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Page 1: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 2: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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DeathDeath & & disabilitydisability((socioeconomicsocioeconomic burdenburden))

Page 3: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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Page 4: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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THROMBUSTHROMBUS EMBOLIEMBOLI

Becauseatherosclerosis

From the heart

Page 5: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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< 4.5 h from onset

Activation of endogenous tPA

clot lysis

Page 6: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

3 days

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2 hours

Angiography

3 days

PerfusionDifussion

rt-PA

Page 7: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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García Berrocoso T et al. AIT. Marge Médica Books, BCN. 2009

neutrophils recruitment

proteases releaseBBB leakage

cell death

INFARCT GROWING

Page 8: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 9: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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2,264 references CK-MB [first publications in 1973]

4,020 references on Troponin [first publications in 1987]

Page 10: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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• Barrier level: BBB and protein release

• Organ level: other diseases than stroke produce acute brain damage

• Cellular level: Neurons, astrocytes…

Page 11: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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Brain SamplesCerebro Spinal Fluid MicrodyalisatePlasma/serum

Page 12: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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)

Page 14: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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Allard et al. Clin.Chem. 2005; 51:2043–51.

3 different cohorts with similar se/sp %

(oxidative stress response)

Page 15: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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PUMPDRAINPIPE

PROBE

CANNULAPI PROBE

IC PROBE

NEUROTRAUMATOLOGÍA

Page 16: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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.

Page 17: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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.

Page 18: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

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.

Page 19: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 20: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 21: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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Searchlight library: 178 human proteins

Page 22: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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

Page 23: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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)

Page 25: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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Anatomía patológica

Page 26: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 27: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 28: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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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

Page 30: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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���(��$������(��$��� �������������������� �������)���)���(���('��)�����'��)�����3 candidates have not been detected in blood samples

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)

Page 31: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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

Page 32: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · 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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no yes

In-hospital mortality

0

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(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

Page 33: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

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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III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

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Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7

Page 36: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · 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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Page 37: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

11,11,?>��1,?>��1,7�7����(���(��������������B�11,B�11,6@�6@����(���(��������������Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7

Page 38: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

Cuadrado E, et al. J Proteome Res. 2009; 8: 3191-7

Page 39: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

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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III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH

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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)

Page 41: Blood Biomarkers in Ischemic Strokeestrellapolar.cnb.csic.es/proteored/Biblioteca... · III SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS - 15-16 December 2011. Barcelona, VHUH, (

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