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

Multi-Analyte Profile (MAP)

The Myriad RBM

Approach

The Approach

Pre-Clinical & Exploratory Phase II & Beyond

Cast a wide net Target key markers

One, validated, highly automated platform throughout drug development

1 2 3

Start with a large Multi-Analyte Profile (MAP)

biomarker panel

Identify key biomarker patterns

Develop a focused, custom panel

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

The object is to find a robust biomarker pattern

These 13 analytes were found to discriminate responders from non-responders.

Custom MAP 13-plex

1. Adiponectin2. EGF3. Eotaxin4. ICAM-15. IL-66. IL-107. IL-158. MCP-19. MMP-910. PAP11. TNF-a12. VEGF13. von Willebrand Factor

Custom MAP

These 13 analytes were used to help the clinicians stratify clinical trial participants

Simponi (golimumab )

Cytokines Acute-Phase

Reactants

MetabolicMarkers

Hormones Inflammatory

markers

Autoimmunity

Cardio-Vascular

CancerMarkers

NovartisEli LillyPfizer

Amgen

Merck GermanyMerck U.S.

CelgeneAstraZeneca

Assay Development ContractsCentocor

NIHSatorisBMS

EU/IMINCI

PsynovaGenentech

Core Competency – Immunoassay development in a multiplexed environment

Validation Parameters

Lowest Detectable Dose / LLOQNormal RangeDynamic RangeImprecisionSpiked RecoveryLinearityCorrelationCross-reactivityMatrix InterferencesStability – Short term storage / Freeze-thaw

CLIA

GLP

Just some of the over 400 users of this biomarker approach that have publicly

acknowledged RBM success

MRBM Bibliography: Publications Citing MAP Services

Publications By Therapeutic Indication

Autoimmune Disease and Arthritis; 6% Bone Disease/Metabolism; 1%

Cancer; 14%

Cardiovascular, 11%

Diabetes and Metabolic Markers;

7%Endocrine; 1%Gastrointestinal; 1%Inflammation and

Immune Response; 37%

Miscellaneous; 9%

Neurological Disease; 18%

Kidney/Tox; 8%

A Few Success Stories

Bone MetastasisSchizophrenia

Myelofibrosis

Pulmonary Fibrosis

Ocular Inflammation

Alzheimer’s DiseaseKidney Disease

Alcohol AbuseCOPD

Expertise in Biomarker Research for Neuroscience

63 Publications30 in NeurodegenerativeDiseases (AD, PD, OD)

Schizophrenia, BD, and MDD

SZP: 18 years

BD: 21 years

MDD: 25 years

Psychiatric vs Neurodegenerative

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.”

We understand much about the terminal pathology

We understand very little about the etiology of AD

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

What are the project’s goals?

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”

Ray et al. Heat Map of the 18 markers

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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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 ”

O’Bryant et al. 2010 – Arch Neurol. 67(9): 1077-1081

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.”

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

TARCC Analyses

AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI)

Demographic data 0.80(0.74-0.86) 0.71(0.62-0.79) 0.78(0.69-0.85)

Clinical variables 0.81(0.75-0.87) 0.74(0.65-0.82) 0.76(0.66-0.84)

Biomarker alone 0.91(0.87-0.95) 0.88(0.80-0.93) 0.82(0.73-0.88)

Combined 0.94(0.91-0.97) 0.89(0.81-0.94) 0.85(0.76-0.91)

O'Bryant et al 2011a

TARCC Analyses

O'Bryant et al 2011a

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

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

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

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

TARCC Analyses

AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI)

Biomarker alone 0.70(0.62-0.78) 0.54(0.45-0.63) 0.78(0.65-0.87)

Biomarker + clinical + demographics

0.88(0.83-0.93) 0.79(0.71-0.86) 0.87(0.75-0.93)

CSF tau/Aβ ratio 0.92(0.87-0.96) 0.84(0.76-0.90) 1.0(0.93-1.0)

O'Bryant et al 2011b

Serum vs. Plasma?

Performance evaluation of a multiplex assay for future use in biomarker discovery efforts to predict body compositionClin Chem Lab Med 2011Beam J., Wright, N., Thompson, P., Hu, C., Guerra, S., and Chen, Z.

”“

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

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

Products Currently in Development:

NeurodegenerativeMAP™CSF MAPMCI/AD Dx and Prognostic (identify rapid converters)AD vs OD Differential

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

NeurodegenerativeMAP1. Adiponectin

2. ACT

3. Alpha 1 Antitrypsin

4. Alpha 1 Microglobulin

5. Angiopoietin 2

6. Angiotensinogen

7. Apolipoprotein A1

8. Apolipoprotein A2

9. Apolipoprotein B

10. Apolipoprotein C-III

11. Apolipoprotein E

12. Apolipoprotein H

13. Anti-thrombin III

14. BLC

15. Beta 2 microglobulin

16. BDNF

17. CD40

18. CEA

19. Clusterin

20. Complement C3

21. Complement Factor H

22. Cortisol

23. EGFR

24. Factor VII

25. FAS Ligand

26. Ferritin

27. Haptoglobin

28. HB-EGF

29. IgM

30. IGFBP 2

31. Interleukin-1 Receptor Antagonist

32. Interleukin-8

33. Interleukin-10

34. Lipoprotein (a)

35. Macrophage Migration Inhibitory Factor

36. Macrophage Inflammatory Protein-1 alpha

37. MMP-2

38. MMP-9

39. Myeloperoxidase

40. Pancreatic Polypeptide

41. RANTES

42. Resistin

43. Sortilin

44. Super Oxide Dismutase

45. Stem Cell Factor

46. Tenascin C

47. Thyroxine Binding Globulin

48. Tissue Inhibitor of Metalloproteinases 1

49. TRAIL-R3

50. Tumor Necrosis Factor Receptor 2

51. Vascular Cell Adhesion Molecule 1

52. Vascular Endothelial Growth Factor

53. Vitamin D Binding Protein

54. Von Willebrand Factor

CSF MAP

Goal : Develop and validate CSF-based biomarkers for Alzheimer's Disease and other neurodegenerative disorders

Processed hundreds of CSF samples on DiscoveryMAP® and other panels

Meta-analysis of datasets and publications

Condensed to the most robust assays

CSF MAP1. Amyloid Beta 402. Amyloid Beta 423. ACT4. Alpha 1 Antitrypsin5. Alpha 2 Macroglobulin6. Apolipoprotein H7. AXL8. EGFR9. FAS Ligand10.Ferritin11.Fetuin A12.HB-EGF13.Interleukin-1 Receptor Antagonist14.Interleukin-8

15.MMP-1016.NCAM17.NT-proBNP18.P-Tau 18119.Placental Growth Factor20.Stem Cell Factor21.Super Oxide Dismutase 122.Stem Cell Factor23.Tau24.TRAIL-R325.Transforming Growth Factor Alpha26.Vascular Cell Adhesion Molecule 127.Vascular Endothelial Growth Factor

Myriad RBM’s Collaboration with the Spinal Muscular Atrophy Foundation

The Spinal Muscular Atrophy Foundation Announces a Biomarker Panel to Guide SMA Therapeutic Development

NEW YORK, NY – April 3, 2012 – The Spinal Muscular Atrophy (SMA) Foundation announced today the launch of a biomarker assay panel for SMA using Myriad RBM’s Multi-Analyte Profiling (MAP) technology platform. The SMA-MAP panel is designed to evaluate the severity of SMA and disease progression and can be used to assess drug efficacy and shorten the duration of clinical trials for SMA therapeutics.

Study Design and Results129 plasma samples from 18 clinical sites

LC/MS + 267 biomarkers from OncologyMAP® and DiscoveryMAP ®

27 biomarker panel developed, including 7 new immunoassays

The Myriad RBM

Advantages

Myriad RBM’s Consultative Services

MRBM has built an extensive database through years of careful sample procurement and collaboration with leading institutions

Successful partnerships with major biopharma companies to develop improved drugs and diagnostics

Data generated using MRBM’s services have been featured in over 330 peer-reviewed journal articles

Myriad RBM’s Companion Diagnostic Programs

Neurodegenerative disease

Psychiatric disorders

Infectious disease

Oncology

Inflammatory disease

Impact of Subpopulation Response

Myriad RBM’s Companion Diagnostic Program with Roche Pharma

Roche Poster, SIRS 2012

Schizophrenia International Research Society, April 14-18, 2012

Myriad RBM Advantages

Cost Effective

Low Sample Volume Requirements

Extensive Biomarker Menu

Assay Precision & Reproducibility

Biomarker Expertise/Database

Myriad RBM’s Product and Services Portfolio

DiscoveryMAP™ 250+ (264 analytes)

DiscoveryMAP™ (189 analytes)

OncologyMAP™ (101 analytes)

HumanMAP® v1.6 (88 analytes)

PsyMAP™ v 1.0 (51 analytes)

CardiovascularMAP™ (50 analytes)

InflammationMAP™ (46 analytes)

MetabolicMAP™ (21 analytes)

KidneyMAP™ (16 analytes)

CustomMAP (any combination of assays from our menu)

RodentMAP® v2.0 (58 analytes)

Rat MetabolicMAP™ (21 analytes)

Rat KidneyMAP™ (12 analytes)

Mouse CytokineMAP A, B & C

CustomMAP

TruCulture™ Tubes

TruCulture™ MAP (46 analytes)

For more information on Myriad RBM’s Companion Diagnostic services, please contact us at

sbs@rulesbasedmedicine.com