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Ralph L. McDade, Ph.D. Strategic Development Officer

Blood-based Biomarkers for Detection of early stage Alzheimer’s Disease: A Successful Multi-Analyte Profiling Approach. Ralph L. McDade, Ph.D. Strategic Development Officer. The Myriad RBM Approach The Platform and Validation Success Stories Alzheimer’s Disease story.

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Ralph L. McDade, Ph.D. Strategic Development Officer

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  1. Blood-based Biomarkers for Detection of early stage Alzheimer’s Disease: A Successful Multi-Analyte Profiling Approach Ralph L. McDade, Ph.D. Strategic Development Officer

  2. The Myriad RBM Approach • The Platform and Validation • Success Stories • Alzheimer’s Disease story

  3. Multi-Analyte Profile (MAP)

  4. The Myriad RBM Approach

  5. The Approach Pre-Clinical & Exploratory Phase II & Beyond Cast a wide net Target key markers 1 2 3 Start with a large Multi-Analyte Profile (MAP) biomarker panel Identify key biomarker patterns Develop a focused, custom panel One, validated, highly automated platform throughout drug development

  6. The Platform • Industrialized form of Luminex xMAP • All liquid handling steps automated using the Tecan Evo platform • Proprietary blockers to handle most matrix effects • Validated to clinical lab standards • GLP and CLIA certified • 56 successful regulatory compliance audits

  7. The object is to find a robust biomarker pattern • These 13 analytes were found to discriminate responders from non-responders.

  8. Custom MAP Custom MAP 13-plex • These 13 analytes were used to help the clinicians stratify clinical trial participants • Simponi (golimumab ) 1. Adiponectin 2. EGF 3. Eotaxin 4. ICAM-1 5. IL-6 6. IL-10 7.IL-15 8.MCP-1 9.MMP-9 10.PAP 11.TNF-a 12.VEGF 13.von Willebrand Factor

  9. Cytokines Acute- Phase Reactants Metabolic Markers Hormones Inflammatory markers Cardio- Vascular Cancer Markers Autoimmunity Core Competency – Immunoassay development in a multiplexed environment Assay Development Contracts EU/IMI NCI Psynova Genentech Novartis Eli Lilly Pfizer Amgen Merck Germany Merck U.S. Celgene AstraZeneca Centocor NIH Satoris BMS

  10. Validation Parameters • Lowest Detectable Dose / LLOQ • Normal Range • Dynamic Range • Imprecision • Spiked Recovery • Linearity • Correlation • Cross-reactivity • Matrix Interferences • Stability – Short term storage / Freeze-thaw CLIA GLP

  11. Just some of the over 400 users of this biomarker approach that have publicly acknowledged RBM success

  12. MRBM Bibliography: Publications Citing MAP Services

  13. Publications By Therapeutic Indication

  14. A Few Success Stories Schizophrenia Bone Metastasis Ocular Inflammation Kidney Disease Pulmonary Fibrosis Alzheimer’s Disease COPD Alcohol Abuse Myelofibrosis

  15. Expertise in Biomarker Research for Neuroscience 63 Publications 30 in Neurodegenerative Diseases (AD, PD, OD)

  16. Psychiatric vs Neurodegenerative • Schizophrenia, BD, and MDD • SZP: 18 years • BD: 21 years • MDD: 25 years • AD, PD, and OD • AD: >65 years • BD: 60 years • OD: >65 years “The blood based biomarker patterns of disease in younger people are easier to see as there are fewer confounders such as underlying diseases like CVD and diabetes. In addition, an average 65 year old in the US is on a regimen of at least five different drugs.”

  17. We understand much about the terminal pathology We understand very little about the etiology of AD

  18. What are the project’s goals? • Early Dx for MCI/AD (blood test for >50 years) • Identify rapid MCI to AD converters (20%) • Differentiate AD from other forms of dementia • Identify responders in drug trials

  19. Tony Wyss-Coray’s group at Stanford Med • Ray Biotech 2-D slide-based array 100+ analytes • First suggestion in literature that a signal for MCI/AD was present in the plasma proteome • “We found 18 signaling proteins in blood plasma that can be used to classify blinded samples from Alzheimer's and control subjects with close to 90% accuracy and to identify patients who had mild cognitive impairment that progressed to Alzheimer's disease 2–6 years”

  20. Ray et al. Heat Map of the 18 markers

  21. EDTA Plasma from 19 AD/22 Controls • HumanMAP v 1.6 (90 analytes) • Attempt to reproduce the Ray, et al findings • Mentions early Rotterdam data with their 1,200 member cohort and our later 152 analyte MAP. • “Furthermore, utilization of other analytes from the 90-analyte panel did show a diagnostic accuracy of approximately 70%”

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  23. CSF from 62 AD; 33 Controls and 25 OD • Pre-DiscoveryMAP (152 analytes) • MAP data added with tau, P-tau181 & Aβ42 >90% accuracy in AD/OD diagnosis • 17 MAP analytes by Random Forest; 32 by PAM • “Two categories of biomarkers were identified: (1) analytes that specifically distinguished AD (especially CSF Aβ42 levels) from cognitively normal subjects and other disorders; and (2) analytes altered in multiple diseases, but not in cognitively normal subjects ”

  24. Serum from 197 AD; 203 Controls (TARC Cohort) • Pre-DiscoveryMAP (152 analytes) • MAP data + clinical data+ ApoE4 genotype = >95% AUC AD vs. normals • 30 MAP analytes by Random Forest; 25 by SAM (minimal overlap with Ray et al: Ang 2 and TNFα) • “The identification of blood-based biomarker profiles with good diagnostic accuracy would have a profound impact worldwide and requires further validation.” O’Bryant et al. 2010 – Arch Neurol. 67(9): 1077-1081

  25. TARCC Analyses • Restricted to only top 30 markers • Added clinical lab values • Total cholesterol, triglycerides, high density lipoproteins, low density lipoproteins, lipoprotein-associated phospholipase [Lp-PLA2], homocysteine, C-peptide) • Retained demographic factors O'Bryant et al 2011a

  26. TARCC Analyses O'Bryant et al 2011a

  27. TARCC Analyses O'Bryant et al 2011a

  28. TARCC Analyses • Need to cross-validate screener in an independent cohort • Alzheimer’s Disease Neuroimaging Initiative (ADNI) • Large-scale study of AD and Mild Cognitive Impairment (MCI) • Has same biomarker panel on subset of AD cases and controls O'Bryant et al 2011b

  29. TARCC Analyses • Problem – ADNI has plasma based proteins while TARCC has serum • There is no consensus as to what blood fraction to look at for AD biomarkers • Many groups look at both serum and plasma, even using same markers • Markers may or may not behave consistently across media O'Bryant et al 2011b

  30. TARCC Analyses • TARCC has plasma-based proteins on 40 AD cases • Looked at serum and plasma results to identify • Proteins that behave consistently across serum and plasma R2>0.75 • Significant (p<0.05) relation to AD status • Identified 11 proteins that met criteria • CRP, adiponectin, pancreatic polypeptide, fatty acid binding protein, IL18, beta 2 microglobulin, tenascin C, I.309, factor VIII, VCAM1, MCP1 O'Bryant et al 2011b

  31. TARCC Analyses • Created RF biomarker risk score based on the 11 proteins using the TARCC serum data • Applied the algorithm (protein risk score, demographics, clinical labs) to the ADNI plasma data O'Bryant et al 2011b

  32. TARCC Analyses O'Bryant et al 2011b

  33. Serum vs. Plasma? • Thoroughly compared serum and plasma from the same donor and bleed with HumanMAP v. 1.6 (90 analytes) • 70 “useful” analytes in “healthy, normal” samples • 29 analytes had a concordance >0.8 and 41 had a concordance of <0.8 between serum and plasma with 24<0.5 • “Serum showed a slight advantage…..” • MRBM recommends serum for any MCI/AD diagnostic “ Performance evaluation of a multiplex assay for future use in biomarker discovery efforts to predict body composition Clin Chem Lab Med 2011 Beam J., Wright, N., Thompson, P., Hu, C., Guerra, S., and Chen, Z. ”

  34. ADNI Cohort of 566 individuals tested for 190 analytes • Focused on 54 controls and 163 MCI to AD converters • 11 analyte signature with APOE • Meta-analysis produced an 8 feature signature with 86% SN and 87% SP • By adding longitudinal data this was improved to over 90% for both SN and SP

  35. Products Currently in Development: • NeurodegenerativeMAP™ • CSF MAP • MCI/AD Dx and Prognostic (identify rapid converters) • AD vs OD Differential

  36. NeurodegenerativeMAP™ • Goal : Develop and validate blood-based biomarkers for Alzheimer's Disease and other neurodegenerative disorders • Processed thousands of samples on our DiscoveryMAP panel from groups including: • Meta-analysis of datasets and publications • Condensed to the most robust assays

  37. NeurodegenerativeMAP • Adiponectin • ACT • Alpha 1 Antitrypsin • Alpha 1 Microglobulin • Angiopoietin 2 • Angiotensinogen • Apolipoprotein A1 • Apolipoprotein A2 • Apolipoprotein B • Apolipoprotein C-III • Apolipoprotein E • Apolipoprotein H • Anti-thrombin III • BLC • Beta 2 microglobulin • BDNF • CD40 • CEA • Clusterin • Complement C3 • Complement Factor H • Cortisol • EGFR • Factor VII • FAS Ligand • Ferritin • Haptoglobin • HB-EGF • IgM • IGFBP 2 • Interleukin-1 Receptor Antagonist • Interleukin-8 • Interleukin-10 • Lipoprotein (a) • Macrophage Migration Inhibitory Factor • Macrophage Inflammatory Protein-1 alpha • MMP-2 • MMP-9 • Myeloperoxidase • Pancreatic Polypeptide • RANTES • Resistin • Sortilin • Super Oxide Dismutase • Stem Cell Factor • Tenascin C • Thyroxine Binding Globulin • Tissue Inhibitor of Metalloproteinases 1 • TRAIL-R3 • Tumor Necrosis Factor Receptor 2 • Vascular Cell Adhesion Molecule 1 • Vascular Endothelial Growth Factor • Vitamin D Binding Protein • Von Willebrand Factor

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