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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
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The Myriad RBM Approach
The Platform and Validation
Success Stories
Alzheimer’s Disease story
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Multi-Analyte Profile (MAP)
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The Myriad RBM
Approach
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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
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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
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The object is to find a robust biomarker pattern
These 13 analytes were found to discriminate responders from non-responders.
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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 )
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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
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Validation Parameters
Lowest Detectable Dose / LLOQNormal RangeDynamic RangeImprecisionSpiked RecoveryLinearityCorrelationCross-reactivityMatrix InterferencesStability – Short term storage / Freeze-thaw
CLIA
GLP
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Just some of the over 400 users of this biomarker approach that have publicly
acknowledged RBM success
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MRBM Bibliography: Publications Citing MAP Services
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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%
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A Few Success Stories
Bone MetastasisSchizophrenia
Myelofibrosis
Pulmonary Fibrosis
Ocular Inflammation
Alzheimer’s DiseaseKidney Disease
Alcohol AbuseCOPD
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Expertise in Biomarker Research for Neuroscience
63 Publications30 in NeurodegenerativeDiseases (AD, PD, OD)
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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.”
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We understand much about the terminal pathology
We understand very little about the etiology of AD
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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?
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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”
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Ray et al. Heat Map of the 18 markers
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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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“
“
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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 ”
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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.”
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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
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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
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TARCC Analyses
O'Bryant et al 2011a
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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
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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
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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
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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
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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
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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
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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
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Products Currently in Development:
NeurodegenerativeMAP™CSF MAPMCI/AD Dx and Prognostic (identify rapid converters)AD vs OD Differential
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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
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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
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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
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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
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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.
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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
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The Myriad RBM
Advantages
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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
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Myriad RBM’s Companion Diagnostic Programs
Neurodegenerative disease
Psychiatric disorders
Infectious disease
Oncology
Inflammatory disease
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Impact of Subpopulation Response
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Myriad RBM’s Companion Diagnostic Program with Roche Pharma
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Roche Poster, SIRS 2012
Schizophrenia International Research Society, April 14-18, 2012
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Myriad RBM Advantages
Cost Effective
Low Sample Volume Requirements
Extensive Biomarker Menu
Assay Precision & Reproducibility
Biomarker Expertise/Database
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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)
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For more information on Myriad RBM’s Companion Diagnostic services, please contact us at