diagnostic biomarkers of alzheimer disease - … biomarkers of alzheimer disease ......

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Diagnostic biomarkers of Alzheimer disease International Congress on Quality in Laboratory Medicine Helsinki, 8 Feb 2018 Maryline Simon, Clinical Science Leader

Agenda

1. What is Alzheimer’s disease?

2. Technical performance of the Elecsys ß-Amyloid (1-42) CSF assay

3. Technical performance of the Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF assay

4. How Biomarkers relate to Alzheimer’s disease diagnosis

WHAT IS ALZHEIMER?

Dementia One of the most growing diseases with limited treatment and diagnosis

2013 → XXmillion people 2030 → 76 million people 2050 → XX million people The field is desperate for advancements to alleviate the heavy burden on patients, caregivers, and healthcare systems.

* WHO: 10 facts of dementia. www.who.int/features/factfiles/dementia/en/ Source: Alzheimer’s Society: www.alzheimers.org.uk/site/scripts/documents_info.php?documentID=412

2013 → 48 million people 2030 → 76 million people 2050 → 130 million people The field is desperate for advancements to alleviate the heavy burden on patients, caregivers, and healthcare systems.

Biomarkers are the earliest signs of Alzheimer’s “Jack curves”

Amyloid-beta accumulation Tau accumulation Brain structure alterations Memory impairment Functional impairment

Time Normal

Abnormal

Magn

itude

Preclinical AD Prodromal AD (=MCI)

AD dementia

>10 years ~5-7 years ~7-10 years

• Subjective symptoms can occur much later than pathology starts • Biomarkers become detectable many years before clinical onset

Clinical function

Memory

Normal

First lesions

Major lesions Bra

in

struct

ure

Jack CR Jr, et al. Lancet Neurol 2010;9:119–28

TECHNICAL PERFORMANCE OF THE BIOMARKERS

Elecsys β-Amyloid (1-42) CSF, Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF

ElectroChemiLuminescence ImmunoAssay (ECLIA) for use with Elecsys and cobas e fully automated analyzers

Easy calibration

Faster result reporting

High precision

Low sample volumes

Wide measuring range

Less hands-on time Fewer redraws/retests Right results, first time

Efficient workflow with Elecsys assays Reagent concept and advanced assay design

Easy to handle

No preparation

Long onboard stability

ELECSYS ß-AMYLOID (1-42) CSF


Design of the Elecsys ß-Amyloid (1-42) CSF assay to measure the ß-amyloid peptide 1-42 with monoclonal antibodies in a sandwich format

Biotinylated monoclonal ß –amyloid (1-42) specific antibody (21F12)

Ruthenylated monoclonal ß-amyloid (1-42) specific antibody (3D6)

9 min

Streptavidin microparticle added

ß-amyloid

Measurement

ß-amyloid Ru 9 min

18 min. turn around time

200 – 1700 pg/mL measuring range

50 L Sample volume

<200 pg/mL LeMR (LOQ)

Ru

Stability Performance data

Analyte 8 hours @ 15-25 °C 24 hours @ 2-8 °C 8 weeks @ -20 °C >1.5 years @ -70 °C (real time) 3 freeze/thaw cycles

Reagent after opening @ 2-8 °C: 8 weeks on the analyzer: 4 weeks

Calibration after 4 weeks when using the same regent lot after 7 days when using the same lot on the analyzer

Robust reagent and calibration stability for weeks

Linearity across a broad range facilitates accurate results

All results > 1700 pg/mL are above cutoff = patients are negative to report higher values is clinically not relevant (but may be interesting scientifically)

LoQ <11.28 pg/mL

Normal measuring range 200 -1,700 pg/mL

Source: Bittner T, et al. Alzheimer’s & Dementia. 2016; 517-526 NB: NOT TO SCALE

No ‘Hook’ Effect up to ~12,000 pg/mL pg/mL

Internal interference studies showed no effects

Compound Concentration tested Interference?

Hemoglobin ≤ 11 mg/dL no

Bilirubin ≤ 0.3 mg/dL no

Lipemia (Intralipid®) ≤ 3 mg/dL no

Biotin ≤ 30 ng/mL no

Serum Albumin ≤ 0.17 g/dL no

Human IgG ≤ 0.02g/dL no

Human IgA ≤ 0.003 g/dL no

Human IgM ≤ 0.002 g/dL no

Rheumatoid Factor 14 IU/mL no

Interference studies: Endogenous interferences study

Interference studies: Common drug and special pharmaceutical interferences:

Common Pharmaceuticals Drug conc [mg/L]

Interference? Special Pharmaceuticals Drug conc [mg/L]

Interference?

N-Acetylcysteine 150 no Atorvastatin 6 no Ampicillin-Na 1000 no Clopidogrel 0.3 no Ascorbic acid 300 no Digoxin 5 no Calcium Dobesilate 200 no Donepezil 30 no Cyclosporine 5 no Escitalopram 15 no Cefoxitine 250 no Esomeprazole 170 no Heparin 5000 U/L no Furosemide 90 no Levodopa 20 no Galantamine 250 no Methyldopa +1.5 20 no Hydrochlorothiazide 120 no Metronidazole 200 no Lisinopril 500 no Phenylbutazone 100 no Memantine 250 no Doxycycline 10 no Metformin 4000 no Acetylsalicylic Acid 1000 no Metoprolol 900 no Rifampicin 60 no Rivastigmine 45 no Acetaminophen 200 no Simvastatin 3 no Ibuprofen 500 no Theophylline 100 no

Internal interference studies showed no effects

Aβ1–42 concentration correlation LC-MS vs Elecsys® assay

Assay standardized to a listed IFCC reference procedure

* International Federation of Clinical Chemistry (IFCC) Certified Reference Material (CRM) and Reference Measurement Procedure (RMP) Source: Shaw LM, et al. Alzheimer’s & Dementia 2016; 12:668

CV

<3.5%

GOAL: Evaluate external reproducibility of the Elecsys® β-Amyloid (1–42) assay under varying conditions

2 sites used all 3 lots and 2 sites

used 2 lots

Within-run variability

Between-run variability

Between-day variability

Between-laboratory variability

Lot-to-lot variability

University of Gothenburg

VU University Medical Center

Amsterdam

Covance Indianapolis

Washington University at St. Louis

external sites

different lots 5 days 2 aliquots

per run 2 runs per day 4 3

* Roche Diagnostics: this product is not yet available in the USA.

Multicenter evaluation study of the fully automated Elecsys® β-Amyloid (1–42) assay*: methods

CV

<3.5%

Parameter variability as %CV

Sample (mean Aβ(1–42) concentration)

Control 1 485pg/mL

Control 2 869pg/mL

CSF 1 342pg/mL

CSF 2 662pg/mL

CSF 3 755pg/mL

CSF 4 935pg/mL

CSF 5 1472pg/mL

Range CSF samples

Within-run (repeatability) 1.02 0.90 1.56 1.35 2.66 1.50 2.04 1.35–2.66

Between-run 0.54 0.47 0.17 0.75 2.46 0.71 0.23 0.17–2.46

Between-day 1.35 1.41 1.75 1.44 0 0.95 1.58 0–1.75

Between-laboratory 0.66 0.76 0 0.46 3.49 0.75 1.75 0–3.49

Lot-to-lot 0.59 1.03 2.25 1.46 0.61 0.89 0.78 0.61–2.25

Total reproducibility†

(95% CI) 1.99

(1.65; 2.49)

2.16

(1.67; 3.05)

3.25

(2.22; 6.04)

2.61

(1.96; 3.88)

5.07

(3.64; 8.33)

2.24

(1.84; 2.86)

3.22

(2.52; 4.46)

2.24–5.07

* Roche Diagnostics: this product is not yet available in the USA. †Total reproducibility includes all factors (estimated from a variance components model): sites, lots, days, runs, within-run

MCE study (Reproducibility) showed high precision

ELECSYS TOTAL-TAU CSF AND ELECSYS PHOSPHO-TAU (181P) CSF


pTau

Biotinylated monoclonal Tau specific antibody/ies

Ruthenylated monoclonal Tau specific antibody (PC1C6)

9 min

Streptavidin microparticle added Measurement

Tau Tau

Bi-pTau (181P) mAb (11H5V1)

Ru

Design of the Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF assays …to measure the total Tau /or phosphorylated (181P)

Tau with monoclonal antibodies in a sandwich format

Ru

Bi-Tau mAbs (5.28.461 and 4.35.411)

tTau

9 min

Stability Performance data

Analyte

5 days @ 15-25°C 14 days @ 2-8°C 3 months @-20°C 6 months @-80 °C 3 freeze/thaw cycles

Reagent after opening @ 2-8 °C: 8 weeks on the analyzer: 4 weeks

Callibration after 4 weeks when using the same regent lot after 7 days when using the same lot on the analyzer

…Stability shown for weeks

Robust reagent and calibration stability

Linearity across a broad range facilitates accurate results …Enough for Clinical routine use

No ‘Hook’ Effect up tTau ~3,000 pg/mL pTau ~300 pg/mL

Normal measuring range

tTau: 80 -1300 pg/mL pTau: 8 -120 pg/mL

MR of Tau assays cover almost 100% of values from IU population

LoQ tTau <62.6 pg/ml pTau < 3.90 pg/mL

Internal interference studies

Common drug and special pharmaceutical interferences:

Compound Concentration tested tTau/(pTau*) Interference?

Rheumatoid factors ≤ 4 IU/mL/ (12 IU/mL*) no

Bilirubin ≤ 6.6 mg/dL /(3 mg/dL*) no

Lipemia (Intralipid®) ≤ 200 mg/dL no

Biotin ≤ 50 ng/mL no

Serum Albumin ≤ 0.7 g/dL no

Human IgG ≤ 0.09 g/dL no

Human IgA ≤ 0.016 g/dL no

Human IgM ≤ 0.004 g/dL no

Hemoglobin ≤ 100 mg/dL no

No effects pTau

Albumin , 0.7g/dL Bilirubin, 6.6mg/dL

Biotin, 60ng/L Haemoglobin, 100mg/dL

IgA, 0.16g/dL IgG, 0.09g/dL

IgM, 0.004 g/dL Rheumatoid factor, 4.2lU/mL

Albumin , 0.7 g/dL Bilirubin, 3 mg/dL

Biotin, 54ng/L Haemoglobin, 100mg/dL

IgA, 0.16g/dL IgG, 0.09g/dL

IgM, 0.004 g/dL Rheumatoid factor, 12lU/mL

tTau

90 95 100 105 110 90 95 100 105 110

A B

Figure 2. Interference of the Elecsys® tTau and pTau Assays by (A) endogenous compounds, (B) common drugs and (C) special drugs

Recovery (%) Recovery (%)

tTau pTau tTau pTau

Internal interference studies No effects

Common drug and special pharmaceutical interferences:

Common Pharmaceuticals Drug conc [mg/L] tTau/(pTau*) Interference? Special Pharmaceuticals Drug conc

[mg/L] tTau/(pTau*) Interference?

N-Acetylcysteine 1660 no Atorvastatin 2 (6) no Ampicillin-Na 1000 no Clopidogrel 0.1 (0.3) no Ascorbic acid 300 no Digoxin 5 (3) no Cyclosporine 5 no Donepezil 10 (30) no Cefoxitine 2500 (2000) no Escitalopram 10 no Heparin 500 U/L (5000 U/L) no Esomeprazole 56 (170) no Levodopa 3 (20) no Furosemide 50 no Methyldopa +1.5 20 no Galantamine 83 no Metronidazole 200 no Hydrochlorothiazide 40 no Phenylbutazone 400 no Lisinopril 500 no Doxycycline 7.5 (50) no Memantine 83 no Acetylsalicylic Acid 1000 no Metformin 3000 (2000) no Rifampicin 60 no Metoprolol 500 (900) no Acetaminophen 200 no Rivastigmine 15 no Ibuprofen 500 no Simvastatin 1 (3) no Theophylline 100 no


Sample (mean tTau concentration)

Control 1 172.4 pg/mL


CSF 1 211.2 pg/mL

CSF 2 289.2 pg/mL

CSF 3 413.5 pg/mL

CSF 4 638 pg/mL

CSF 5 1084 pg/mL

Range CSF samples

Within-run (repeatability)

1.20 1.15 1.53 1.63 1.31 1.74 1.81 1.31–1.81

Between-run 1.98 1.90 2.25 2.19 2.02 2.14 2.04 2.02–2.25

Between-day 2.31 2.22 2.73 2.73 2.41 2.76 2.73 2.41–2.76

Between-laboratory 2.67 2.15 0.883 1.09 1.02 1.13 1.55 0.883–1.55

Lot-to-lot 5.02 4.28 5.76 5.57 5.35 5.61 5.92 5.35–5.92

Total reproducibility† 6.14 5.28 6.43 6.30 5.95 6.35 6.70 5.95–6.43

MCE Study (Reproducibility Study) Elecsys tTau shows high precision

CV

<3.5%


Sample (mean pTau concentration)



CSF 1 19.79 pg/mL

CSF 2 28.82 pg/mL

CSF 3 40.69 pg/mL

CSF 4 61.93 pg/mL

CSF 5 102.4 pg/mL

CSF 6 108.9 pg/mL

Range CSF samples

Within-run (repeatability)

1.57 1.32 1.40 1.33 1.43 1.75 1.49 1.53 1.33–1.75

Between-run 1.31 1.45 1.87 1.99 2.19 1.59 2.11 2.31 1.59–2.31

Between-day 2.04 1.96 2.34 2.39 2.62 2.36 2.59 2.77 2.34–2.77

Between-laboratory 2.23 2.38 1.05 1.37 2.47 2.39 2.50 2.62 1.05–2.62

Lot-to-lot 3.33 2.06 1.76 1.98 2.78 3.05 2.97 2.78 1.76–3.05

Total reproducibility† 4.50 3.70 3.11 3.39 4.55 4.54 4.67 4.72 3.11–4.72

MCE Study (Reproducibility Study)

Elecsys pTau shows high precision

Enhanced QC Concept

Ensuring high precision and accuracy

To ensure high precision and accuracy of Abeta42, tTau and pTau measurements in all laboratories world-wide, we have elaborated an „Enhanced Quality Control Concept“ (eQC Concept), where each laboratory establishes it‘s own measurement bias and CV.

The eQC Concept is based on Total Error Requirements, which were established through clinical considerations.

The maximal allowable Total Error (TEa) for tTau and pTau assays is: TEa (%) = relative bias (%) + 1.65 x CV (%) = 26.5% with: relative bias ≤ 10% and CV ≤ 10%

Key featuresw tTau pTau Abeta42

Measuring range 80 - 1300 pg/mL 8 - 120 pg/mL 200 - 1700 pg/mL

Limit of Quantification (LoQ) 63 pg/mL 4 pg/mL 11 pg/ml

Within-run precision CV 0.7 – 1.2 % CV 1.5 – 2.7 % CV 0.8 – 1.6 %

Within-lab precision CV 1.4 – 2.0 % CV 1.9 – 3.5 % CV 1.6 – 2.9 %

External reproducibility (3 sites) CV 5.9 - 6.4 % CV 3.1 - 4.7 % CV 2.2 - 5.1 %

Lot-to-lot consistency (3 lots) r=0.998 (Pearson) r=0.999 (Pearson) r=0.996 (Pearson)

Platform consistency (e411 vs. e601) r=1.00 (Pearson) r=1.00 (Pearson) n.a.

Correlation to Innotest r=0.98 (Spearman) r=0.96 (Spearman) r=0.95 (Spearman)

Reagent stability 4°C > 25 months > 25 months > 25 months

Onboard stability 20°C > 4 weeks > 4 weeks > 4 weeks

No cross reactivity for n.a. non-p181P-Tau Abeta(1-38) & (1-40)

Reference Method/Material Weighted tTau Roche Ref. Material

Weighted pTau Roche Ref. Material LC-MS (Leinenbach et al. 2014)

…to ensure minimal batch variation

Analytical Performance tTau, pTau and Abeta42 assays Summary

Alzheimer’s Association Quality Control (AAQC) Program for CSF biomarkers

CSF=cerebrospinal fluid; CV=coefficient of variation Source: CPS MSA after Roche Dx – Roche Rx update medical (Nov. 2016)

Aim: to standardize biomarker measurements between laboratories (research and clinical)

Design: - Aliquots of pooled CSF samples sent to participating laboratories - 2 unique samples per round for comparisons

between laboratories - PLUS QC sample maintained over multiple rounds

for longitudinal comparisons - 3 rounds per year - ~90 laboratories participate - Final report for each round includes:

- Biomarker concentrations for individual laboratories - Mean biomarker concentrations and %CV

for all laboratories - Longitudinal stability (percentage deviation over time)

CSF=cerebrospinal fluid; CV=coefficient of variation Source: CPS MSA after Roche Dx – Roche Rx update medical (Nov. 2016)

Between laboratory CV (percent): Between laboratory CV (percent):

INNOTEST@

β-Amyloid (1-42) Fujirebio

Euroimmune / ADx

β-Amyloid (1-42)

AlzBio3


Meso-Scale Human Aβ42

IBL Aβ 1-42

Elecsys@

β-Amyloid (1-42) Roche Diagnostics

Lumipulse@


Round ELISA ELISA Luminex ECL V-PLEX ELISA Fully automated Fully automated

2014-14A 18 16 low n 2,9

2014-14B 21 18 low n 4,4

2014-15A 15 7,1 12 4,6

2014-15B 17 14 12 3,4

2014-16A 27 57 40 13 3

2014-16B 17 19 30 11 2,5

2015-17A 19 6,5 17 21 1,9

2015-17B 14 8,2 15 20 3,2

2015-18A 13 22 25 10 7,2

2015-18B 13 16 13 9,4 4,7

2015-19A 13 13 15 10 3

2015-19B 13 13 14 13 1,5

2016-20A 17 18 ND 11 2

2016-20B 21 15 ND 14 Level out of range

2016-21A 15 18 22 8,1 10 6,3

2016-21B 11 15 29 7,4 5,2 4

2016-22A 14 12 28 39 5,9 7,5

2016-22B 19 13 24 37 6,7 9,4

2017-23A 15 7,3 12 16 1,6 21

2017-23B 16 13 18 19 3 8,6

2017-24A 15 8,9 too few 29 13 3 13

2017-24B 13 9,9 too few 25 3,1 5,6 14

2017-25A 13 9,0 23 20 6 11 10

2017-25B 14 9,5 24 21 8,7 12 14

Mean 16,0 15,2 21,6 17,2 7,7 4,7 10,8

The Alzheimer’s Association QC program – results 2014-2018

Between laboratory CV (percent): Between laboratory CV (percent):

INNOTEST@

Total tau Fujirebio

Euro Immune Total tau

AlzBio3

Total tau Fujirebio

Elecsys@

Total tau Roche Diagnostics

Lumipulse@

Total tau Fujirebio

INNOTEST@

Phospho tau Fujirebio

AlzBio3

Phospho tau Fujirebio

Elecsys@

Phospho tau Roche Diagnostics

Round ELISA ELISA Luminex Fully

automated

Fully

automated Round ELISA Luminex

Fully

automated

2014-16A 19 6 16 2014-16A 16 25

2014-16B 20 21 18 2014-16B 17 22

2015-17A 28 17 20 2015-17A 16 34

2015-17B 20 <LLOQ 19 2015-17B 13 6

2015-18A 14 10 13 2015-18A 10 32

2015-18B 12 7,2 13 2015-18B 10 7

2015-19A 15 23 18 2015-19A 12 58

2015-19B 14 29 11 2015-19B 11 35

2016-20A 14 12 7 2016-20A 14 54

2016-20B 15 9,4 13 2016-20B 17 14

2016-21A 17 20 18 2016-21A 12 48

2016-21B 20 5,1 14 2016-21B 11 20

2016-22A 15 6,6 15 2016-22A 7,0 60

2016-22B 19 4,4 23 2016-22B 14 52

2017-23A 14 14 11 2017-23A 10 44

2017-23B 12 18 8,5 2017-23B 9,9 33

2017-24A 13 5,7 too few 3,8 2017-24A 12 too few 20

2017-24B 13 19 too few 3,6 2017-24B 13 too few 1,7

2018-25A 14 18 5,2 1,3 4 2017-24A 9,1 28 1,3

2018-25B 13 22 36 2,2 6,5 2017-24B 11 36 2,2

Mean 16,0 14,1 15,5 2,7 5,3 Mean 12,3 33,7 1,8

The Alzheimer’s Association QC program – results 2014-2018

CLINICAL VALIDATION & UTILITY

Elecsys β-Amyloid (1-42) CSF, Elecsys Total-Tau CSF and Elecsys Phospho-Tau (181P) CSF

Patients with cognitive decline

or

For the detection of amyloid

Patients with cognitive decline

…adapt therapy for personalized healthcare CSF biomarkers can be used to

Identify patients at higher risk of progression

3.0

2.5

2.0

1.5

0 6 12 24 Visit

CDR-

SB

…Patient identification

Biomarker status Abeta42, tTau and pTau PET concordance and progression claim (CE)

CSF biomarkers concordance with visual read amyloid-PET

Alternative to amyloid-PET (PET concordance) Identification of subject likely to progress (progression)

Aβ421000 pg/mL Aβ421000 pg/mL

tTau/Aβ420.28 tTau/Aβ420.28

pTau/Aβ420.024 pTau/Aβ420.024

tTau300 pg/mL* pTau27 pg/mL*

pTau

pg/m

L

Bio

FIN

DE

R

pTau vs Abeta42

N = 277

25

50

75

ADNI

tTau

pg/m

L

tTau vs Abeta42

N = 277

250

500

750

pTau

pg/m

L

N = 646

25

50

75

tTau

pg/m

L

N = 646

600

800

200

400

1000 2000 3000 1000 2000 3000

1000 2000 3000 1000 2000 3000

Abeta42 pg/mL Abeta42 pg/mL

100 1000

100

PET visual read

Negative

Positive

Cut-off

Abeta42=β-Amyloid(1–42); ADNI=Alzheimer's Disease Neuroimaging Initiative; CI=confidence interval; CSF=cerebrospinal fluid; NPA=negative percentage agreement; OPA=overall percentage agreement; PET=positron emission tomography; PPA=positive percentage agreement; pTau=phosphorylated Tau; tTau=total Tau.A

* tTau and pTau have no values as alternatives to amyloid-PET as they are not relevant.

CSF Biomarkers identify MCI-patients at higher risk of progression

Cut-off: 0.028 N: 300 (BM-) N: 319 (BM+)

0 6 12 24

1.5

2.0

2.5

3.0

Figure 2. Change in CDR-SB over time according to pTau/Abeta42 status (ADNI progression cohort, n=619).

Poster presentation CTAD 2017

Single assay or in Ratio

pTau/ Albeta 42, tTau/

Abeta 42

Patients assessed for AD

Patient (SCD, MCI and AD)

No plaques Cognitive issue

due to other non AD cause

Presence of amyloid plaques, Informs HCP’s on likelihood that

symptoms are caused by an underlying AD pathological process.

Abeta42 Positive

No or low amyloid plaques

Cognitive impairment probably not due to AD

Positive Negative Negative

Patients with confirmed MCI

Single assay or in Ratio

Lower risk of progression within next 2 years

higher risk of progression within next 2 years

implications for patients, their relatives & and their carers

Patient (MCI)

Negative

Positive

Biomarkers Abeta42, tTau and pTau, ratio recommended Summary utility

Doing now what patients need next

diagnostic biomarkers of alzheimer disease - … biomarkers of alzheimer disease ......

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