HC, IC, UC) 92.22% 37.50% 96.38% 43.33% IBD (UC, CD) vs ( IC, HC) 88.75% 82.92% 90.42% 86.25% 7 CM10 2663 CD vsUC <10 –12 2681 CD vsUC <10 –12 5822 CD vsUC <10 –12 8604 IBD vs IC <10 –12 et CD vsUC=1.33 10 –6 • Q10 : • CDvsUC = 1.26 10 –8 • CD vs IC <10 –12 et UCvs IC <10 –12 • 4827 CDvs UC <10 –12 et IC = 6.10 –10 • 12608 CDvsUC = 8.10 –6 et IBD vsIC = 1.76 10 –5 Q 10 : Intensity mean of peaks per categories: CM10: Intensity mean of peaks per categories: Importance of each value of m/z on each surface and for every comparisons: * Validationof our « models » on a new batch of samples (10 of each categories) * Correlation with existing tests (ANCA - ASCA – CRP) *Purification of the « more powerful » proteins in terms of discrimination and their identification by MS-MS. 8 PERSPECTIVES : m/z Values = peaks = proteins are not so powerful in discriminating taken independently. All together combined in « models », theyare a lot more efficient and discrimination of samples is obtained with good scores of specificity and sensitivity. Combination of the 2 models corresponding to the 2 surfaces : Q10 and CM10 would may be increase specificity and sensitivity !?!