Biomarkers for Alzheimer’s disease - hstalks.com · • Sens. for MCI-AD 95% • Spec. against...

Biomarkers for Alzheimer’s diseaseProf. Henrik Zetterberg

1The screen versions of these slides have full details of copyright and acknowledgements

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Biomarkers for Alzheimer’s Disease

Henrik Zetterberg, MD, PhDProfessor of Neurochemistry

The Sahlgrenska Academy, University of Gothenburg

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Alzheimer’s disease

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Neurofibrillary tangles

Neuropathological criteriafor Alzheimer’s disease

Senile plaques

General neuronal loss

Modified from: Blennow et al., Lancet 2006



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Distribution of brain changesin Alzheimer’s disease

Mild cognitive impairment

Mild dementia Moderate dementia

Severe dementia

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Molecular pathology

• Senile plaques are composed of a 42 amino acid long aggregation-prone protein called amyloid b 42 (Ab42)

• Neurofibrillary tangles are composed of hyperphosphorylated isoforms of the intraaxonal protein tau (P-tau)

• Neuroaxonal degeneration is reflected by release of intraaxonal tau proteins (all isoforms, total-tau; T-tau)into the brain interstitial fluid that communicates freely with CSF

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Brain

Proximity to disease

Accessibility

Concentration of analyte

Proteolytic degradation

BloodCSF

Choosing the sample for biomarker analysis



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Cerebrospinal fluid

Total volume 150 mL, production rate 20 mL per hourStandard volume of sampling, 10-12 mL

Epidural vein

ChoroidPlexus

Choroid Plexus

Choroid Plexus

Circulation of CSF CSF in gray,

Venous blood in orange

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Is lumbar puncture dangerous?

No. of cases: 395Post LP headache: 2.1 %Meningitis / hematoma: 0

No. of cases: 241Post LP headache: 4.1%Meningitis / hematoma: 0

Andreasen et al., 2001

No. of cases: 342 (428 LP)Post LP headache: 0.9 %Meningitis / hematoma: 0

Peskind et al., 2005

Consecutive studies on complications after LP

No. of cases: 1089Post LP headache: 2.6 %Meningitis / hematoma: 0Zetterberg et al., Eur Neurol, 2010

Blennow et al., 1993

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Core CSF biomarkersfor Alzheimer’s pathology

Senile plaquesAb42

Neurofibrillary tanglesP-tau

Axonal injuryT-tau



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CSF biomarkersfor Alzheimer’s pathology - candidates

InflammationCell count

IgG/IgM productiona1-ACT, MCP-1, TNF-a, TGF-b

Oxidative stressF2-isoprostanes

Blood-brain barrier damageCSF/serum albumin ratio

Amyloid pathologyAb42 / Ab40

b-sAPP / a-sAPPBACE1 activity

Neurofibrillary tanglesP-tau

Axonal injuryT-tau

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Different uses of biomarkers in the evaluation of patients with memory problems

1. To diagnose neuroinflammatory and neuroinfectious conditions CSF cell count

Albumin ratio

Intrathecal IgG and IgM production

Specific inflammatory markers

2. Specific markers of AD neuropathology

T-tau, P-tau and Ab42 as supporting diagnostic markers in the clinic

T-tau, P-tau and Ab42 as additional inclusion criteria in studies of anti-AD drugs

3. Specific markers for drug effect monitoring Reduced levels of T-tau after treatment indicate less intense axonal degeneration

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CSF biomarkers for AD: Ab1-42

a-secretase Plasmamembrane

g-secretase

b-secretase BACE1



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Study design: • Consecutive AD cases from a community-based sample• AD (n=53), healthy controls (n=21)

CSF biomarkers for AD: Ab42

Andreasen et al., Arch Neurol 56: 673, 1999

AD Controls

CS

F-Ab

42

0

500

1000

1500

2000

2500 Sensitivity 92%Specificity 95%

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Data adjusted for:• Age at death

• Education

• Dementia severity

• ApoE4

• Time from diagnosis until death

• PM interval

• Time until Ab42 analysis

• CAA severity

Plaque count

CSF

-Ab42

(pg/

mL)

0 5 10 15 200

20

40

60

80

Non-dementedDemented

Strozyk et al. Neurology 60: 652-656, 2003

Study design:• 155 autopsy cases• Plaque counts - Bielschowsky stain of neocortex and hippocampus• Post-mortem CSF samples

CSF Ab42 reflects plaque pathology

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CSF Ab42 reflects plaque pathology (2)

General idea verified in PIB-PET studies:• Negative correlation between CSF-Ab1-42 and PIB retention in the brain

Fagan AM et al., Ann Neurol 2006Forsberg A et al., Neurobiol Aging 2008

Posterior cingulum

PIB

(RO

I/ref

)

Ab1-42

MCI non-converters MCI converters



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Diagnostic value

>100 papers

High total-tau and phospho-tau and low Ab1-42 in CSF =

85-95% sensitive and specific for AD in AD-control and longitudinal MCI studies

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Comb. of Ab42/P-tau and T-tau:

• Sens. for MCI-AD 95%

• Spec. against other MCI 87%

Prediction of incipient ADin MCI cases using CSF biomarkers

Hansson et al., Lancet Neurol 2006

Study design: • Follow-up study on MCI (>4 years)• Lumbar puncture and analysis of Aβ42 and variants of tau in CSF• MCI n=134 57 MCI → AD

56 MCI → MCI21 MCI → other dementias

Months

18 Buchhave et al., Arch Gen Psychiat 2012

Combination of Ab42/P-tau and T-tau:• T-tau > 350 pg/mL• Ab42 / P-tau ratio < 6.5

• Sens. for MCI-AD - 88%• Spec. non MCI-AD - 94%

• PPV - 94%• NPV - 89%

Prediction of AD within 10 yearsin patients with MCI

Cum

sur

viva

l

Time under risk for AD (months)



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CSF biomarkers for Alzheimer’s disease –diagnostic performance in a homogeneous

mono-center population, 2010

• AD patients: n=32

• Stable MCI: n=13

• Other dementias: n=15

• Controls: n=20

Johansson P et al., Journal of Alzheimer's Disease 24(3): 537-546, 2011

Sens

itivi

ty%

100% - Specificity%

20Sensitivity 83% Specificity 88%

Ab42

/P-ta

u 181

ratio

MCI-AD vs. controls MCI-AD vs. stable MCI + MCI-other

CSF T-tau (ng/L)

Sensitivity 83% Specificity 72%

Mattsson et al., JAMA 2009

Prediction of incipient AD in MCI cases using CSF biomarkers in a multi-center study

Ab42

/P-ta

u 181

ratio

CSF T-tau (ng/L)

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The Alzheimer’s Association QC programfor CSF biomarkers

• Goal 1 - Establish a standardized protocol for lumbar puncture and CSF processing

• Goal 2 - Monitor analytical variability

• Goal 3 - Establish a detailed protocol for laboratory proceduresfor AD CSF biomarkers

• Goal 4 - Improved quality and stability of assays

Please sign up if you perform Alzheimer biomarker analyses: [email protected]



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Can CSF markers predict development of ADin pre-symptomatic individuals?

Study design:• Population-based study, 55 healthy individuals

• LP at baseline, mean age 72

• Follow-up 8 years later: 45 remained normal, 10 AD with dementia or drop in MMSE > -5

Gustafson et al., JNNP 2007

Normal →Dementia/

MMSE drop

0

200

400

600

800

1000

1200

p = 0.006 r = 0.40 p< 0.05

Change in MMSE

Bas

elin

e C

SF

Ab42

-12 -9 -6 -3 0 3

1400

1200

1000

800

600

400

Normal →Normal

Bas

elin

e C

SF

Ab42

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Can CSF markers predict development of ADin pre-symptomatic individuals? (2)

Study design:• Clinical study on 57 healthy elderly individuals

• LP at baseline

• Follow-up during 3 years

Stomrud et al., Dement Geriatr Cogn Disord 2007; 24: 118-124

CSF Ab42 is lower in healthy elderly who will develop cognitive disurbances

Subjective memory impairment affecting quality of life (memQoL) at follow-up

No YesNo YesNo Yes

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Can we measure Ab oligomers in CSF?

Xia W et al., Arch Neurol. 2009 Feb; 66(2): 190-9Fukumoto H et al., FASEB J. 2010 Aug; 24(8): 2716-26

Y

Y

Abet

a

Y

YAb

eta

Abeta

Epitopeblocked

= 82E1, N-terminus-specific MAbY= biotin= avidin-HRP = chemiluminescent substrate

Abeta = b-Amyloid



25 Hölttä et al., in preparation

Can we measure Ab oligomers in CSF? (2)

Controls AD

Con

c pg

/mL

Oligomer in-house assay

P<0.0001

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Are CSF biomarkers dynamic?

27 Zetterberg et al., Arch Neurol 2006

Are CSF biomarkers dynamic? (2)

After a Bout (7-10 d)

After Rest (3 mo)


After Rest (3 mo)


After Rest (3 mo)

Glia

l Fib

rilla

ry A

cidi

c P

rote

in, n

g/L

Tota

l Tau

, ng/

L

Neu

rofil

amen

t Lig

ht P

rote

in, n

g/L



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AD drug development

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If you are interested in details of this, please read:

Zetterberg, H. et al., (2010) Alzheimers Res Ther 2(6): 32

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Biomarkers in studies of inhibitionof Ab aggregation

Change from Baselineat Week 12

CS

F A

b 42(p

g/m

L)C

SF

Ab 4

0(p

g/m

L)

Trial design:• Randomized, double-blind,• placebo-controlled, parallel study• 12 weeks oral treatment

with 0 mg (placebo), 50mg and 250mg of PBT-2

CS

F A

b 40(p

g/m

L)C

SF

Ab 4

2(p

g/m

L)



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CSF biomarkers in active Ab immunotherapy trials

Gilman S, et al. Arch Neurol 2005

• Reduction with treatment (in antibody reponders) on the downstream biomarker T-tau

• No clear effect on b-amyloid (Ab42)

Placebo Placebo Antibody responders

Antibody responders

CSF Ab42 CSF T-tau

Cha

nge f

rom

bas

elin

e (pg

/mL)

Cha

nge f

rom

bas

elin

e (pg

/mL)

CSF substudy in the AN1792 trial (active β-amyloid immunization)• Paired CSF samples (baseline and 1 year) from AD cases:

antibody responders n=11, placebo n=10

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CSF biomarkers in passive Abimmunotherapy trials

Blennow, Zetterberg et al. Arch Neurol 2012

Phase II bapineuzumab trials:• CSF samples from 27 bapineuzumab and 19 placebo cases

• CSF samples taken at baseline and 1 year

p=0.0013

CSF T-Tau

Cha

nge

at W

eek

54 (p

g/m

l)

-150

-100

-50

0

50

100

CSF P-Tau

Cha

nge

at W

eek

54 (p

g/m

l)

-15

-10

-5

0

5 p=0.027

p=0.0305

Bapineuzumab Placebo Bapineuzumab Placebo

CSF Ab1-42

Cha

nge

at W

eek

54 (p

g/m

l)

Bapineuzumab Placebo -30

-20

-10

0

10

20

Cha

nge

a t W

eek

54 (p

g/m

l)

0

50

100

150

p=0.0159

Cha

nge

at W

eek

54 (p

g/m

l)

-600

-400

-200

0

200

400

600

Bapineuzumab Placebo Bapineuzumab Placebo

CSF Abx-42 CSF Ab1-40

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Phase III clinical trails on bapineuzumab

Sperling R, presented at EFNS 2012

Phase III bapineuzumab APOE e4 trial:• Multicenter, randomized, double- blind, placebo-controlled trial

• Mild-moderate AD (MMSE 16-26)

• Bapineuzumab n=658

• Placebo n=432

Main outcome: change in ADAS-Cog over 18 months



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CSF biomarkers in the Phase III bapineuzumab trial

Sperling R, presented at EFNS 2012

Phase III bapineuzumab APOE e4 trial:• Multicenter, randomized, double- blind, placebo-controlled trial

• Mild-moderate AD (MMSE 16-26)

• Bapineuzumab n=658

• Placebo n=432

Secondary outcome: change in CSF phospho-tau over 18 months

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The current situation in AD drug development:

• Promising biomarker changes but no clinical benefit

• Too little, too late?

• Studies on preclinical AD needed

• Are we missing something?

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Summary

• CSF biomarkers can monitor the neuropathology of AD:– T-tau is a marker of cortical axonal degeneration

– P-tau is a marker of neurofibrillary tangle pathology

– Ab1-42 is a marker of plaque pathology

• These markers have high diagnostic accuracy and have been implemented in the recently revised clinical criteria for Alzheimer’s disease (http://www.alz.org/research/diagnostic_criteria/)

• Current research is aiming at establishing biomarkers for microglial activation and synapse loss

Biochemical markers together with neuroimaging and clinical evaluation allow for making a pre-dementia diagnosis of AD and

can be used to detect biochemical treatment effects in clinical trials of anti-AD drug candidates



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Clinical Neurochemistry Laboratory

Sahlgrenska University Hospital

Mölndal/Gothenburg, Sweden

Thanks for listening!

Funding: The Swedish Research Council, Swedish Brain Power, JPND and the Alzheimer’s Association

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