Transcript ADNI Biomarker Core Report Leslie M Shaw & John Q Trojanowski Magdalena Korecka Michal Figurski Teresa Waligorska Magdalena Brylska Leona Fields Sarah Pan.

ADNI Biomarker Core Report
Leslie M Shaw & John Q Trojanowski
Magdalena Korecka
Michal Figurski
Teresa Waligorska
Magdalena Brylska
Leona Fields
Sarah Pan
ADNI Biomarker core 2013-early 2014
• Studies reported in the ADNI/LONI website & studies newly
approved by RARC/NIA/ADNI using ADNI biofluids
• Biofluid report update
• 2014 ADNI II batch analyses of CSF underway
• mrm/tandem mass spectrometry reference method for
Ab1-42, progress report
• Review of anchoring to 2007 ADNI BL & cutpoints for CSF
biomarkers in ADNI
• Planning for ADNI III
ADNI Biomarker core 2013-early 2014:
Add-on studies reported on the ADNI/LONI website & add-on
studies newly approved by RARC/NIA/ADNI using ADNI biofluids
Reported
– mrm/tandem mass spectrometry of 567 tryptic peptides associated with
221 proteins, Caprion/FNIH/ADNI/PPSB in ADNI I CSF
Approved/shipped or to be shipped
– YKL40 & Vilip1 in longitudinal ADNI CSF, WashU, Anne Fagan, 4th qtr 2013
– Metabolic Pathways & Networks in ADNI I BL serum, DukeU, Rima
Kaddurah-Douk, 2nd qtr 2014
– DDE* in ADNI serum, EmoryU, Allan Levey, 2nd qtr 2014
– Neurogranin in BASELINE CSF of ADNI I subjects, UGothenberg, Sweden,
Kaj Blennow, 2nd qtr 2014
– Tau in plasma of ADNI I BASELINE subjects, UGothenberg, Sweden, Kaj
Blennow, 2nd qtr 2014
– Phosphorylated a-synuclein in ADNI I BL CSF, UWash, Jing Zhang, 2nd qtr
2014
*DDE-a major breakdown product of the insecticide DDT that is highly fat
soluble, very long biological half-life, potential risk factor for
neurodegeneration. JAMA Neurology 2014.
Subjects providing CSFs at Baseline in Each ADNI Study Phase
Time from Sample Collection to Freezing for ADNI GO and ADNI 2 CSF and PLA
N*
Average Time (min)
Median Time (min)
95% CI (min)
180
160
CSF Through March 2014
1077
43.62
28.00
40.19 - 47.04
*N is the samples for which collection time and
freezing time data is available as of April 1, 2014 .
140
Number of Subject Samples
Plasma Through March 2014
3629
72.14
55.00
70.14 - 74.14
PLA_bl
PLA_m06
PLA_m12
120
PLA_m24
100
PLA_m36
80
PLA_m48
60
PLA_m60
40
PLA_m72
20
PLA_m84
0
PLA_m96
CSF_bl
CSF_m24
CSF_m36
CSF_m48
Sample & Follow-Up Month
Time (Minutes)
X = Time
CSF_m60
CSF_m72
CSF_m84
ADNI GO and 2
Number of Biofluids Collected as of April 1, 2014
Number of Aliquots in Bank
All ADNI
Number of Biofluids Collected as of April 1, 2014
Number of Aliquots in Bank
Total (CSF) Total (PLA + SER) Grand Total as of 4/1/2014 Grand Total as of 3/11/2013
1,178
7,422
8,600
5,329
35,510
109,932
145,442
94,290
Total (CSF) Total (PLA + SER) Grand Total as of 4/1/2014 Grand Total as of 3/11/2013
2,140
15,061
17,201
13,930
65,568
221,852
287,420
236,268
CSF_m96
Aliquot counts by study subject
•
•
•
•
•
Updated as of 4/14 2014
Aliquot counts for plasma, serum, CSF, urine
Total volume and # of pristine aliquots available
Aliquots utilized for Biomarker Core measurements
Aliquots sent to investigators who had
RARC/NIA/ADNI-approved studies
2014 batch analyses of ADNI II CSFs
• N=406 ADNI II CSFs + 15 pristine randomly selected
•
•
•
replicates assayed using the AlzBio3 RUO immunoassay
253 BASELINE samples; 153 have a paired BL(2013) + 24
month sample
Results will be ready for reporting on ADNI/LONI
website end of May.
QC data to date in next ppt.
2014 batch analyses of ADNI II CSFs
(Interim analysis CSF pool qc data)
2013
Sample
t-tau
CSF abnormal
pool #53
CSF normal
pool #54
Ab1-42
CSF abnormal
pool #53
CSF normal
pool #54
p-tau181
CSF abnormal
pool #53
CSF normal
pool #54
N
25
25
25
25
25
25
Mean ± SD
2014
Sample
N
Mean ± SD
t-tau
CSF abnormal
pool #53
7
120±4.9
65.9±6.0
CSF normal
pool #54
7
58.0±3.3
148±9.4
CSF abnormal
pool #53
7
147±6.4
236±18
CSF normal
pool #54
7
241±4.7
26.5±1.4
CSF abnormal
pool #53
7
27.7±0.7
CSF normal
pool #54
7
19.7±1.1
128±10.4
19.1±1.2
Ab1-42
p-tau181
Comments on:
(1) “anchoring” & (2) cutpoint determination
Anchoring ADNI 2008 & later batch datasets to 2007 BASELINE dataset:
• 2007 ADNI BASELINE data: generated at the same time as non-ADNI UPenn ADRC AD(autopsy-based) &
living controls using the same lot # of immunoassay reagents
•
•
Cutpts determined in the ADNI-independent cohort;ROC(balanced sens/spec) & mixture modeling used
The anchoring process used a linear regression best fit eqn – no effect on interpretation of the data using
cutpts determined in the “anchored”-to-2007 datasets vs the corresponding “RAW” datasets
Cutpoint determination:
• In the ADNI-independent cohort with pathologic diagnosis in the AD group: ROC, with balanced
•
sensitivity/specificity/test accuracy, and mixture modeling in this cohort
very similar cutpt values
Diagnosis-independent mixture modeling, first shown to provide a cutpoint in the ADNI 1(NC, LMCI, AD)
population that was equivalent to that in two ADNI-independent path-based AD populations, the UPenn
autopsy(AD) & Belgian autopsy(AD) cohorts. This confirmed the 192 pg/mL cutpoint for Ab1-42
•
If data not anchored to 2007, ie, if “RAW” data used, 192 pg/mL corresponds to ~250 pg/mL in 2008
onward
•
for prospective studies the choice of cutpoint very much depends on:
– specificity vs sensitivity needs(eg, if goal in a treatment trial is to miss as few subjects with amyloid plaque
–
–
•
burden then greater sensitivity and less specificity may be required=higher cutpt for Ab1-42; if goal in a more
clinical setting is to provide confirmation of a high level of clinical likelihood of AD diagnosis, greater specificity
may be required=lower cutpt for Ab1-42)
Analytical method selected and any biases associated with the method, such as precision performance, accuracy,
lot-to-lot variance
Reference population, eg, clinically based AD compared to pathologically based AD diagnoses
The addition of t-tau or p-tau181 to Ab1-42 results in improved detection of AD pathology
Anchoring 2008 CSF BASELINE dataset to 2007
N=328 BL 2007 CSF pristine aliquots
analyzed In 2008 as part of the ADNI
grant-defined longitudinal dataset, BL+yr1.
*a modification of the assay buffer in 2008
resulted in a modest linear difference
across measured concentrations of Ab1-42.
Linear regression*
Model for anchor
MM cutpt=241pg/mL
MM cutpt=202 pg/mL
217 AD + NC
217 AD + NC
“RAW” data
“transformed”data
Linear regression
Model for anchor
No effect of “anchoring” on the
distribution of the data, but this
“rescales” the dataset to the 2007
baseline dataset.
*Data and regression analyses as described, 4/27/2008, Seattle ADNI SC meeting.
No bias produced by “anchoring”on interpretation of CSF Ab1-42
ADNI 2012/13, anchored to 2007;
CSF Ab1-42 concordance with FBP using Mixture Modeling (MM) cutpt
concord
discord
FBP-/Ab-
FBP+/Ab+
FBP-/Ab+
FBP+/Ab-
concordance
n=143
116
27
78
38
23
4
0.811
LMCI n=135
123
12
34
89
9
3
0.911
EMCI n=243
209
34
115
94
25
9
0.860
AD
n=79
75
4
4
71
4
0
0.949
All
n=600
523
77
231
292
61
16
0.872
NC
●●ADNI subjects are all 2012/13 AD+NC who had both lp & FBP scan●●
ADNI 2012/13, RAW data;
CSF Ab1-42 concordance with FBP using MM cutpt
concord
discord
FBP-/Ab-
FBP+/Ab+
FBP-/Ab+
FBP+/Ab-
n=143
116
27
78
38
23
4
0.811
LMCI n=135
123
12
34
89
9
3
0.911
EMCI n=243
209
34
115
94
25
9
0.860
AD
n=79
75
4
4
71
4
0
0.949
All
n=600
523
77
231
292
61
16
0.872
NC
concordance
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•
•
•
•
•
•
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High conc Guanidine HCl(5 M) to release Ab1-42 from aggregates, oligomers
Mixed bed ion exchange 96 well format for 1st step sample cleanup
Use of a surrogate matrix with equivalent performance to CSF as a calibration matrix: defined all
constituents and sources of these
Use high quality Ab1-42 standard and cross-checked performance of several lots of this material; use
uniformly N15-labelled IS; cross-check performance against an international standard preparation
Waters ACUITY 2D HPLC + API 5000 tandem mass spectrometer
Employ quality controls: aCSF (4 mg/mL BSA + electrolytes + Ab1-42) & CSF pools throughout
Evaluated all major analytical parameters as defined in US FDA Guidance and CLSI guidelines
Analyzed AD(autopsy-confirmed) & control CSF samples provided by the UPenn ADRC
ADNI 2: Biomarker Core
mrm LC/MSMS method for Ab peptides in CSF
Xevo TQ-S tandem mass spectrometer
Ab1-40
Ab1-42
Ab1-38
Eppendorf
Oasis MCX
LoBind Tubes mElution Plate
ACQUITY UPLC
Trapping column
Injection position of
switching valve - 2D
chromatography
Time(min)
Analytical column
•
UPLC BEH-300
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C18 2.1x150mm, 1.7 µm
m/z 1129.0
(Ab1-42 precursor [4+] ion)
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•
•
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m/z 1078.8
( product [4+] ion)
Further improved using a Xevo TQ-S tandem mass spectrometer; CSF aliquot size 100 mL
Accuracy-based measurement of Ab1-42, Ab1-40 & Ab1-38 for all ADNI CSF samples
Round Robin study completed(4 centers) in collaboration with Kaj Blennow, manuscript in press
Will help answer questions about potential contribution of other metabolites,
eg, Ab1-40 , Ab1-38 to utility of Ab1-42 and other APP species, tau fragments.
Method comparison in autopsy-proven AD subjects & living controls (n= 41 each)
P=0.2229, deLong’s test
Support of standardization efforts
• ADNI-longterm commitment to standardization of all methods
• Alz Assn Global Biomarker Standardization Consortium
– Analytical methods standardization--strong support for improved performance of existing and new
immunoassays for CSF biomarkers
– Support for mrm/tandem mass spectrometry for direct measurement of absolute Ab1-42 concentration
– IFCC/IRMM project to develop reference Ab1-42 peptide material and using mrm/msms and large pools
of CSF with accurately measured Ab1-42
– Need same for t-tau
• CAMD(Coalition Against Major Diseases) has made a substantial commitment to
support use of HV and CSF AD biomarkers in treatment trials
– Hippocampal volume
– CSF AD biomarkers
• Close collaboration with Japan ADNI on a joint effort to standardize lab-to-lab method
performance of AlzBio3 immunoassay
– Completed development and testing of a unified test procedure
– A follow up study now just underway for a final assessment in AD and normal CSF samples
Collaboration with Japan ADNI
• Completed first pilot study using a jointly developed “Unified” test procedure (U-SOP) that was
described at the AAIC meeting in Boston, 2013
• Will shortly complete the follow-up study that includes 20 AD and 20 controls, each run twice in
separate runs. A total of 4 CSF qc pools are included and each run in quadruplicate in a total of 4
analytical runs (n=16 per pool).
Plate 1 (Day 1)
1
2
3
4
5
6
A
Blank
Blank
ConA
B
St1
St1
ConB
C
St2
St2
J-Pool_1
D
St3
St3
E
St4
F
7
8
9
10
ConA
US-Pool_10
US-Pool_10
J-CSF 7 aliquot 1
J-CSF 7 aliquot 1
US-CSF 9 aliquot 1
ConB
US-CSF 1 aliquot 1
US-CSF 1 aliquot 1
J-CSF 8 aliquot 1
J-CSF 8 aliquot 1
US-CSF 10 aliquot 1 US-CSF 10 aliquot 1
J-Pool_1
US-CSF 2 aliquot 1
US-CSF 2 aliquot 1
J-CSF 9 aliquot 1
J-CSF 9 aliquot 1
J-Pool_1
J-Pool_1
J-CSF 1 aliquot 1
J-CSF 1 aliquot 1
US-CSF 3 aliquot 1
US-CSF 3 aliquot 1
J-CSF 10 aliquot 1
J-CSF 10 aliquot 1
J-Pool_2
J-Pool_2
St4
J-CSF 2 aliquot 1
J-CSF 2 aliquot 1
US-CSF 4 aliquot 1
US-CSF 4 aliquot 1
US-Pool_15
US-Pool_15
US-Pool_10
US-Pool_10
St5
St5
J-CSF 3 aliquot 1
J-CSF 3 aliquot 1
US-CSF 5 aliquot 1
US-CSF 5 aliquot 1
US-CSF 6 aliquot 1
US-CSF 6 aliquot 1
US-Pool_15
US-Pool_15
G
St6
St6
J-CSF 4 aliquot 1
J-CSF 4 aliquot 1
J-Pool_2
J-Pool_2
US-CSF 7 aliquot 1
US-CSF 7 aliquot 1
ConA
ConA
H
St7
St7
J-CSF 5 aliquot 1
J-CSF 5 aliquot 1
J-CSF 6 aliquot 1
J-CSF 6 aliquot 1
US-CSF 8 aliquot 1
US-CSF 8 aliquot 1
ConB
ConB
US-CSF 9 aliquot 1
11
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BIOMARKER CORE AIMS FOR THE ADNI-3 RENEWAL
Leslie M. Shaw and John Q. Trojanowski

It is important to take into account the heterogeneity of AD in the ADNI-3 Biomarker Core
in ADNI3. Many studies emphasize this including ADNI data showing that that a very small
minority (4/22) of ADNI subjects with clinical AD/MCI only had AD plaque and tangle pathology
at autopsy, while 82% had plaques and tangles in addition to TDP-43 and/or alpha-synuclein (asyn) inclusions as well as hippocampal sclerosis in some cases (Toledo et al, ANP Commun,
1:65, 2013). These findings are echoed in a larger study of non-ADNI Penn subjects (Toledo et
al, ANP, 124:23-35, 2012).

Further, we have used a CSF total a-syn assay in ADNI CSF samples that may enable
detection of co-morbid LBs in MCI/AD subjects in ADNI3 (Toledo et al, ANP, 126:683-697,
2013) and we also have access to a phospho-a-syn immunoassay that could be incorporated
into ADNI-3 (Wang et al, Sci Trans Med, 4:121-20, 2/15/2012).

TDP-43 biomarkers are not yet available so we work with Hugo Vanderstichele at ADx and
Andreas Jeromin at Quanterex on TDP-43 ELISA based assays, but it is not yet certain if a
TDP-43 immuno-assay will be ready for use in ADNI-3. We also need to address the issue of
co-morbid cerebrovascular disease (CVD), but information on CVD may come from imaging
rather than chemical biomarker studies.
With this as background, the Aims of the Biomarker Core in ADNI 3 are as follows:
1. Continue to collect, aliquot, store, curate and track all biofluid samples collected from subjects in ADNI1, ADNI-GO, ADNI-2 and ADNI-3 and continue regular reconciliation & reviews with the clinical core at
UCSD .
2. Continue longitudinal studies of existing ADNI biomarkers that are most informative in ADNI studies
including CSF t-tau, p-tau181 and Aβ1-42 as well as possibly total a-syn and phospho-a-syn while VILIP1
and YKL40 data should be available later this year from WashU.
3. Implement other validated and promising new biomarker immunoassays such as those that measure
other species of tau and Aβ in CSF, but especially in plasma, including fragments of tau and Aβ as well
as oligomers thereof in addition to other biomarkers such as VILIP1 and YKL40. Ongoing studies will
determine the likelihood of incorporating these new analytes into ADNI-3.
4. Continue modeling assay data studies for interconversions, eg, lot-to-lot; analytical platform to platform
5. Pending the outcome of ongoing studies, incorporate CSF and plasma proteomic, metabalomic and
lipidomic biomarkers.
6. Use our newly validated mrmMass Spec assay to identify and measure additional species of Aβ (e.g.
Aβ 40, 38, etc) as well as other species/fragments of tau.
7. Partner with the PPSB and other investigators who pursue additional approved Add-On studies of AD
biomarkers in plasma and/or CSF in order to bring these assays on-line for use in ADNI 3
Examples Of Analytes And Biomarker Platforms To Consider In ADNI 3
At the Keystone AD/PD Meeting, Dennis Selkoe described
detection of Aβ oligomers in CSF and will apply to study ADNI
CSF samples with this immunoassay; Mary Savage has recently
described validation of a new Ab oligomer immunoassay & we
plan to collaborate on studies using UPenn ADCC CSF from AD,
PD, ALS, FTLD and controls.
Kaddurah-Daouk will study ADNI serum with the platforms
described here and Federoff plans further studies with his
platform
It takes a great team effort!
John Q Trojanowski
Magdalena Korecka
Magdalena Brylska
Teresa Waligorska
Michal Figurski
Leona Fields
Sarah Pan
Virginia M-Y Lee
Chris Clark*
Steve Arnold
Hugo Vanderstichele
*Deceased
Margaret Knapik-Czajka
Ravi Patel
Pawel Zero
William Hu
Ju Hee Kang
Jon Toledo
Anne Fagan
Uwe Christians
Kaj Blennow
Henrik Zetterberg
Holly Soares
Adam Simon
Robert Dean
Eric Siemers
Piotr Lewczuk
William Potter
Rand Jenkins
Erin Chambers
Supported by the NIH/NIA and families
of our patients
ADNI investigators include: (complete listing
available at www.loni.ucla.edu\ADNI\
Collaboration\ADNI_Manuscript_Citations.pdf).