This presentation focusses on the importance of diagnostic biomarkers for Alzheimer's disease. MRI, amyloid PET and CSF biomarkers are discussed in detail.

1. Biomarkers in Alzheimer’s Disease
Dr Pramod Krishnan
Consultant Neurologist,
HOD Neurology
Manipal Hospital, Bengaluru

2. AD is the most common form of dementia1
13%
17%
70%
*Other types of dementia include dementia of undetermined aetiology, dementia with Lewy bodies and a group of diseases that contribute to
frontotemporal dementia
1. Knapp. et al. (2014). Dementia UK: Update. © Alzheimer’s Society 2014; 2. https://www.mayoclinic.org/diseases-conditions/alzheimers-
disease/expert-answers/alzheimers-and-dementia-whats-the-difference/faq-20396861
AD is still used interchangeably with dementia, but significant advances in understanding its
pathophysiology and epidemiology have been made, making this interchangeable use inaccurate2
62%
Alzheimer's Disease
Vascular Dementia
Mixed Dementia
Dementia with Lewy Bodies
Parkinson Disease
Frontotemporal Dementia
Other

3. Changes associated with AD start before
symptoms manifest
MCI, mild cognitive impairment
Aggarwal NT, et al. Indian J Med Res 2015;142:369–82
Birth 40 60 80 Death
Life course
Total loss of
independent function
Normal age-related
memory loss
Cognitive decline
accelerates after AD
diagnosis
MCI: memory problems;
other cognitive functions
OK; brain compensates for
changes
AD brain changes start
decades before
symptoms show
Healthy aging MCI Clinically diagnosed AD

4. Amyloid and tau accumulation are key pathological features of
AD
Accumulation of both amyloid and tau starts 15 years prior
to symptom onset
Time
Normal
Magnitude
Preclinical AD AD dementia
>10 years ~5-7 years
Prodromal
AD (=MCI)
~7-10 years
Amyloid-beta
accumulation
Tau
accumulation
Brain structure
alterations
Memory
impairment
Functional
impairment
Dynamic biomarkers
of the Alzheimer’s
pathological
cascade.28
Accumulation of
pathological amyloid
plaques and tau
tangles starts 15 years
prior
to symptoms
onset29,30

5. Presentation to GP
Individual or family
member notices clinical
symptoms and presents
to GP.
GP
GP assessment
Clinical history
Physical examination
Blood tests (thyroid/vitamin)
Cognitive screening test
Specialist
Referral to AD
specialist
Neuropsychological test
battery
MRI
Biomarker
testing
Biomarker testing
Amyloid PET
CSF biomarkers
Diagnosis
Diagnosis and
treatment
Amyloid testing in
conjunction with other
tests confirm MCI due to
AD or other causes.
Appropriate treatment
and care provided.
Intervention
Symptom
management
CSF and amyloid PET rarely ordered today
and are primarily only ordered for difficult
differential diagnosis
Occasionally conducted for differential
diagnosis with frontotemporal or Lewy body
dementia (~10–15% of people with suspected
AD)
Typical patient pathway
Bottleneck at referral from GP to AD
Specialist
*Grey denotes gaps and hurdles in
diagnostic pathway

6. Patients face a mountain of diagnostic challenges to
receive an AD diagnosis
CSF, cerebrospinal fluid; PET, positron emission tomography
References: 1. Lang L, et al. Prevalence and determinants of undetected dementia in the community: a systematic literature review and a meta-analysis. BMJ Open. 2017;7:e011146. 2. Alzheimer Europe. European carers’ report 2018: Carer’s experiences of
diagnosis in five European countries. 2018 [Internet; cited 2021 Feb 2]. Available from: https://www.alzheimer-europe.org/Publications/E-Shop/Carers-report/European-Carers-Report-2018. 3. Löppönen M, et al. Diagnosing cognitive impairment and dementia in
primary health care - a more active approach is needed. Age Ageing. 2003;32:606–12. 4. Boustani M, et al. Screening for dementia in primary care: A summary of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med. 2003;138:927–37. 5.
Valcour VG, et al. The detection of dementia in the primary care setting. Arch Intern Med. 2000;160:2964–8.
Many countries do not yet have clear recommendations for how to diagnose MCI/AD.1
Difficulties arise due to varied diagnostic pathways, uncertain prognoses,
and a long timeline to reach a diagnosis.
The average time from symptom onset to diagnosis is ~26 months.2
50%-75% of people with dementia do not have an
official diagnosis of MCI or AD.1,3-5
Primary care physicians are often not equipped to make referral decisions.1

7. Importance of early diagnosis of
MCI-AD and AD

8. • Between 50% and 75% of people with
dementia have no formal diagnosis8-11
- Rates of undocumented/undetected
diagnosis vary by severity and age8
• 53% of carers reported that an earlier
diagnosis of AD would have been preferred12
Patients experience a range of delays during diagnosis;
factors contributing to delays may be related to; the carer
(40.9%), the HCP (39.5%), the person with dementia (37.8%),
the healthcare system (25.0%).12
More than 50% of people with dementia have no formal
diagnosis
Family members reported that an earlier diagnosis would
have been preferable
Percentage (%) undocumented
Percentage (%) of patients with undetected dementia
67%
54%
27%
Mild Moderate Severe
17%
22%
28%
64–74 years 75–84 years 85+ years

9. Diagnosing AD at an earlier stage
• The ALCOVE project proposed that diagnosis should generally occur earlier than is
currently common practice
• This is a time when patients and their family first notice changes in cognitive function
• The information can be used by patients and their family to plan for the future
ALCOVE, Alzheimer’s Cooperative Valuation in Europe
Dubois B et al. J Alzheimers Dis 2016; 49: 617–31
Timeline of AD progression and diagnosis points on the disease continuum
T1
Earliest possible
diagnosis in the
event that reliably
predictive
bookmarkers are
developed
T4
Current
‘late-stage’
diagnosis
T2
Earliest
possible
diagnosis
using
currently
available
technology
T3
‘Timely’ diagnosis
responding to
patient and carer
concerns rather
than proactively
screening for the
disease
Onset of neuropathology
Subjective impairment/help seeking
Reliably predictive biomarkers
Onset of cognitive decline
Onset of disability

10. Accurate diagnosis of early AD is critical for optimal care
Identification of amyloid pathology at earlier stages has the potential to benefit physicians and patients through…
Confirmation of
AD pathology may
increase physician
confidence to
diagnose and
manage patients
appropriately
Use of objective
tools may lead to
reduced
resource use and
faster time to
diagnosis
Timely treatment
initiation may
improve patient
outcomes
Confirmation of
amyloid positivity
may enable
clinical trial
participation and
potential
treatment
Knowing their
amyloid status
helps patients and
carers plan for
their future
Increased
diagnostic
confidence
Optimized
diagnostic
procedures
Timely patient
management
Access to
clinical trials
and DMTs
Fulfilled
desire for
diagnosis
10

11. Biomarkers enhance diagnostic accuracy and physician
confidence
A prospective national study in
France investigated the impact
of abeta 42, pTau and tTau CSF
biomarker in the care of MCI
patients in memory clinics.5
The study reported that, after
measurement of Abeta 42,
pTau and tTau in CSF.5
28.8%
of patients
diagnosis
changed
46.6%
of patients
management
was modified
A French study by Cognat et al
(2019) reported that diagnostic
confidence significantly increased
after conducting a lumbar
puncture (p<0.0001) and that CSF
results modified management in
46.4% (71/156) of patients with
23.5% enrolled in clinical trials
(Cognat, E et al. 2019).
This study clearly demonstrated
the value of CSF testing in
informing clinical decisions and
optimizing patient management.

12. Confirmation of amyloid positivity enhances diagnostic
accuracy and physician confidence
Confirmation of amyloid positivity is crucial for confident diagnosis of AD, including at MCI stage1,2
*CSF assays have demonstrated concordance with amyloid PET imaging3
For more information, please see Hansson 2018 publication summary slides.
References: 1. Kim 2018; 2. Cognat 2019; 3. Hansson 2018.
• In a prospective national study of MCI diagnosis, based
on physician questionnaire responses, diagnostic
confidence significantly increased (p<0.0001) after
conducting analysis of AD CSF biomarkers:2
• CSF results modified management in 46.4% of MCI
patients, including 23.5% who were enrolled in clinical
trials and 19.6% who received AChEi treatment
6.73 ± 1.8
pre-CSF
analysis
8.3 ± 1.4
post-CSF
analysis
Diagnostic confidence (10-point scale)
vs.
• A review of studies published between 2012 and 2018
examined the diagnostic impact of amyloid PET* on
AD diagnosis in clinical practice:1
– All studies reported increased diagnostic
confidence or diagnostic certainty after
amyloid PET imaging. However, limited number
of MCI patients was included1
– Overall change in diagnosis after amyloid PET
ranged from 9% to 68% of cases1
– Most common change in management was
initiation or discontinuation of planned AD
symptomatic therapies
12

13. Confirmation of amyloid positivity may lead to reduction
in some diagnostic procedures
• A review of studies investigating the clinical utility of amyloid PET* reported a 24.4% reduction in planned
structural imaging across 19 clinical centers in the US following confirmation of amyloid positivity2
• A UK-based study reported reductions in the number of AD assessments following introduction of amyloid
PET testing2
Changes in diagnostic testing and referrals after amyloid PET in the UK3
2,379
1,971
478
1,230
198
850
377
44
698
1,136
1,020
173 101 135
656
239
57
669
0
500
1,000
1,500
2,000
2,500
Neuropsychological
testing
referral
Any imaging Genetic tests CSF tests Serological tests EEG Polysomnography Other tests Other specialist
referrals
(e.g. psychiatrist,
sleep medicine)
Number
of
testing
and
referrals
Pre-PET recommended (n) Post-PET implemented (n)
*CSF assays have demonstrated concordance with amyloid PET imaging1
For more information, please see Hansson 2018 publication summary slides.
References: 1. Hansson 2018. 2. Kim 2018; 3. Rabinovici 2019.
13

14. 14
✔ Enable families to plan for the future (advanced care planning) and spend more time
together (e.g. activities that provide happiness and fulfilment)1
✔ Begin health measures to preserve existing cognitive function to preserve daily
activities1
✔ Time for assembly of medical and caregiving teams to prevent, treat and manage
coexisting medical conditions commonly observed with AD1
✔ Initiate currently available symptomatic drugs2
✔ Consider participation in clinical trials for researching new treatments2
✔ Initiate DMT when they become available to change the course of the disease2
There are valuable medical, emotional and societal benefits
of a timely and accurate diagnosis of AD
AD, Alzheimer's disease; CSF, cerebrospinal fluid; DMT, disease-modifying therapy; MCI, mild cognitive impairment
1. Alzheimer’s Association. 2018 Alzheimer’s disease facts and figures. Available at: https://www.alz.org/media/HomeOffice/Facts%20and%20Figures/facts-and-figures.pdf; Accessed
May 2021
2. Dubois B et al. J Alzheimers Dis 2016; 49:617–31
Medical
Emotional and Societal

15. Amyloid positivity confirmation is an inclusion criterion in
trials of AD therapies
1 2 3 4 5 6 7
a b c d e
AD biomarkers are accepted as surrogate markers for the presence of AD pathology; confirmed diagnosis of AD (e.g.
by confirming amyloid positivity) may be required for prescription of future therapies indicated for AD
• The FDA recently granted accelerated
approval for Aduhelm (aducanumab)
based on the surrogate endpoint of
reduction of amyloid beta plaque in
the brain in patients with MCI and mild
AD, with amyloid PET confirmed AD
pathology1
• Clinical trials for other AD therapies are
ongoing2
Clinical and patient value
References: 1. FDA 2021c; 2. Cummings 2020.
Treatment
Stage of development (year of study
completion)
Aduhelm
(aducanumab)
FDA Fast Track approval (June 2021), under
evaluation with EMA and PMDA (as of June
2021)
Solanezumab (pre-
symptomatic)
Phase 3 (2022)
Gantenerumab Phase 3 (2023)
BAN2401
(lecanemab)
Phase 3 (2024)
Example of therapies approved, under-review, or under
clinical development with amyloid positivity as inclusion
criterion2
15

16. 16
The majority of clinical trials evaluating DMTs are enrolling
individuals in the MCI or early AD disease stage
AD, Alzheimer's disease; DMT, disease-modifying therapy; MCI, mild cognitive impairment
1. Cummings J, et al. Alzheimers Dement (N Y) 2020;6:e12050; 2. Liss JL, et al. J Intern Med 2021 doi: 10.1111/joim.13244
Phase 2 and 3 DMT trials in AD in 20201
5
10
7
4
0
5
10
15
20
25
Preclinical AD* Clinical AD
Mild to moderate AD
Mild/early AD
MCI due to AD/prodromal AD
Preclinical AD
Should these trials be successful, many
DMTs for AD will apply specifically to MCI
due to AD and early dementia,
underscoring the importance of
biomarker confirmation in the
identification of AD in the clinical setting2

17. Biomarkers for the early
detection of amyloid pathology

18. Definition of Biomarker
• A biomarker is a characteristic that is objectively measured and evaluated as an
indicator of normal biological processes, pathologic processes, or biological
responses to a therapeutic intervention.
• Biomarkers help characterize the baseline state, a disease process, or a response to
treatment.
• Biomarkers include measures of genes, “omics” technologies (genomics,
transcriptomics, proteomics, metabolomics, lipidomics), imaging, blood or
electrophysiological studies.

19. Validated biomarkers that are proxies for AD pathological
changes already exists
• Several studies have reinforced that certain imaging and cerebrospinal fluid (CSF) biomarkers are valid
proxies for neuropathological changes of AD31
Imaging-to-autopsy comparison studies have established that amyloid positron emission tomography (PET) is a valid in vivo
surrogate for amyloid deposits31
It is also widely accepted that CSF biomarkers such as amyloid β(1-42) and phosphorylated tau (181P) are a valid
indicator of the abnormal pathologic state associated AD.31
• By contrast ,additional research has highlighted the fact that measures of neurodegeneration or neuronal
injury that are commonly used in AD research – magnetic resonance imaging (MRI), fluorodeoxyglucose
(FDG) PET and CSF total tau, are not specific for AD31

20. Clinical diagnosis of amyloid pathology
• Clinical diagnosis has modest performance and provides no information on
histopathological causes of dementia e.g.
• Modest sensitivity (71%-81%) and specificity (approximately 70%) for AD.

21. 21
Aβ42, pTau and tTau are measured through biochemical or
imaging techniques to support a clinical diagnosis of AD
* CSF Aβ40 is not a biomarker of AD pathology but serves as a proxy for ‘total’ Aβ levels
Aβ, amyloid beta; AD, Alzheimer's disease; CSF, cerebrospinal fluid; PET, positron emission tomography; pTau, phosphorylated tau; tTau, total tau
Figures created with biorender.com. PET scan image courtesy of University of Pittsburgh available from: https://commons.wikimedia.org/wiki/File:PiB_PET_Images_AD.jpg.
1. Lashley T, et al. Dis Model Mech 2018;11:dmm031781; 2. Blennow K & Zetterberg H. J Intern Med 2018;284:643–63
Levels of Aβ42, Aβ40*, pTau and tTau in
the CSF are measured using
immunoassays1,2
CSF is obtained via
lumbar puncture1
PET scans are used to visualize amyloid plaques in the brain1
PET scan of a person with AD
(left) vs a healthy control (right)
Amyloid tracers such as 18F-florbetapir or
11C-Pittsburgh compound B bind to amyloid
plaques and are detected by the PET
scanner. Increased amyloid load in the
brain is visualized as red colored areas on
the scan1

22. 22
MRI in MCI and Alzheimer’s disease

23. 23
MRI features of Alzheimer’s disease
• Initial atrophy is noted in the hippocampus and entorhinal cortex, followed
by parahippocampal gyrus, fusiform gyrus and temporal pole.
• This predicts conversion to AD in MCI patients.
• This differentiates AD from other dementia types.
• Additional limbic structures including the amygdala, olfactory bulb tract,
cingulate gyrus, and thalamus are impacted in AD.
• Subsequently, atrophy becomes more diffuse.
• Volumetric analysis (voxel based morphometry) can be used to quantify the
atrophy.

24. 24
T1-weighted MRI imaging using an MPRAGE (Magnetisation Prepared Rapid Gradient Echo) sequence
shows decreased grey matter volume in an AD patient compared to a healthy control, and intermediate grey
matter decline in a patient with MCI.

25. 25

26. 26

27. 27
The MTA-score should be rated on
coronal T1-weighted images at a
consistent slice position.
Select a slice through the corpus of the
hippocampus, at the level of the
anterior pons.
> 75 years : MTA-score 3 or more is
abnormal (i.e. 2 can still be normal at
this age)

28. 28
The score is based on a visual rating of the width
of the choroid fissure, the width of the temporal
horn, and the height of the hippocampal
formation.
score 0: no atrophy
score 1: only widening of choroid fissure
score 2: also widening of temporal horn of lateral
ventricle
score 3: moderate loss of hippocampal volume
(decrease in height)
score 4: severe volume loss of hippocampus
< 75 years: score 2 or more is abnormal.
> 75 years: score 3 or more is abnormal.

29. 29

30. 30
MRI features in Alzheimer’s disease
• Periventricular white matter hyperintensities are more common in AD, and
predict conversion from MCI to AD.
• However, they are less common compared to vascular dementia patients.

31. 31
On MR, white matter hyperintensities (WMH)
and lacunes - both of which are frequently
observed in the elderly - are generally viewed
as evidence of small vessel disease.
The Fazekas-scale provides an overall
impression of the presence of WMH in the
entire brain.
It is best scored on transverse FLAIR or T2-
weighted images.
Score:
Fazekas 0: None or a single punctate WMH
lesion
Fazekas 1: Multiple punctate lesions
Fazekas 2: Beginning confluency of lesions
(bridging)
Fazekas 3: Large confluent lesions

32. 32
T2-weighted MRI imaging using a FLAIR (Fluid Attenuated Inversion Recovery) sequence shows
increased WMHs in an AD patient compared to a healthy control and intermediate levels of WMHs in a
patient with MCI.

33. 33
Advanced MRI techniques
• Diffusion tensor imaging
• Arterial spin labelling
• Magnetic resonance spectrography
• Functional MRI
• Machine learning and Artificial intelligence techniques using MRI.

34. 34
Amyloid PET scan in AD

35. 35
Amyloid PET in Alzheimer's disease
• Approved for clinical use by the USFDA, EMA and other agencies.
• Allows the noninvasive detection of amyloid plaques, a core
neuropathologic feature that defines the disease.
• However, amyloid pathology can also be found in cognitively unimpaired
older adults and in patients with other neurodegenerative disorders.
• It is expensive and not yet available in India.
• It is the preferred test to detect amyloid in clinical trials for therapeutic
interventions in AD.

36. 36
Examples of negative and positive Aβ PET findings using different tracers.

37. 37
Evolution of amyloid PET positivity across AD spectrum.
(A) Positive 11C-PiB scan of cognitively normal (CN) participant, in which significant binding
is observed in precuneus, posterior cingulate cortex, and medial prefrontal areas.
(B) Positive 11C-PiB scan of MCI patient, in which significant and moderate binding is
observed throughout cortex.
(C) Positive 11C-PiB scan of AD patient, in which significant and severe binding is observed
throughout cortex.

38. 38
Utility of Amyloid PET scan
Amyloid PET can detect
• cerebral Aβ deposition with precision
• has good specificity for AD neuropathology,
• can inform on the presence of contributing amyloid co-pathology in other
diseases,
• will inform eligibility for emerging anti-Aβ therapeutics.

39. 39
FDG PET and SPECT in AD

40. 40
SPECT in Alzheimer's disease
• It is a nuclear medicine imaging modality in which a gamma-emitter
radiotracer is injected into the patient and tomographic images of its
distribution are then obtained.
• SPECT allows for measurement of cerebral perfusion.
• Perfusion is often reduced in the brain of patients with dementia, either due
to decreased blood supply (e.g.vascular dementia) or decreased demand
secondary to neuronal dysfunction or cell death (e.g. AD)
• HMPAO SPECT is the most frequently used techniques.

41. 41
SPECT, 18F-FDG PET, and 11C-PIB PET of two patients.
Upper row is of a 63-year-old female with MCI. SPECT (A) and 18F-FDG PET (B) showed hypoperfusion and
hypometabolism in the bilateral parietal cortex. Amyloid PET (C) showed significant cortical amyloid deposits.
Lower row is of a 55-year-old female with suspected AD. SPECT (D) and 18F-FDG PET (E) showed a left posterior
parietal hypoperfusion/hypometabolism suggestive of AD. 11C-PIB PET (F) confirmed cortical amyloid deposit.

42. 42
Utility of SPECT in AD
• To differentiate between AD and other types of dementia.
• Evaluation of suspected AD in the context of mild cognitive impairment.
• In Alzheimer’s disease, perfusion and metabolic images typically show
decreased activity in the temporal, parietal, and prefrontal cortices, with
preservation of the primary sensorimotor and occipital area.

43. CSF Biomarkers in Alzheimer’s
Disease

44. CSF Aβ42, pTau and tTau are core validated biomarkers of
AD pathology and associated neurodegeneration
Aβ, amyloid beta; AD, Alzheimer's disease; CSF, cerebrospinal fluid; pTau, phosphorylated tau; tTau, total tau
Figure created with biorender.com
Blennow K & Zetterberg H. J Intern Med 2018;284:643–63
Intracellular
neurofibrillary tangles
Extracellular amyloid
plaques
Amyloid pathology
Tau pathology
Neuronal/axonal
degeneration
Synaptic
dysfunction
Microglial
differentiation and activation
Neurodegeneration
High CSF pTau indicates increased
levels of tau phosphorylation, the key
component of neurofibrillary tangles
Elevation of CSF tTau indicates the
intensity of neurodegeneration or
severity of neuronal damage
Reduced CSF Aβ42 reflects the
aggregation and deposition of this
aberrant amyloid protein in the
brain

45. 45
CSF Aβ42, pTau and tTau can support early diagnosis of
AD as abnormalities in their levels precede clinical
symptoms
Aβ, amyloid beta; AD, Alzheimer's disease; CSF, cerebrospinal fluid; FDG, fluorodeoxyglucose (18F); MCI, mild cognitive impairment; MRI, magnetic resonance imaging; PET, positron
emission tomography; pTau, phosphorylated tau; tTau, total tau
Figure adapted from 1. Jack CR Jr, et al. Lancet Neurol 2013;12:207–16; 2. Blennow K & Zetterberg H. J Intern Med 2018;284:643–63; 3. Barthélemy NR, et al. Nat Med 2020;26:398–
407
Recent data have shown some
species of tau, pTau(217) and
pTau(181), to be present in the
CSF 20 years before tau
pathology manifests in the brain3
Change in CSF tau levels
precede the symptomatic onset of
AD1,2
Time
CSF Aβ42
Amyloid PET
CSF tau
MRI + FDG PET
Cognitive
impairment
Normal
M
C
I
Detection
threshold
Min
.
Max
.
Biomarker
abnormality
Change in CSF Aβ42 is the earliest
biomarker signal of AD pathology1,2

46. 46
Subjects with MCI-AD and AD have elevated levels of CSF
tau and decreased levels of Aβ42 versus control subjects
*Random effects meta-analysis using the method of DerSimonian and Laird (P-values ≤0.05 were considered significant)
Aβ, amyloid beta; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; pTau, phosphorylated tau; tTau, total tau
Olsson B, et al. Lancet Neurol 2016;15:673–84
Data from a systematic review of 231 articles and meta-analysis
comprising 15,699 subjects with AD and 13,018 control subjects
CSF pTau
CSF tTau
CSF Aβ42
1.72 times higher (P<0.001)*
1.76 times higher (P<0.001)*
0.67 times lower (P<0.001)*
CSF pTau
CSF tTau
CSF Aβ42
1.88 times higher (P<0.001)*
2.54 times higher (P<0.001)*
0.56 times lower (P<0.001)*
Subjects with
stable MCI
Subjects with
MCI due to AD vs Healthy
controls
Subjects
with AD vs

47. 47
– Concordance with amyloid PET
– Discrimination of AD in different populations
– Predicting progression to AD in subjects with MCI
Rationale for using CSF pTau/Aβ42 and tTau/Aβ42
ratios to support the clinical diagnosis of AD
Aβ, amyloid beta; AD, Alzheimer's disease; MCI, mild cognitive impairment; PET, positron emission tomography; pTau, phosphorylated tau; tTau, total tau

48. 48
CSF tau/Aβ42 ratios have been evaluated in the two best-
characterized and representative cohorts in the field
Aβ, amyloid beta; AD, Alzheimer's disease; ADNI, Alzheimer’s Disease Neuroimaging Initiative; BioFINDER, Biomarkers For Identifying Neurodegenerative Disorders Early and Reliably
study group; CDR-SB, clinical dementia rating – sum of boxes; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; MMSE, Mini-Mental State Examination; PET, positron emission
tomography
1. Elecsys® phospho-Tau (181P) CSF method sheet ms_07357036190 v1.0. Roche Diagnostics GmbH. 2017; 2. http://biofinder.se/the_biofinder_study_group/; 3. http://www.adni-
info.org/. Websites accessed May 2021
Concordance with amyloid PET visual read
Study cohort BioFINDER1,2
Inclusion criteria Patients with mild cognitive symptoms (MCS)
with CSF and PET imaging available
Primary analysis population
(N)
277
Subjective cognitive decline (n) 120
MCI (n) 153
No assignment (n) 4
Methodology • Amyloid PET scans read independently by
three trained readers with majority voting
to assign positive or negative status
• Cut-off ratios established as the value that
optimized concordance with amyloid PET
Identification of patients at risk of cognitive decline in ≤2
years
Study cohort ADNI1,3
Inclusion criteria Early or late MCI with CSF and clinical
assessment scores (CDR-SB and MMSE)
available at baseline
Primary analysis population
(N)
619
Early MCI (n) 277
Late MCI (n) 342
Methodology • Linear mixed-effects models used to
measure the biomarkers’ ability to separate
patients at lower vs. higher risk of cognitive
decline (change in CDR-SB or MMSE)
within 2 years
• Models were adjusted for age, sex,
education time and baseline value of the
respective clinical score.

49. 49
CSF pTau/Aβ42 and tTau/Aβ42 ratios are highly concordant
with amyloid PET
Aβ, amyloid beta; AD, Alzheimer's disease; BioFINDER, Biomarkers For Identifying Neurodegenerative Disorders Early and Reliably study group; CI, confidence interval; CSF, cerebrospinal fluid; MCI, mild
cognitive impairment; NPA, negative percentage agreement; OPA, overall percentage agreement; PET, positron emission tomography; PPA, positive percentage agreement; pTau, phosphorylated tau;
tTau, total tau
1. Hansson O, et al. Alzheimer’s Dement 2018;14:1470–81; 2. Elecsys® phospho-Tau (181P) CSF method sheet ms_07357036190 v1.0. Roche Diagnostics GmbH. 2017;
3. Elecsys® total-Tau CSF method sheet ms_07356994190 v2.0. Roche Diagnostics GmbH. 2018
Biomarker cut-offs2,3
Cut-off (+) Cut-off (-)
pTau/Aβ42 >0.024 ≤0.024
tTau/Aβ42 >0.28 ≤0.28
Performance of CSF biomarker cut-offs vs amyloid PET1-3
PPA
(sensitivity)
% (95% CI)
NPA
(specificity)
% (95% CI)
OPA
% (95% CI)
pTau/Aβ42 90.9
(83.9–95.6)
89.2
(83.5–93.5)
89.9
(85.7–93.2)
tTau/Aβ42 90.9
(83.9–95.6)
89.2
(83.5–93.5)
89.9
(85.7–93.2)
pTau/Aβ42, tTau/Aβ42 ratio positive/negative result is
concordant with a positive/negative amyloid PET scan.
BioFINDER cohort1
Disease stage: MCI, AD
PET visual read negative
PET visual read positive
pTau/Aβ42 cut-off
N=277
pTau versus Aβ42 by visual PET status
10
0
75
50
25
pTau
pg/mL
Aβ42
pg/mL
1000 2000 3000

50. 50
pTau/Aβ42 and tTau/Aβ42* ratios are highly concordant
with amyloid PET across the disease spectrum
*Data not shown for tTau vs Aβ42
Aβ, amyloid-beta; AD, Alzheimer's disease; ADNI, Alzheimer's Disease Neuroimaging Initiative; CI, confidence interval; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; NPA,
negative percentage agreement; PET, positron emission tomography; PPA, positive percentage agreement; pTau, phosphorylated tau; tTau, total tau
Hansson O, et al. Alzheimers Dement 2018;14:1470–81 (Supplementary materials)
PPA, %
(95% CI)
NPA, %
(95% CI)
PPA, %
(95% CI)
NPA, %
(95% CI)
PPA, %
(95% CI)
NPA, %
(95% CI)
PPA, %
(95% CI)
NPA, %
(95% CI)
PPA, %
(95% CI)
NPA, %
(95% CI)
82.1
(63.1–93.9)
93.1
(87.3–96.8)
66.7
(44.7–84.4)
92.9
(84.1–97.6)
79.4
(70.5–86.6)
94.5
(89.9–97.5)
90.2
(82.7–95.2)
88
(75.7–95.5)
99.1
(95.2–100)
85.7
(57.2–98.2)
Scatterplots of pTau versus Aβ42 per sub-population in ADNI cohort
(n=819)
Significant
memory concern
Early
MCI
Late MCI AD
Cognitively
unimpaired
n=160 n=95 n=277 n=155 n=132
Visual PET status ● Negative ▲ Positive ■ Missing
100
75
50
25
0
pTau
pg/mL
100
75
50
25
0
pTau
pg/mL 100
75
50
25
0
pTau
pg/mL
100
75
50
25
0
pTau
pg/mL
100
75
50
25
0
pTau
pg/mL
Aβ42 pg/mL
1000 2000 3000
Aβ42 pg/mL
1000 2000 3000
Aβ42 pg/mL
1000 2000 3000
Aβ42 pg/mL
1000 2000 3000
Aβ42 pg/mL
1000 2000 3000

51. 51
The high discriminatory sensitivity and specificity of CSF
tau/Aβ42 ratios has been verified across cohorts
Aβ, amyloid beta; AD, Alzheimer’s disease; AIBL, Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing; CSF, cerebrospinal fluid; FTD, frontotemporal dementia; MCI, mild
cognitive impairment; NPA, negative percentage agreement; PET, positron emission tomography; PPA, positive percentage agreement; pTau, phosphorylated tau; tTau, total tau;
WU ADRC, Washington University Alzheimer's Disease Research Center
1. Schindler SE, et al. Alzheimers Dement 2018;14:1460–9; 2. Doecke JD, et al. Alzheimers Res Ther 2020;12:36
WU ADRC (N=198)1
Cognitively unimpaired, very mildly or
mildly cognitively impaired participants
AIBL (N=202)2
Cognitively unimpaired, MCI, AD or
FTD participants
pTau/Aβ42
92%
83%
85%
97%
Cohorts tTau/Aβ42
PPA NPA
92% 89%
90% 91%
PPA NPA
CSF
Amyloid
PET

52. 52
CSF tau/Aβ42 ratios consistently outperformed individual biomarkers
in discriminating between amyloid-positive and negative scans
Aβ, amyloid beta; AD, Alzheimer’s disease; AIBL, Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing; CSF, cerebrospinal fluid; FTD, frontotemporal dementia; MCI, mild
cognitive impairment; PET, positron emission tomography; pTau, phosphorylated tau; tTau, total tau; WU ADRC, Washington University Alzheimer's Disease Research Center
1. Schindler SE, et al. Alzheimers Dement 2018;14:1460–9; 2. Doecke JD, et al. Alzheimers Res Ther 2020;12:36
WU ADRC (N=198)1
Cognitively unimpaired, very mildly or
mildly cognitively impaired participants
78% 79%
AIBL (N=202)2
Cognitively unimpaired, MCI, AD or
FTD participants
89%
79% 75% 91%
87%
91%
Cohorts Overall percent agreement (OPA)
CSF
Amyloid
PET
77%
81%
tTau/Aβ42
pTau/Aβ42
pTau
Aβ42 tTau

53. 53
Hulstaert F, et al. (1999)1
Multicenter study
Clinical diagnosis of probable AD (N=150) vs
healthy controls (N=100)
Sensitivity Specificity
Aβ42
tTau
tTau/Aβ42
Clinical diagnosis of probable AD (N=150) vs
other neurologic disorders (N=84)
Sensitivity Specificity
Aβ42
tTau
tTau/Aβ42
78%
79%
85%
81%
70%
87%
71%
71%
85%
CSF tau/Aβ42 ratios outperform individual biomarkers when
discriminating subjects with AD in different populations
All studies performed with ELISA assays (Innogenetics®)
Aβ, amyloid beta; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; FTD, frontotemporal dementia; ELISA, enzyme-linked immunosorbent assay; pTau, phosphorylated tau; tTau, total
tau
1. Hulstaert F, et al. Neurology 1999;52:1555–62; 2. Cruz De Souza L, et al. J Neurol Neurosurg Psychiatry 2011;82:240–6; 3. Santangelo R, et al. Curr Alzheimer Res 2019;16:587‒95
63%
89%
86%
Cruz De Souza L, et al. (2011)2
Single-center study
Clinical diagnosis of probable AD (N=60) vs
FTD (N=27)
Sensitivity Specificity
Aβ42
pTauAβ42
tTau/Aβ42
Clinical diagnosis of probable AD (N=60) vs
semantic dementia (N=19)
Sensitivity Specificity
Aβ42
pTauAβ42
tTau/Aβ42
98%
98%
95%
68%
84%
84%
68%
92%
95%
85%
93%
85%
Santangelo R, et al. (2019)3
Retrospective single-center study
Clinical diagnosis of probable AD (N=277) vs
other types of dementia (N=249)
Sensitivity Specificity
Aβ42
pTau
tTau
pTauAβ42
tTau/Aβ42
71%
72%
72%
74%
81%
64%
80%
72%
81%
76%

54. 54
pTau/Abeta 42 ratio is more robust than Abeta42/Abeta 40 ratio
Clear separation is observed in amyloid-PET positive
and amyloid-PET negative patients for pTau/Abeta42
pTau/Abeta42
0.022
75
50
25
Elecsys
®
pTau,
ng/mL
1000 2000 3000
Elecsys® β-Amyloid (1-42)
100
Abeta42/Abeta 40
40000
Elecsys®
β-Amyloid
(1-40)
30000
20000
10000
1000 2000 3000
Elecsys® β-Amyloid (1-42)
0.05
Visual PET status  Positive  Negative Cut-off
The Elecsys® CSF Aβ40 assay used in this study is for research use only.
• Study analysis on 277 patients samples of
BioFINDER cohort was performed
• Results show that Abeta 42/40 has high
concordance with amyloid PET but is less robust
compared with pTau/Abeta42 ratio 38

55. Role of CSF biomarkers in
predicting progression of MCI to AD

56. 56
CSF pTau/Aβ42 ratio predicted clinical decline in patients
with MCI over the course of 2 years*
*As assessed by the ability of biomarker groups to predict changes in clinical scores (CDR-SB) from baseline to 24 months in the ADNI MCI cohort (N=619)
Aβ, amyloid beta; ADNI, Alzheimer’s Disease Neuroimaging Initiative; CDR-SB, Clinical Dementia Rating – sum of boxes; CSF, cerebrospinal fluid; LS, least squares; MCI, mild cognitive
impairment; pTau, phosphorylated tau; ; SE, standard error
Hansson O, et al. Alzheimers Dement 2018;14:1470–81 3. Palmqvist 2017.
Biomarker-positive patients
progressed 1.4–1.6 points
(CDR-SB score)
Biomarker-negative patients
had a significantly smaller
change in CDR-SB of less than
0.5 points
Time course of pTau/Aβ42 in patients with MCI
Time (months)
0 6 12 24
1.
5
2.
0
2.
5
3.
0
LS-Mean
CDR-SB
score
(±
SE)
Baseline biomarker status
Positive
Negative
Clinical progression predicted by predefined CSF biomarker cut-offs in the ADNI MCI cohort (N=619)
Over the course of
2 years of follow-up
• There is also evidence to suggest CSF biomarkers may become abnormal prior to PET abnormality in some patients, enabling even
earlier detection of AD pathology3

57. 57
CSF pTau/Aβ42 and tTau/Aβ42 ratios are highly predictive
of risk of progression to AD*
*As assessed by time-to-event analyses for the outcome time-to-dementia diagnosis.
Aβ, amyloid beta; AD, Alzheimer’s disease; BioFINDER, Biomarkers For Identifying Neurodegenerative Disorders Early and Reliably study group; CI, confidence interval; CSF, cerebrospinal fluid; MCS, mild
cognitive symptoms; pTau, phosphorylated tau; tTau, total tau
Blennow K, et al. Sci Reports 2019.9:19024 (adapted and licensed under CC BY 4.0)
Biomarker
Hazard ratio (95%) CI
Dementia AD
pTau/Aβ42 3.38 (2.35–4.87) 11.48 (6.04–21.81)
tTau/Aβ42 3.38 (2.35–4.86) 10.31 (5.55–19.13)
Aβ42 2.63 (1.83–3.78) 6.00 (3.38–10.65)
pTau 1.94 (1.39–2.72) 3.86 (2.51–5.95)
tTau 1.67 (1.20–2.33) 3.00 (1.98–4.55)
pTau/Aβ42 tTau/Aβ42
Kaplan–Meier survival analysis for all-cause dementia diagnosis within 6 years – BioFINDER cohort (MCS;
N=431)
Hazard ratios (Cox proportional regression) for
conversion to dementia or AD by CSF biomarker
status
%
Dementia-free
(BioFINDER)
0 12 24 36 48 60 72
Time (months)
0.75
1.00
0.50
0.25
0
0 12 24 36 48 60 72
0.75
1.00
0.50
0.25
0
Biomarker positive
Biomarker negative

58. 58
CSF pTau/Aβ42 and tTau/Aβ42 ratios are highly predictive
of risk of progression to AD*
*As assessed by time-to-event analyses for the outcome time-to-dementia diagnosis.
Aβ, amyloid beta; AD, Alzheimer’s disease; ADNI, Alzheimer’s Disease Neuroimaging Initiative; CI, confidence interval; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; pTau, phosphorylated tau; tTau, total tau
Blennow K, et al. Sci Reports 2019.9:19024 (adapted and licensed under CC BY 4.0)
Biomarker
Hazard ratio (95%) CI
Dementia
pTau/Aβ42 4.76 (3.22–7.04)
tTau/Aβ42 5.20 (3.48–7.78)
Aβ42 4.41 (2.89–6.72)
pTau 2.73 (2.02–3.70)
tTau 2.12 (1.59–2.84)
pTau/Aβ42 tTau/Aβ42
Kaplan–Meier survival analysis for all-cause dementia diagnosis within 6 years – ADNI cohort (MCI; N=619)
Hazard ratios (Cox proportional regression) for conversion to
dementia by CSF biomarker status
72
Time (months)
0 12 24 36 48 60
0.75
1.00
0.50
0.25
0
Time (months)
%
Dementia-free
(ADNI)
0 12 24 36 48 60 72
0.75
1.00
0.50
0.25
0
Biomarker positive
Biomarker negative

59. 59
CSF Aβ42/pTau ratio has been shown to have good sensitivity
and specificity in predicting progression from MCI to AD
See slide notes for further information.
Aβ, amyloid beta; AD, Alzheimer’s disease; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; pTau, phosphorylated tau; tTau, total tau
Ferreira D, et al. Front Aging Neurosci 2014;6:287
79%
63%
72%
85% 86%
0%
50%
100%
72% 76%
70%
79%
60%
Aβ42 pTau tTau Aβ42/pTau Aβ42/tTau
Meta-analysis examining the capacity of CSF biomarkers to predict progression from MCI to AD
Sensitivity Specificity
n=10
studies
n=5
studies
n=8
studies
n=6
studies
n=5
studies
n=10
studies
n=5
studies
n=8
studies
n=6
studies
n=5
studies
Aβ42/pTau ratio was the most sensitive and specific biomarker for determining likelihood of progression from
MCI to AD

60. Clinically validated cut-offs simplify and standardized
worldwide interpretation of the results
• Universal cut-offs concentrations are already applied
for many biomarkers in clinical routine (i.e HbA1c in
diabetes mellitus)
• The next step is to apply the same concept for AD
biomarkers to ensure universal interpretation of
results25
• CSF assays have clinically validated
cut-offs that allow easier adoption by the lab and
between lab comparison.
Cut-off (+) Cut off (-)
Abeta 42 ≤ 1030 pg/mL > 1030 pg/mL
pTau > 27 pg/mL ≤ 27 pg/mL
tTau > 300 pg/mL ≤ 300 pg/mL
pTau/ Abeta 42 > 0.023 ≤ 0.023
tTau/ Abeta 42 > 0.28 ≤ 0.28
CSF assays cut-off for concordance with Amyloid PET were
established using PET visual readouts and then validated for
clinical progression claim.26,28

61. Blood biomarkers in
Alzheimer’s disease

62. Amyloid beta as biomarker
• Plasma Aβ42/Aβ40 ratio may be useful in predicting and/or diagnosing AD, but the
results have been inconsistent.
• This may be because conventional immunoassays are inadequate in measuring low
blood concentrations of Aβ42 and Aβ40.
• The recent use of fully automated and/or ultrasensitive methods, such as ECL-
based assays, SIMOA, and IP-MS technology, has clearly revealed that a decrease in
the plasma Aβ42/Aβ40 ratio is an indicator of brain amyloidosis.
• It shows a strong correlation with amyloid PET, and CSF Aβ42/Aβ40 ratio.
• IP-MS technology seems to be the most-accurate analytical tool at present.

63. Tau as a biomarker in Alzheimer’s disease
• A series of reports suggested that plasma phosphorylated tau at threonine
181 (pTau181) and threonine 217 (pTau217) are useful AD biomarkers.
• The quantities of pTau181 and pTau217 in plasma correlate with their
corresponding levels in the CSF and with amyloid PET scan.

65. Other AD associated protein biomarkers (non-amyloid and non-tau)
Biomarker Correlation Comment
Neurofilament (NfL) Indicate neuronal damage Non-specific
Glial fibrillary acidic protein
(GFAP)
Indicate astrocyte activation and
degeneration
Non-specific
Neurogranin (NGRN) Indicates synaptic dysfunction Promising marker
MicroRNA (miRNA) miR-125b, miR-455-3p, and miR-501-3p Promising marker

66. Current Limitations
• Consensus regarding the best technique for measurement.
• Reliable cutoff values for individual biomarkers must be determined.
• A large biomarker gray zone between normal and abnormal values.
• Need to factor in the effect of age, comorbidities, ethnicities etc in
interpreting the report.
• Pre-analytical errors that profoundly affect assay results must be addressed.

67. Salivary biomarkers in Alzheimer’s disease
• Equivalent to serum.
• Easy to collect, non-invasive.
• Inexpensive.
• Easy to reproduce results.
• Nonspecific
• Methodology is yet to be standardised.

68. Other potential biomarkers
• EEG
• Urine biomarkers
• Genetics (Apo E, presenilin)
• Deep learning and Machine learning techniques using MRI, EEG and other
investigations.

71. Conclusion
• An accurate diagnosis is critical to alleviating anxiety, and ensuring the right
care/support pathway for individuals, and their families.
• Biomarker testing of amyloid pathology can increase diagnostic accuracy and
shorten time to diagnosis.
• Amyloid PET and CSF pTau/Aβ42 and tTau/Aβ42 ratios are highly
concordant.
• Early conformation of amyloid pathology will be a pre-requisite for initiating
future disease modifying therapies.

72. THANK YOU