blood biomarkers in ischemic strokeestrellapolar.cnb.csic.es/proteored/biblioteca... · iii...
TRANSCRIPT
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Blood Biomarkers in Ischemic StrokeA window to the brain"
III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH
III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH
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DeathDeath & & disabilitydisability((socioeconomicsocioeconomic burdenburden))
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III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH
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THROMBUSTHROMBUS EMBOLIEMBOLI
Becauseatherosclerosis
From the heart
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< 4.5 h from onset
Activation of endogenous tPA
clot lysis
3 days
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2 hours
Angiography
3 days
PerfusionDifussion
rt-PA
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García Berrocoso T et al. AIT. Marge Médica Books, BCN. 2009
neutrophils recruitment
proteases releaseBBB leakage
cell death
INFARCT GROWING
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Stroke Plasma Biomarkers
Stroke Biomarkers
In total 5,190 references, only 1,043 for plasma biomarkers (2011/12/07)
But...any biomarker has not yetbeen applied to the clinical practice
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2,264 references CK-MB [first publications in 1973]
4,020 references on Troponin [first publications in 1987]
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• Barrier level: BBB and protein release
• Organ level: other diseases than stroke produce acute brain damage
• Cellular level: Neurons, astrocytes…
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Brain SamplesCerebro Spinal Fluid MicrodyalisatePlasma/serum
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2-DE of Post-mortem CSF 2-DE of Ante-mortem CSF
(n=4) (n=4)
Differentially expressed proteinsMass spectrometry
identificationMSMS of GST A1 C112S, m/z 1342
400 600 800 1000 1200 1400 1600 1800m/z0
100
%
1358.31358.1
1357.8
1277.8
1267.1
457.2
328.2
1166.7
1140.6685.3
581.31062.6
827.0
1358.6
1386.8
1387.1
1487.4
1579.9 1810.4 1897.8
Immunoassay validation(Plasma)
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Lescuyer P, et al. Proteomics 2004;4:2234–41.
Calcyphosin (CAYP)
Cathepsin D (CATD)
Protein DJ-1 (PARK7)
Fatty Acid Binding Protein (FABP)
Glial Fibrillary acidic protein (GFAP)
Glutathione S-transferase P (GSTP-1)
Nucleoside Diphosphatase Kinase A (NDKA)
Peroxiredoxin-5 (PRDX5)
Peptidyl-prolyl cis-trans isomerase A (PPIA)
Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1)
Ubiquitin fusion degradation protein 1 (UFD1)
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Allard et al. Clin.Chem. 2005; 51:2043–51.
3 different cohorts with similar se/sp %
(oxidative stress response)
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PUMPDRAINPIPE
PROBE
CANNULAPI PROBE
IC PROBE
NEUROTRAUMATOLOGÍA
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6 MDs x 2 zones (IC, PI, CL)
Dayon L, et al. J Proteome Res. 2011;10:1043-51.
Protein Abbrev.
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Serum levels
(ELISA)
p = 0.0001
Brain microdialysate levels
(MS quantification)
Dayon L, et al. J Proteome Res. 2011;10:1043-51.
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So, a very interesting list of potential strokebiomarkers coming from close to the human brainhas been generated through proteomics techniques.
The detection in higher levels in blood samplesfrom stroke patients of these proteins pointed out its promising use to the biochemical monitoring ofstroke patients.
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Normal 1st ischemic stroke 2nd ischemic stroke
Cer
ebro
vasc
ular
dam
age White matter lesions
Risk prediction
Lp-PLA2
DIAGNOSIS Treatment effectiveness
PROGNOSIS
Recurrence
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• Stroke has to be distinguished from ‘mimics’ QUICKLY
• % ‘mimics’ depends on the healthcare system level
Community Hospital Internal Medicine Neurology Stroke Unit
or Ambulances
50%30%
20%5%
more specialized assistance
wrong
diagnosis
But...few publications so far comparingstrokes with mimics, but with healthy controls
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Searchlight library: 178 human proteins
178 proteins
9 Stroke
2 Mimics
4 Healthy controls
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22 first candidates
146 Stroke
61 Mimics
23 Healthy controls
6 DIAGNOSTIC BIOMARKERS FOR
STROKE
SCREENINGCANDIDATES VALIDATION
www.lin-bcn.com
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0.0078.116 (1.795-36.697)Dyslipidemia
<0.00126.72 (4.766-149.805)Atrial Fibrillation
0.033.467 (1.125-10.687)Hypertension
<0.0018.699 (2.877-26.299)TNFR1
0.00734.552 (2.613-456.953)IGFBP3
0.0029.193 (2.264-37.323)BNGF
0.008114.766 (3.496-3767.611)IL17
P-valueOdds Ratio (I.C. 95%)Clinical Factors +
Biomarkers
0.0078.116 (1.795-36.697)Dyslipidemia
<0.00126.72 (4.766-149.805)Atrial Fibrillation
0.033.467 (1.125-10.687)Hypertension
<0.0018.699 (2.877-26.299)TNFR1
0.00734.552 (2.613-456.953)IGFBP3
0.0029.193 (2.264-37.323)BNGF
0.008114.766 (3.496-3767.611)IL17
P-valueOdds Ratio (I.C. 95%)Clinical Factors +
Biomarkers
Now we’re preparing a multicenter study which will include 1,500 stroke suspicionsto validate this DIAGNOSTIC model
Montaner J et al. Manuscript in preparation
Protein A
Protein B
Protein C
Protein D
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Emergencydepartment
Stroke unit
All strokes
Worse
Stable
Better
Biomarker test
• Patient and relatives information
• Risk/benefit of treatment options
• Rationing decisions to safe sources (patient allocation)
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Anatomía patológica
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Tannu NS, Hemby SE. Nat Protoc. 2006;1:1732-42.
About 1,500 spots in 2DE-DIGE gels.
132 spots differentially expressed between areas.
42 spots identified by MALDI-TOF/TOF MS, corresponding to 39 different proteins
Proteomics
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Cuadrado E, et al. J Neuropath Exp Neurol 2010;69:1105-15.
Proteins mainly involved in:
• Immunity pathways
• Axonal growth
• Energy and structure
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20 spots identified by ESI LTQ-OT MS, corresponding to12 NEW proteins
García-Berrocoso T et al. Manuscript in preparation
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51 proteins identified in brain samples
8 protein candidates
I
PI
CL
Overexpressed in IC
Underexpressed in IC
60 Stroke blood samples (ELISAs) - Clinical data
- Neurological outcome (NIHSS)
- Functional state (mRS) (3rd month)
- Biological function
- Levels in IC
- Commercial ELISA
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Proteins A & B are higher expressed in patients functionally disabled at 3rd monthProtein D is lower expressed in patients functionally disabled at 3rd month
García-Berrocoso T et al. Manuscript in preparation
no yes
Disability 3rd month
0
5
10
15
20
Bas
elin
e G
elso
lin (
ng/m
L)
no yes
Disability 3rd month
0
10
20
30
Bas
elin
e C
ysA
(ng/
mL)
no yes
Disability 3rd month
0
10
20
30
Bas
elin
e R
ho G
DI-1
(ng/
mL)*
p=0.052
no yes
Disability 3rd month
10
20
30
40
Bas
elin
e C
NTN
-1 (n
g/m
L)
no yes
Disability 3rd month
1
2
3
4
Bas
elin
e D
PY
SL2
(ng/
mL)
*
Bas
elin
eA
(ng/
mL)
Bas
elin
eB
(ng/
mL)
Bas
elin
eC
(ng/
mL)
Bas
elin
eD
(ng/
mL)
Bas
elin
eE
(ng/
mL)
1 - Specificity
Sen
sitiv
ity
Age, DMAge, DM, protein AReference line
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Protein A > 19,87 ng/mL improves significantly the predictive value ofthe clinical data for the prognosis of disability at 3rd month.
0,788 – 0,9850,0500,887Age, DM, protein A
0,685 – 0,9730,0740,829Age, DM
95% CISEAUCModel
García-Berrocoso T et al. Manuscript in preparation
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no yes
In-hospital mortality
0
10
20
30
Bas
elin
e C
ysA
(ng/
mL)
p = 0.112
Protein B > 34,35 ng/mL improvessignificantly the prediction ofin-hospital mortality in comparison with only clinical data
0,919 – 1,0160,0250,968NIHSS, previous stroke, protein B
0,776 – 0,9760,0510,876NIHSS, previous stroke
95% CISEAUCModel
García-Berrocoso T et al. Manuscript in preparation
Bas
elin
eB
(ng/
mL)
NIHSS, previous strokeNIHSS, previous stroke, protein BReference line
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To explore the presence of hundreds of proteins (low scale) directly in the plasma has allow us to create a Diagnostic model for stroke that hopefully will help clinicians in the future.
At a large scale, the screening of the proteome of brain tissue has also given very good candidates for the prognosis of stroke when measured in blood within the first hours after the symptoms’onset.
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P-1TIM
P-2
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0MMP-1
3TIM
P-1TIM
P-2
MMP-1MMP-2MMP-3MMP-8MMP-9MMP-1
0MMP-1
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Contralateral Hemisphere
Infarct Core
pgpr
otei
n/ µ
g to
tal p
rote
in
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Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7
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Vesse
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130 kDaCD31
GFAP
NeuN 48 kDa46 kDa
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Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7
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11,11,?>��1,?>��1,7�7����(���(��������������B�11,B�11,6@�6@����(���(��������������Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7
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Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7
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To study the cellular proteomes might help in finding the origin of some protein candidates to be biomarkers for stroke, but also finding new proteins that were hidden within the dynamic range of whole brain homogenates usually used.
Now we’re working on this cellularapproach at proteomics large scale
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Now we know better what we want from biomarkers to stroke:
- Specific from the disease
- Highly sensitive to help in decision-making processes
- Measurable in blood
- Enough power to improve clinical models
We need:
- Go-on discovering new blood biomarkers
- Verification of known biomarkers (multicenter & multinacional studies)
- Put all pieces together (e.g. metanalysis)
J. Montaner
T. García-Berrocoso
A. Penalba
C. BoadaI. Fernández-Cadenas
P. Delgado
I. Riba
C. Nafría
X. Castañé
I. Fernández
M. Hernández
C. Merino
A. Rosell
A. Morancho
V. Barceló
J. Gibert
L. García-Bonilla
M. Campos
J. Montaner
N. Corbeto
D. Giralt
L. Ortega
J. Álvarez-Sabín M. Ribó C. Molina J. Pagola A. Flores D. Rodríguez-Luna
M. Rubiera E. Santamarina M. Quintana O. Maisterra G. OrtegaUNIDAD DE ICTUS
S. Ramón y Cajal A. Ortega E. MartínezAnatomía patológica
www.lin-bcn.com
J-C Sanchez L. DayonF. Canals N. Colomé
III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH
Proteomics