Standardization of CSF biomarkers assays-preanalytical and analytical aspects of CSF Biomarkers assays. Alexandroupoli EEKX-KB October 13, 2018

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1 Standardization of CSF biomarkers assays-preanalytical and analytical aspects of CSF Biomarkers assays Alexandroupoli EEKX-KB October 13, 2018

2 Dementia FTD VaD AD AD AD a-syn

3 Ab 42 N b-secretasi Membrana plasmatica C b-sapp b-secretasi C99 Ab-42 -secretasi Ab-40

4 Tau and p-tau Formichi 2006

5 Differential diagnosis of dementia Antonio Ligabue

6 Alzheimer s Disease

7 Alzheimer s disease Slowly progressive memory loss and dementia Neuropathology: plaques β-amyloid tangles phosphorylated tau protein neuronal and synaptic degeneration Very rare (< 0.1%) familial form - very common sporadic and age-related form USA 2014 Around patients with Alzheimer s A new patient every minute Around patients < 65 years Costs for society around 214 billion USD per year more than costs of cancer, heart disease and stroke together

8 Sperling 2011

9 The three established ELISA methods for AD CSF biomarkers HT7 BT2 Mol Chem Neuropathol 1995 AT % increase in AD 3D6 1 b-amyloid 42 21F12 Arch Neurol % decrease in AD AT270 T T T T T T T T T T T S SS S SS S SS S S S S S SS SS SS SSS S HT % increase in AD

10 IWG Criteria (FDA and EMA approved) Clinical signs and symptoms (amnestic syndrome of the hippocampal type) CSF biomarkers (Ab 1-42, Tau, P-tau) PET amyloid tracer retention (PiB and others) PET bilateral parieto-temporal hypometabolism (FDG) MRI medial temporal/hyppocampus atrophy From the asymptomatic through prodromal and most severe stages of dementia From clinocopathological to clinico-biological entity

11 How do amyloid PET and CSF Aβ42 compare? Study design: 118 patients with cognitive complaint examined for both CSF biomarkers - as part of clinical routine 2 years and amyloid 18 F-flutemetamol PET Cut-offs: CSF Aβ42 < 647 pg/ml 18 F-flutemetamol PET > 1.42 Original cohort n= 118 Validation cohort n= 38 Positive PET+CSF or Negative PET+CSF 92 % Positive PET+CSF or Negative PET+CSF 97 % Palmquist S, et al, JAMA Neurol 2014

12 IWG-2 criteria Diagnostic Marker Pathophysiological marker Reflects in-vivo pathology Is present at all stages of the disease Observable even in the asymptomatic state Might not be correlated with clinical severity Indicated for inclusion in protocols clinical trials Dubois 2014

13 IWG-2 criteria Progression marker Topographical or downstream marker Poor disease specificity Indicates clinical severity (staging marker) Might not be present in early stages Quantifies time to disease milestones Indicate for disease progression Dubois 2014

14 International Work Group terminology Preclinical AD Asymptomatic at risk for AD (*asymptomatic) Presymptomatic AD AD patients Prodromal AD (*predementia) AD dementia (*dementia) *High *Intermediate *Unlikely Atypical forms of AD Logopenic aphasia Posterior atrophy Frontal variant AD *National Institute on Aging, Alzheimer s Association Terminology

15 Pre-analytical confounding factors The kind of needle used for CSF collection Hemorragic CSF (14-20%) Post lumbar puncture headaches

16 Pre-analytical confounding factors Polypropylene tubes (PP) perfom better than glass or Polystirene tubes (PS) Different kind of PP Different treatment into the wall Volume/surface ratio Hydrophobic/hydrophilic balance of the analyte Total protein content of the sample Multiple tube transfers

17 Leitao MJ 2015

18 Fourier A 2015

19 Recommendations 1 Time of the day? Fasting? Spinal cord intervertebral space? Spining after collection (within 2 h max), at 2000 x g, 4 C, avoid multiple tube transfer Tubes should be filled up close to total tube capacity and stored at -80 C Avoid more than three freeze-thaw cycles Max. tested stability 2 years at -80 C

20 Large variation in Alzheimer CSF Aβ42 levels between laboratories Large CSF Aβ42 variability across laboratories

21 The Alzheimer s Association QC program for CSF biomarkers Principle for the QC program: For each round, 3 QC samples (pooled CSF) are sent out 2 unique samples - for comparisons between labs 1 identical sample - for comparisons over time > 90 labs Frequency: 3 times per year Variability between labs and between ELISA batches - need of standardization efforts: pre-analytics analytical procedures assay manufacturing

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23 Variability linked to kit providers Mattson 2013

24 Mattson 2013

25 Fourier A 2015

26 Recommendations 2 RT standardized at 25 C Run no more than a half plate in ELISA method Definition of the criteria of acceptance (calibration curve parameters, CVs for sample run in duplicate) QC samples? (native CSF pools, spiked CSF with standards, peptides range of concentrations)? Cut off of the Center (biochemical, clinical, imaging and neuropsychological findings)

27 The IFCC Work Group for CSF proteins Reference Measurement Procedure (RMP) High precision Intended use Mass spectrometry-based technique Certified calibrator (amino acid analysis) Tested in Round Robin studies Set level in the Certified Reference Material Golden standard method for absolute quantification Certified Reference Material (CRM) Large aliquoted CSF pool Exact level set using RMPs Commutability between assays tested Tested for long-term stability etc. Golden standard CSF with exact levels Intended use Distribution to kit vendors and large labs Harmonize CSF levels between assay formats Assure stability between production lots

28 Reference method for CSF Aβ42 - Validated Golden standard method Antibody-free Single Reation Monitoring (SRM) Triple Quad mass spec method for CSF Aβ isoforms CSF + internal Aβ standards Guanidine HCl denaturation SPE antibody-free purification Elute + dry + redissolve in ACN and NH 4 OH Separation of Aβ by HPLC Aβ1-40 Endogenous Aβ Aβ standards Aβ1-38 Aβ1-42 Vantage Triple Quad mass spectrometry Time (min) Quantification of Aβ isoforms Isotope labelled Aβ calibrator added to the CSF sample (and thus processed identically) No antibodies involved absolute quantification without interference (matrix effects)

29 The final destination: CSF biomarker assays on fully automated clinical analyzers Fully automated - minimize variations due to differences in laboratory procedures Will allow uniform cut-off levels - reduced between-run, between-batch and between-lab variations Single sample analysis fast results (< 30 min) to the clinician Roche Diagnostics Cobas Fujirebio - Lumipulse

30 Amyloid b1-42 T-tau Phospho tau 181 Ref. Cutoff values 450 > < < 52 Blennow 2001 Mattsson 2013 Mulder 2010 Duits 2014 Prodromal AD (predementia AD) (MCI developing AD) Ab42/40 Tau/Ab42 Blennow 2003 Hansson 2006 Snider 2009 Shaw 2009 Visser 2009 Mattsson 2009 Brys 2009 Riemenschneider 2002 Preclinical AD (Asymptomatic AD) N N Tau/Ab42 P-tau/Ab42 Skoog 2003 Gustafson 2007 Stomrud 2007 Fagan 2007

31 Tau protein and combined markers in differential diagnosis between AD and Healthy controls References Specificity T-tau 92% 81% Sensitivity 78% 82% Bibl 2012 Duits 2014 P-tau 91% 81% Bibl 2012 Ab42+T-tau 88% 88% Kang 2013 Tau/Ab42 90% 85% Duits 2014 P-tau /Ab42 88% 85% Duits 2014 Innotest Amylod/Tau Index Schoonenboom formula 86% 85% Hulstaert 1999 Riemenschneider % 91% Schoonenboom 2012 Mattsson formula 90% 80% Mattsson 2009

32 Can CSF biomarkers identify prodromal AD? Follow-up study on MCI (>4 years) CSF samples taken at baseline MCI n= MCI AD 56 MCI MCI 21 MCI other dem. Controls n = 37 Cut-off: T-tau >350 pg/ml + Ab42 / P-tau ratio < 6.5 Sensitivity MCI AD 95 % Specificity MCI MCI + other 87 % Hazard ratio : 25.5 ( )

33 Fronto Temporal Dementia

34 Microtubule-binding protein tau (tau) Predominant behaviour/social disorders TAR DNA binding protein-43 (TDP-43) Fused-in sarcoma proteins (FUS) Tau negative TDP negative Ubiquinated inclusions (UPS) FTD Without significant inclusions (ni) Primary language disturbances (PPA) Progranulin (GRN) Tau (MAPT) C9 orf 72 (Genetic forms)

35 Amyloid b1-42 T-tau Phospho tau 181 Other markers Ref. T-tau/b1-42 Lower in FDT vs AD Sjogren 2000 Bian 2008 Clark 2003 Irwin 2013 FTD N N N Progranulin (PGRN) Low in FTD from PGRN mutation Ghidoni 2011 Finch 2009 Hsiung 2011 ptau 181 /T-tau FTD TDP-43 < 0,37 FTD Tau > 0,37 Hu 2013 Mixed FTD+AD Bian 2007 Toledo 2012 Higher TDP-43 levels in plasma or CSF levels of patients with AD, FTD and ALS vs healthy controls

36 Vascular Dementia

37 Vascular Cognitive Impairment Single ischemic lesion Recurrent transient ischemic attaks Multiple silent ischemic attaks Dementia Cognitive impairment

38 Vascular comorbidity may be present in 30-60% of AD patients.and conversely. AD pathology may be present in 40%-80% of VaD patients Amyloid b1-42 T-tau Phospho tau 181 Ref. Mixed VaD+AD Vascular Cognitive Impairment N Lower than in AD N Waldemar 2007 Leszek 2013 Nagga 2002 Stefani 2005 Paraskevas 2009 Ravaglia 2008

39 Synucleinpathies

40 a-sinucleinopathies Parkinson s disease (PD) Parkinson s disease with dementia (PDD) Dementia with Lewy bodies (DLB) Multiple Systemic Atrophy (MSA)

41 Amyloid b1-42 T-tau Phospho tau 181 Other markers Ref. PD a-syn * <1,6 pg/ml a-syn/tau ratio Mollenhauer 2011 Kasuga 2010 DLB a-syn Parnetti 2001 Vanderstichele 2006 Mollenhauer 2011 PDD Compta 2009

42 Prion Disease

43 Human Prion Diseases Sporadic or of unknown cause Creutzfeld-Jacob disease (scjd) Acquired through ingestion or transplantation of contaminated tissues Genetic caused by mutation in the prion protein gene (PRNP)

44 Role of in differential diagnosis Other neurodegenerative diseases Specificity 95-97% Non-neurological conditions 91-97% Acute neurological events with rapid dementia 82-85% Vascular dementia (10,7%) AD (5,8%) DLB (5,3%) FTD (4,6%) CNS inflammation (19,2%) Stroke (15,2%) Epilectic fit (17%) CNS tumors (18,4%) Stoeck 2012

45 Epilectic fits (24%) CNS tumors (22%) Stroke (18,4%) Stoeck 2012 Amyloid b1-42 T-tau Phospho tau 181 Other markers Ref. CJD CSF Buerger 2006 Coulthart 2011 >1400 pg/ml T-tau/p-tau 181 >25 Blennow 2005

46 Novel Candidates approaches Gino Rossi

47 Approch pathogenesis driven (amyloid, oxidation, inflammation, dyslipidemia) Approach screening technology driven (proteomicic, metabolomics)

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49 Protein/Peptide Method References Neural Cell Adhesion Molecule 1 Alpha dystroglycan Neuronal Pentraxin Receptor Neuronal Cell Adhesion Molecule Chitinase3-like 1,Chromogranin A Carnosinasi 1 2-DE LC/MS Yin GN, Brain Res DE LC-MS RJ Perrin, PloS One 2011 ApoA1, ApoE, Transthyretin 2-DE LC-MS Castano E,, Neurol Res 2006 Daviddson P Neuroreport 2002 PurchadesM,Brain Res 2003 Korolainen MA Clin Chem 2007 Pattern of CSF protein in FAD with PSEN1 and APP mutations vs relatives non carrier LC/MS non labeled Ringman JM, Arch Neurol 2012 Pattern of Age related changes LC/MS Zhang J, Neurobiol Aging 2005

50 Protein Method References Pattern of Ab peptides in CSF Pattern of Ab isoforms and peptides during treatment with disease modifyng drugs SNAP 25 in CSF Neurogranin Peptides pattern Immunoaffinity/M S Immunoaffinity/M S Immunoaffinity/M S Immunoaffinity/M S E. Portelius, Curr Pharm Des 2011 G Brinkmalm 2012 E Portelius, Alzhiemers Res Ther 2010 G Brinkmalm, Mol Neurodegener 2014 Kvartsberg H, Alzheimer Dementia 2014 Quantification of ApoE MRM-MS Han SH, Mol Cell Prot 2014 Quantitation of ab42, ab40, Retinol Binding Protein, Cystatin C MRM-MS YS Choi, J Chormatogr. B 2013 Neurogranin Immunoaffiity/MS Thorsell A, Brain Res 2010 Quantification of ab42, ab40, ab38 MRM-MS J Pannee, J Alz Dis 2013

51 Concerns 1. Number of partecipants to the studies and stratification of partecipants 2. Different panels of metabolites 3. Low through-put proteomics methods 4. Relative abundancy of candidate biomarkers in the analytical samples 5. Harmonization of the preanalytical and analytical steps (different analytical platforms) 6. Limited dynamic range 7. Quality of the study design (STARD) 8. Interpretation of the Big Data

52 AL Baird, 2015

53 AL Baird, 2015

54 Variability of results Different Analytical Patforms Different matrices Different panel of metabolites Different Clinical Cohorts Preanalytical confounding factors Standardisation/ Harmonisation Big data without big integration big confusion Integration of the data from the Routine Laboratory with other laboratory techniques (Proteomics, Genomics, Flow Cytometry) and diagnostic tools (Radiology, Pathology)

55 Biomarkers and clinical trials as early as possible Umberto Boccioni

56 A big challenge! But we cannot leave them alone