An Update on Blood Biomarkers for Stroke

NEUROLOGY/NEUROSURGERY GRANDROUNDS

Naresh Mullaguri MDRe s i d e nt P hy s i c i a n

D e p a r t m e n t o f N e u ro l o g yU n i ve rs i t y o f M i s s o u r i

AN UPDATE ON BLOOD BIOMARKERS FOR

05/02/2023 University of Missouri - Neurology/Neurosurgery Grand rounds

DISCLOSURES

• NONE


OBJECTIVES• Discuss various categories of available

biomarkers• Mechanism of their release into the

circulation• Predictive value• Supporting evidence for each Biomarker• Advantages over conventional testing• Gaps to fill


Why do we need a Biomarker?• Discovery of biomarkers for cardiac myocyte injury revolutionized

Cardiology in managing Coronary artery disease.

• As Cerebrovascular diseases were following the trend of Cardiovascular diseases in terms of acute management with improved outcomes, there is a tremendous need to search for the blood biomarkers to aid in diagnosis, management, identification and prevention of complications and prognostication in Stroke patients.

But WHY?


What is a biomarker?• In 1998, the NIH Biomarkers Definitions Working Group defined a biomarker as “a

characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention.”

• WHO classification : Any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence or outcome of disease


Ideal Biomarker

• Reliable• Rapidly measured• Readily available• Assist with diagnosis• Determination of Stroke subtype or

mechanism• Prediction of outcome or response to therapy

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Challenges in the development of blood biomarkers for stroke

• Heterogeneity of stroke• Presence of blood brain barrier• Complexity of Brain injury• Individual markers lack sufficient

sensitivity and specificity

University of Missouri - Neurology/Neurosurgery Grand rounds


Brain Natriuretic Peptide(BNP)• Identified in Brain as a neurohormone but secreted by the cardiac

ventricles in response to increased wall tension and volume overload• Physiologically it has diuretic, natriuretic and hypotensive effects and acts

by inhibiting both sympathetic and RAAS.• Measurement of BNP in the setting of Cryptogenic stroke is helpful in

assessing the likelihood of a Cardioembolic source including the presence of Atrial fibrillation(cut off values were undefined)

• Elevated BNP levels suggest the need for intensive cardiac evaluation including echocardiography and prolonged cardiac rhythm monitoring.

BNP and Cardioembolic stroke:Observational studies and systematic review and meta-analysis suggest the use of BNP levels in identifying patients with stroke due to Cardioembolic source.


BNP? NT-ProBNP?


Role of BNP

• Elevated Plasma BNP values are associated both with Heart failure and Atrial fibrillation, two important Cardioembolic mechanisms with specific management implications.

• Paroxysmal A.fib if transient, infrequent and largely asymptomatic is difficult to catch unless monitored for a long time for several weeks with Event-loop recorders in patients with cryptogenic TIA/Ischemic infarct.

• Can be an useful adjunct to determine which patients are most likely to harbor occult AF.


Observational Studies supporting association of elevated plasma BNP and Cardioembolic Stroke


Post-hoc analysis of WARSS• WARSS is a RCT compared Warfarin to Aspirin in the prevention of

recurrent ischemic stroke in patients with presumed non-cardioembolic Stroke.

• 1028 patients in WARSS have blood samples and those with NT-proBNP concentrations in top 5 percent(>750pg/mL) had a significant reduction in the composite endpoint of Stroke or death favoring Warfarin(HR 0.3 CI 0.12-0.84)

• No benefit of Warfarin over Aspirin was seen in those with NT-proBNP levels <750pg/mL

• However, WARSS was performed prior to widespread use of prolonged cardiac monitoring for acute Stroke and occult A.fib may have been under diagnosed.

• Taken in its entirety, the data support the role of BNP as a predictor of Cardioembolic stroke particularly due to Heart failure and Atrial fibrillation.

• Despite the usage of different assays like BNP and NT-proBNP in various studies, the likelihood of cardioembolism appears to linearly rise with the BNP.


NT-ProBNP and Incident Stroke risk

• Elevated BNP is associated with increased risk of incident stroke/TIA

• In 3346 subjects without heart failure in Framingham Heart study, those in the highest tertile of NT-proBNP levels had a 5X increased risk of TIA/stroke compared with those in the lowest tertile.

• In a subset of 1502 subjects in the population based REGARDS study who had baseline NT-proBNP levels measured, those in the highest quartile of NT-proBNP levels

had nearly 3X increased risk of first stroke compared to those in the bottom quartile, even after adjusting for traditional vascular risk factors. The elevated levels were particularly associated with Cardioembolic stroke with a 9X greater risk for those in the top compared with the bottom quartile of NT-proBNP levels.

• In a Danish prospective study of 537 patients without cardiovascular disease at baseline, those in the top quintile of NT-proBNP levels compared with those in the lower quintiles had a >3X greater risk for Stroke or TIA after the adjustment for traditional cardiovascular risk factors, left ventricular dysfunction and renal function.


• In 6189 patients with available blood samples in the RE-LY trial which randomized patients with A.fib to Warfarin or Dabigatran, subjects with plasma NT-proBNP levels in the highest quartile(>1402ng/L) had an annual stroke risk of 2% while those in the lowest quartile(<387ng/L) had an annual stroke risk of 0.8%. NT-proBNP remained a predictor of stroke even after thromboembolic risk adjustment using the CHADS2-VASc risk model score.

• In 14,892 patients with available blood samples in the ARISTOTLE trial, which randomized patients with atrial fibrillation to Warfarin or Apixaban, subjects with NT-proBNP levels in the highest quartile(>1250ng/L) had an annual risk of stroke or systemic embolism of 2.2 %, while those in the lowest quartile (<363ng/L) had an annual stroke risk of 0.7%. Addition of NT-proBNP to the CHA2DS2-VASc score significantly improved prediction of embolic events.

Prediction of elevated NT-proBNP levels and stroke risk in patients on anticoagulation


BNP and Stroke outcome

• Elevated BNP is associated with increased post-stroke mortality, although the incremental clinical utility of BNP measurement for predicting death after stroke is likely small.

• In a meta-analysis of individual data from 2258 patients with stroke, the addition of NT-proBNP to clinical variables resulted in a small improvement in prediction of mortality after the stroke, reclassifying 8% of patients into more accurate risk categories.

• Data on the association between BNP and functional outcome after stroke are conflicting.


C-Reactive Protein• Acute phase reactant predominantly produced

by Liver and regulated by inflammatory cytokines.

• It generally increases in response to injury, infection or inflammatory and has been studied extensively as a predictor of cardiovascular disease and an indicator of underlying systemic atherosclerosis.


Diagnosis of Atherosclerosis


CRP and Carotid Artery Atherosclerosis

• In a longitudinal study of 179 patients, CRP was an independent predictor for the development and progression of early carotid atherosclerosis. Progression intensified with increasing CRP levels even within the normal range, implicating possible direct pro-inflammatory and pro-thrombotic vascular interaction.

• However, CRP concentration is a measure of early carotid atherosclerotic activity and development than of the extent of atherosclerosis or degree of carotid stenosis.

• Elevated CRP levels have not been shown to be useful as a diagnostic marker for identifying stroke or TIA patients with significant Carotid stenosis(>50%)


CRP and Stroke risk

• Overall, available data do not suggest a clinically useful role of CRP measurement in estimating future stroke risk for either first or recurrent stroke.

D-dimer

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D-dimer cont..• It is a fibrin degradation product generated during

Fibrinolysis, and levels are elevated in the setting of active clot formation and turnover

• In clinical practice, D-dimer levels were incorporated into diagnostic algorithms used to identify patients with DVT and PE

• In addition, measurement of D-dimer in the setting of Cryptogenic Stroke is helpful to assess the likelihood of cancer-related hypercoagulability, though optimal cutoff values for individual assays are undefined.


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D-dimer cont..• D-Dimer and Cerebral Venous Thrombosis:An elevated D-dimer level supports the diagnosis of CVT, but a normal level does not exclude the diagnosis in patients with suggestive symptoms and predisposing factors.==================================================================

A 2012 meta-analysis included 14 studies that evaluated D-dimer in 1134 patients for the diagnosis of confirmed CVT. In seven studies that evaluated patients with suspected CVT , D-dimer was elevated in 145 of 155 patients in whom CVT was confirmed and was normal in 692 of 771 patients in whom CVT was ruled out, yielding a sensitivity and specificity of 94% and 90 % respectively.

- D-dimer levels were performed less in the other seven studies that enrolled subjects with already diagnosed CVT, the sensitivity and specificity of the test is 89% and 83% respectively.

- The sensitivity of the D-dimer for CVT was also lower in patient with isolated headache as the presenting symptom(82%), in those with subacute or chronic clinical presentations of CVT(83%) and in those with single affected venous sinus(84%)


Journal of Thrombosis Hemostasis 2012


D-dimer cont..• In a subsequent study of 233 patients with suspected

CVT and symptom onset less than seven days , D-dimer demonstrated a sensitivity and specificity of 94 and 98% respectively for predicting CVT.

• It is proposed to use the same threshold levels as used in the diagnostic protocols for DVT(>500ng/mL of Fibrinogen equivalent units)

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Mechanism of Cancer Hypercoagulability

University of Missouri - Neurology/Neurosurgery Grand rounds Hematologist 2011


D-dimer and Stroke mechanism:

While relatively infrequent, cancer associated hypercoagulability is an important stroke mechanism. A number of studies suggested a potential role for D-dimer as a marker of cancer associated hypercoagulability in the diagnostic evaluation of Stroke.

Studies to support:140 patients with ischemic Stroke and active cancer, D-dimer levels were significantly higher in patients with no identifiable cause of stroke aside from the potential cancer hypercoagulability compared with those who had a determined, conventional stroke mechanism (8.4 vs 3.9mcg/mL)


• In a case-control study of 204 patients with ischemic stroke, D-dimer levels were significantly higher in 104 patients with active cancer compared with 100 patients with inactive cancer (5.7 vs 1.0mcg/mL, versus 0.6mcg/mL in 408 control patients with no stroke or history of cancer).

• In the active cancer group, elevated D-dimer levels were associated with absence of a conventional stroke mechanism and presence of multiple ischemic lesions in different vascular territories.


D-dimer and stroke mechanism• In a retrospective report, the group of 71 patients with cryptogenic

stroke and active cancer had significantly elevated plasma D-dimer levels than the group of 277 patients with cryptogenic stroke without cancer or control group of 33 patients with active cancer but no stroke (10.7 vs 0.5 vs 0.7mcg/mL).

• Using a data-derived D-dimer cutoff of 2.15 mcg/mL, the sensitivity and specificity for identifying cancer hypercoagulability as the stroke mechanism was 74% and 97% respectively.

• Among 10 patients without known active cancer who had multifocal lesions in different vascular territories and who exceeded the D-dimer cutoff, all had occult malignancy on further work up.

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D-dimer and Stroke mechanism• In a prospective study of 74 patients with active cancer and otherwise

cryptogenic ischemic stroke, elevated D-dimer levels were associated with increased micro embolic signals in the Transcranial Doppler monitoring (odds ratio 1.08 per 1mcg/mL increase; 95% CI 1.01-1.15), indicating active ongoing embolization presumed due to hypercoagulability. (HITS : High Intensity Transient Signals)

University of Missouri - Neurology/Neurosurgery Grand rounds; Marta Rubiera et al. Stroke. 2010


D-dimer and Stroke mechanism

• D-dimer is also elevated in patients with stroke due to Cardioembolic source, though levels of elevation are usually more modest than those seen in cancer.

• In a study of 707 prospectively evaluated patients with acute ischemic stroke, D-dimer levels were significantly higher in stroke classified as Cardioembolic(1.1mcg/mL), lacunar(0.6mcg/mL) or undetermined(0.8mcg/mL) and similar results have been seen in other smaller studies.

• Given this, the ability of D-dimer to discriminate between Cardioembolic and non-Cardioembolic stroke in individual patients is limited.

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D-dimer and Stroke outcome• Early Stroke progression and recurrence has been associated with

increased thrombin generation and fibrin turnover, suggesting that D-dimer levels may help to identify patients at high risk.

• In two studies(54 and 153 patients respectively), elevated D-dimer levels were associated with stroke progression or early recurrent ischemic lesions on MRI, and another report of 180 patients with acute ischemic stroke found that D-dimer was an independent predictor of poor outcome. BUT OTHER STUDIES HAVE NOT SHOWN A CONVINCING ASSOCIATION BETWEEN D-DIMER LEVELS AND STROKE OUTCOME.

• For example, a report of 382 patients with stroke and Atrial fibrillation found no significant difference in D-dimer levels between those with and without stroke progression.

Cerebrovascular diseases 2009, Acta. Neurology Scandinavia 2008


Fibrinogen• Fundamental precursor for Fibrin clot• Elevated levels correlate with stroke risk, increased stroke severity and

poor outcome after Stroke.• A fibrinogen depletion coagulopathy may also play a role in hemorrhagic

complications following the use of intravenous thrombolytic therapy for acute ischemic stroke.


Fibrinogen and Stroke Risk

• In a meta-analysis of 31 prospective studies that included data from over 150,000 subjects without a prior history of stroke or Cardiovascular disease, Fibrinogen had moderately strong association with risk of any stroke(HR 2.06 for every 1g/L increase in Fibrinogen(95% CI 1.83-2.33), with only a modest attenuation of the effect after adjustment of the vascular risk factors. The relationship is strongest in young participants(Age 40-59 years) – JAMA 2005

• Association b/n Fibrinogen and recurrent Stroke risk is less clear. In 472 patients with IS included in a sub study of the PROGRESS trial, those in top tertile of the Fibrinogen levels(>4.04g/L) compared to the bottom tertile(<3.32g/L) had a significantly increased recurrent stroke risk(OR 1.34, 95% CI 1.01-1.78). However in the Edinburgh Stroke study of 817 patients, no clear relationship was seen between Fibrinogen and risk of recurrent Stroke – Stroke 2005


Fibrinogen and Stroke outcome• Levels were associated with poor outcome after the stroke, additive value to usual

clinical predictors remains uncertain.Evidence:- In a report of 900 patients with AIS, Fibrinogen>3.5g/L was independently assoc.

with one year mortality (HR 1.69, 95% CI 1.12-2.55)- In 817 patients with largely sub acute ischemic stroke in Edinburgh Stroke study,

mortality was significantly higher in those in the top quartile(>5.41g/L) compared with those in the bottom quartile(<3.81g/L); HR 1.45, 95% CI 1.24-1.72. Assoc. was not specific for vascular death.

- In 465 patients in the NINDS tPA trial, a higher fibrinogen level at 24hrs after thrombolysis was associated with a significantly increased risk of death at 90 days of approximately 40%.

- In 870 placebo treated patients with AIS from the STAT and ESTAT trials, the chance of good functional outcome decreased significantly across increasing quartiles of baseline Fibrinogen levels. Good functional outcome was observed in STAT for the bottom and top quartiles was observed in 36 Vs. 26 percent of pts. Respectively, corresponding fig. in ESTAT were 54 Vs. 25%.


Fibrinogen and complications from Thrombolysis

• Fibrinogen levels decrease and FDPs increase significantly after 2 hours of administration of IV thrombolysis for AIS. This cause an early Fibrinogen degradation Coagulopathy. While the baseline Fibrinogen levels do not predict symptomatic ICH following thrombolysis for ischemic stroke, changes in “F” and FDP may predict hemorrhage.

EVIDENCE:• In a study of 547 patients with AIS treated with rt-PA, a >200mg/dL

decrease in Fibrinogen over the first 6hrs after thrombolysis, compared with a smaller decrease was associated with a 4X increased risk of symptomatic ICH – Neurology 2013

• In a similar cohort of 157 patients, an increase in FDP>200mg/L at two hours after thrombolysis was associated with a nearly 5X increase in the risk of early(within 24hrs)parenchymal ICH. Early hematomas were significantly predictive of poor prognosis at three months – Stroke 2004

These data suggest a potential role of serial fibrinogen measurement for assessing risk of post-thrombolysis hemorrhage, it is unclear how this might impact treatment.

Lipoprotein associated Phospholipase A2 (LP-PLA2) Lp-PLA2 is an enzyme expressed predominantly by

Leukocytes that is involved in the metabolism of LDL to pro-inflammatory mediators.

Vascular specific inflammatory bio-marker highly expressed in the necrotic core of atherosclerotic plaque and is linked to plaque inflammation and instability.

Both Lp-PLA2 mass and activity can be measured in the serum and plasma.

Diagnostic testing for Lp-PLA2 mass is available and used clinically for long-term cardiovascular risk prediction, though it is not clinically used in clinical practice for Stroke evaluation.

Although some studies suggest better reproducibility and reliability in vascular risk prediction over time with Lp-PLA2 mass, the practical performance characteristics are less well defined.

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Lp-PLA2



Lp-PLA2 and Stroke Mechanism

• 2 separate studies of patients with acute TIA, each approx. 165 patients, evaluated Lp-PLA2 activity was associated with large artery atherosclerotic mechanism responsible for the CVA. Stroke 2009

• The discriminatory value of Lp-PLA2 measurement was not sufficient to allow its use as a diagnostic test for this purpose.


Lp-PLA2 and Stroke risk• A meta-analysis of 79,036 subjects in 32 prospective studies

found that elevations in Lp-PLA2 mass and activity were associated with an increased risk for ischemic stroke after adjustment for the other predictors of vascular risk. Lancet 2010

• There was strong association with Lp-PLA2 mass; for each standard deviation increase in the mass level, the risk of ischemic stroke increased by 14%. Circulation 2005


Lp-PLA2 association with race and ethnicity

• A population based longitudinal study with over 1900 participants and a median follow up of 11 years found that the association between Lp-PLA2 level and stroke risk varied by race and ethnicity.

• The Lp-PLA2 was associated with an increased risk of atherosclerotic stroke in non-Hispanic white subjects but not in Hispanic or black subjects. More studies are needed to determine the relationship

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Lp-PLA2 Utility

Lp-PLA2 may help guide the primary prevention strategies such as antiplatelet or statin therapy, in patients who are otherwise at intermediate risk of Stroke where uncertainty about the belief of these strategies exist.

A Lp-PLA2 mass level of 235ng/mL has been suggested as a reasonable threshold for identifying those at increased cardiovascular risk. Cardiology 2006

The levels will not help if checked in low risk patients (no or minimal vascular risk factors) or high risk patients (those with known Cardiovascular risk factors including prior stroke) as it would be unlikely to change management.



Lp-PLA2 and recurrent cerebrovascular events

Elevated levels may predict short-term risk of recurrent CVA, even after adjustment for clinical predictors. However, it is unlikely to impact treatment decisions for secondary prevention strategies in patients with prior stroke.

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Neuron Specific Enolase


• Enzyme released after neuronal damage, has been studied as a marker for brain injury including cerebral infarction, HIE, evaluation and prognosis

• A systematic review of 12 studies including 597 patients concluded that measurement of NSE was of limited value for diagnosis of AIS, even though the serum levels were elevated in patients compared to controls, correlated with infarct volume and correlated with degree of neuronal deficit. Cerebrovascular Disease 2005

• Major problems, with NSE as a biomarker include delayed release into the systemic circulation after brain injury, lack of specificity of cerebral infarction. Sensitivity and specificity of NSE for the diagnosis of Stroke is 53% and 64% in a study of 72 patients.

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Cellular Fibronectin• Glycoprotein primarily produced by vascular endothelial cells.• Prospective studies have reported that high pretreatment c-Fibronectin

levels predict the risk of hemorrhagic transformation after the IV thrombolysis for AIS.

• Rapid assay is not available for decision making prior to thrombolysis.


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Copeptin Secretion


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Copeptin

• Copeptin is a peptide consisting of C-terminal part of pro-Arginine vasopressin(AVP) which is released with AVP from the Pituitary in response to hemodynamic or Osmotic stimuli.

• Increased levels may predict recurrent cerebrovascular or other vascular events after TIA/AIS.

• In several prospective, it was an independent predictor of functional outcome and mortality after Ischemic stroke. Annals of Neurology 2009

• Multicenter CoRisk study evaluated 788 patients with AIS and measured Copeptin levels within 24 hours of symptom onset. The median Copeptin levels for those with a favorable and unfavorable outcome were 9.6 and 32.2pmol/L. Neurology 2013

• A 10 fold increase in Copeptin levels independently predicted unfavorable outcome(OR 2.17, 95%CI 1.46 to 3.22 and mortality (HR 2.4, 95% CI 1.6-3.6) at 90 days, irrespective of acute treatment including thrombolysis. Stroke 2010

• Better risk prediction than NIH stroke scale and age for Copeptin showed in a risk reclassification analysis.



Copeptin • Copeptin also appears to be a marker of Post stroke complications,

particularly infection.

• In the CoRisk cohort, although not powered to evaluate individual complications, elevated levels independently predicted the development of in-hospital complications(OR 1.93, 95% CI 1.33-2.80)


GLIAL FIBRILLARY ACIDIC PROTEIN• Predominantly produced by Astrocytes, making it a potential Brain-specific

biomarker• Significantly higher levels in intracerebral hemorrhage compared to

Ischemic stroke when measured in the acute phase 2-6hrs after symptom onset. Role in determining Stroke sub-type.

• Data is too limited to advocate clinical use in this scenario• Has been evaluated as a prognostic marker for ischemic stroke. In one

report it significantly correlated with the degree of neurologic deficit, the infarct volume and functional outcome at 90 days. (European Journal of Neurology – 2006)


Matrix Metalloproteinase 9

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MMP 9


• MMP 9 is a proteolytic enzyme that specifically degrades the major components of the basal lamina surrounding the cerebral blood vessels.

• In a systematic review of 22 studies and 3289 patients with acute ischemic stroke, higher MMP 9 levels correlated with greater stroke severity, Larger infarct volume and poor functional outcome.

• In several small studies, MMP9 was an independent predictor of hemorrhagic transformation associated with acute ischemic stroke, both in patients treated with IV tPA and in those not treated with thrombolysis. (Stroke 2001 and 2003)


S100 Calcium Binding Protein B• Astroglial protein that has been studied as a serum marker for cerebral

injury and disruption of blood brain barrier.• Single center studies have suggested S100B levels measured at multiple

points after Stroke were predictive of a malignant course and larger infarct volume and poor outcome.

• Independent factor for hemorrhagic deterioration after thrombolysis in ischemic stroke patients and as a diagnostic marker for stroke. In both scenarios, accuracy is too low to be clinically useful.

An Update on Blood Biomarkers for Stroke

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Transcript of An Update on Blood Biomarkers for Stroke