WO2021009074A1

WO2021009074A1 - Novel markers as early predictors of alzheimer's pathology

Info

Publication number: WO2021009074A1
Application number: PCT/EP2020/069648
Authority: WO
Inventors: Charlotte Teunissen; Hugo Vanderstichele
Original assignee: Adx Neurosciences Nv
Priority date: 2019-07-12
Filing date: 2020-07-10
Publication date: 2021-01-21

Abstract

The present invention generally relates to brain neuroscience field and relates to a new marker for evaluating cerebral beta-amyloid (Aβ) accumulation in the brain and methods for analysis thereof. In particular, the invention introduces a marker to be used in pre-symptomatic diagnosis, in clinical trial enrichment settings, and evaluation of drug efficacy of therapeutic and prophylactic drugs in Alzheimer's disease.

Description

NOVEL MARKERS AS EARLY PREDICTORS OF ALZHEIMER'S PATHOLOGY

TECHNICAL FIELD

[0001] The present invention generally relates to brain neuroscience field and relates to a new marker for evaluating cerebral beta-amyloid (Ab) accumulation in the brain and methods for analysis thereof. In particular, the invention introduces a marker to be used in pre-symptomatic diagnosis, in clinical trial enrichment settings, and evaluation of drug efficacy of therapeutic and prophylactic drugs in Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is characterized by global cognitive decline, and defining historical features including two distinguishing pathologies: amyloid plaques, which are extracellular deposits consisting mainly of aggregated beta-amyloid (Ab) peptide, and, neurofibrillary tangles which are deposits consisting predominantly of hyperphosphorylated tau (pTau) protein. The underlying disease pathology precedes the onset of cognitive symptoms by many years (Jack et al, 2018).

[0003] Detection of abnormal Ab accumulation has been incorporated as a criterion in the clinical AD diagnosis and is essential for the identification of clinical trial participants, early drug intervention and for therapeutic effectiveness monitoring (Albert et al. 2011).

[0004] In patients with AD, Ab accumulation can be visualized in vivo with positron emission tomography (PET) using ligands that bind to Ab fibrils or through the detection of reduced levels of the Ab42 peptide in cerebrospinal fluid (CSF). Neurological decline can be assessed using MRI techniques. However, those methods are unsatisfactory since they apply imaging equipment and thus are not highly suitable for routine analysis. AD affects 10% of people over the age of 65 and is expected to affect >100 million by 2050 (Association As, 2017; Brookmeyer et al., 2007). Although PET is effective for detecting Ab accumulation, it requires high examination cost and long time for executing the examination, and thus is not a diagnostic method that allows for a majority of elderly people to easily undergo the examination. Consequently, there is a great need for more cost-effective and minimally invasive methods that can detect early Ab accumulation in a routine setting and detect early-onset of neurodegeneration.

[0005] Attention went to the development of blood-based biomarkers of Ab. However, the use of Ab as plasma biomarker for AD faces the problem that the concentrations of the Ab peptides (Ab40 and Ab42) in serum or plasma are extremely low or much lower than in CSF, so that there is a need for improved assays which were sensitive enough so as to allow reliable detection of said peptide species. As AD is a multifactorial disease with different phenotypes occurring in parallel, blood-based biomarkers other than Ab might help in more accurate biological staging of disease severity and in monitoring of disease progression. Promising results have been obtained for plasma tau (Chatterjee et al 2018; Dage et al., 2016; Deters et al., 2017), neurofilament light (NfL) (Mattsson et al., 2017; Lewczuk et al., 2018., Zhou et al., 2017) and the Ab42/Ab40 ratio (Fandos et al., 2017; Janelidze et al., 2016).

[0006] Indeed, blood neurofilament light (NfL), a protein that disintegrates from axons and neurons upon neuronal damage (Mattsson et al., 2017), might be a potential biomarker as recent studies in AD showed that blood NfL levels increase over the cognitive continuum from cognitively normal to mild cognitive impairment (MCI) to AD dementia (Mattsson et al., 2017; Lewczuk et al., 2018. Zhou et al., 2017) and levels are associated with time of symptom onset (Weston et al., 2017).

[0007] Studies have also demonstrated that Ab42/Ab40 ratio correlates with brain Ab and can differentiate patients with AD from healthy control participants (Fandos et al., 2017; Janelidze et al., 2016). A critical equilibrium seems to exist between the two major Ab species and the amount between free and protein-bound Ab, and the relative ratio of Ab peptides is more crucial in neurotoxicity that the absolute amounts of the peptides.

[0008] Additional markers, such as total tau (tTau), NfL, and b-secretase enzyme (BACE1) were evaluated as improvement to predicting amyloid deposition in the brain, but several studies only show marginal improvements of the plasma Ab42/Ab40 ratio (Shi et al.,2019; Vergallo et al; 2019; Mielke M et al;, 2019; Epub, PMID 31182505; Palmqvist et al., 2019)

[0009] A further protein, glial fibrillary acidic protein (GFAP), the principal intermediate filament cytoskeletal protein of astrocytes, has been proposed as useful candidate marker for brain injuries. Increased GFAP reflects an increase in reactive astrocytes, i.e. astrogliosis, which is the brain's response to injury. Increased GFAP expression was shown in areas surrounding Ab plaques as well as with increasing severity of tauopathy in AD brain (Hondius et al., 2016). While GFAP plasma immunoassays are available, this biomarker is understudied in AD (Bogoslovsky et al., 2017). One recent report showed that blood GFAP levels are increased in AD compared to control, and controls and AD could be differentiated with reasonable sensitivity and high specificity using this marker (Oeckl et al., 2019). In diseases other than AD but involving neuronal damages such as traumatic brain injury and multiple sclerosis it was shown that although plasma NfL and GFAP levels were correlated, GFAP had added value to NfL in predicting severity of these diseases (Oeckl et al, 2019; Hogel et al., 2018).

[0010] Current assays fail to accurately detect cerebral Ab accumulation state in blood or derivatives thereof, such as serum or plasma. There is thus generally a need for an improved marker for determining a cerebral Ab accumulation state in a living body-derived sample from a test subject. A lot of attention has gone to the improvement of the accuracy of Ab42 and Ab40 assays. In this respect, several patents describe amyloid measurements and their improved detection in plasma.

WO2011149947 and AU2017200029 encompasses a method for determining Ab turnover in blood and uses hereto a labeled amino acid that is administered to a patient prior to collecting a blood sample from the patient. The turn over of Ab is measured by determining the amount of labeled Ab and unlabeled Ab.

W02011070174 describes a method that introduces a change in ionic strength and in the molecular interactions within a sample leading to the release of Ab40 and Ab42 bound to plasma proteins and other components. This allows for the estimation of the total level of Ab in plasma.

W02014081851 relates to methods for modeling the in vivo kinetics and metabolism of amyloid-b (Ab) isoforms. In particular, methods for determining one or more kinetic parameters of Ab42 and at least one other Ab peptide are described. Determination whether a subject has amyloid pathology is based on a difference between the two kinetic parameters.

Several antibodies have been described to detect other Ab isoform peptides and to be used in immunological assays. For instance, WO2012140296 describes a highly specific antibody that recognizes Ab17 in a specific manner without showing any substantial cross-reactivity towards other Ab species such as Ab15, Ab16, Ab38, Ab40 or Ab42.

W02015111430 describes an improved method for measuring APP cleavage peptides (also referred to as Ab-like peptides) in blood using a antibody-immobilized carrier to capture APP cleavage peptides, dissociating the APP cleavage peptides from the carrier and detecting them using matrix-assisted laser desorption/ionization mass spectrometry. The method measures APP cleavage peptides is particularly suitable for small blood samples or in samples where the peptides are present in trace amount in the blood sample.

WO2015178398 discloses marker panels and methods for determining a cerebral Ab accumulation status based on measuring a combination on measuring a combination of ratio's of Ab-like peptides WO2017047529 discloses marker panels and methods for determining a cerebral Ab accumulation status based on measuring a combination of a ratio of Ab peptides (Ab39/ Ab42 or (Ab40/ Ab42), and the ratio of Ab petide and Ab-like peptide (ARR669-711/Ab42).

[0011] Unlike Ab, the GFAP biomarker is understudied in AD. One patent in connection to GFAP measurement in plasma includes W02005029087 which describes the use of serum or plasma GFAP as a diagnostic marker for intracerebral hemorrhage. The invention especially relates to methods for the very early assessment of intracerebral hemorrhage. Plasma GFAP level is measured using Elecsys technology.

[0012] With the establishment of various new high-sensitive analytical platforms, the development of a valid and robust Ab blood assay seems more within reach, as with those techniques convincing results for translating PET and CSF amyloid biomarkers into blood amyloid biomarkers were obtained. For example, for the Elecsys immunoassays it has been shown that cut-offs established in one European cohort could with high accuracy detect Ab positivity in an independent cohort in the US (area under the ROC curve [AUC] 0.96, 95% Cl 0.95-0.98) (Flanssson et al., 2018). Simoa is another one of these techniques, and has the advantage to be translatable to daily clinical practice due to automation of procedure with high throughput, and to be easily transferrable to and implementable in all neurochemical laboratories. Using Simoa technology, studies showed that plasma amyloid levels decrease over the Alzheimer's continuum (Janelidze et al., 2016; Verberk et al., 2018; Vergallo et al., 2019; Shi et al., 2019 ) and with reasonable accuracy amyloid abnormal and normal individuals can be discriminated (Janelidze et al., 2016), even at the pre-symptomatic phase (Verberk et al., Shi et al., 2019).

[0013] Despite all recent biomarker research and improved technologies for evaluating Ab accumulation in the brain there is still a need for clinical accurate non-invasive and cost- efficient methods that detect Ab accumulation in subjects at the very early stage of the disease, either before subjects will become MCI or during MCI phase. Such methods would be applicable in clinical trial enrichment settings, in the evaluation of drug efficacy of therapeutic and prophylactic drugs in neurodegenerative diseases.

SUMMARY OF THE INVENTION

[0014] The present invention generally concerns an improved marker for determining a cerebral Ab accumulation state in a living body-derived sample from a test subject. The invention as set out in the claims shows for the first time that the combination of Ab42/Ab40 ratio and GFAP measurement in blood or plasma samples improves the accuracy for detecting abnormal Ab accumulation, even before any clinical symptoms of cognitive function decline can be observed in the test subject.

[0015] Accordingly, the invention provides in a first aspect a marker for determining a cerebral Ab accumulation state in a living body-derived sample from a test subject, the marker comprising a combination of the analytes Ab42/Ab40 ratio and GFAP.

[0016] The invention also provides a syndrome diagnosis marker for determining cognitive impairment in a test subject, the marker comprising a combination of the analytes Ab42/Ab40 ratio and GFAP.

[0017] In another aspect is provided, an analytic method for determining a cerebral Ab accumulation state in a test subject, the method comprising:

o subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40 and GFAP, to obtain measurement levels of Ab42, Ab40 and GFAP ; o a calculation step of calculating the ratio of Ab42 level to Ab40 level: Ab42/ Ab40; and o an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level of the test subject is higher than the standard level which is the GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

In a further preferred aspect, said analytic method is characterized in that the cerebral Ab accumulation state is determined in the test subject before said test subject shows any clinical symptoms of cognitive function decline, or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). In still another preferred aspect, the cerebral Ab accumulation state is determined in the test subject before said test subject shows any clinical symptoms of cognitive function decline, or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subjective cognitive decline (SCD). In still a further aspect, the cerebral Ab accumulation state is determined in the test subject before said test subject shows any clinical symptoms of cognitive function decline. The marker and method optionally incorporate additional analytes besides Ab42/Ab40 ratio and GFAP.

[0018] The marker and methods of the present invention are particularly beneficial for patients with neurodegenerative disease or patients susceptive of neurodegenerative disease, such as, for example, for patients with early-onset of AD, even before any clinical symptoms can be observed, or for patients with AD. The marker and methods are particularly advantageous in a clinical trial enrichment setting of who would be referred to a specialized center for a PET scan and who should not. It also finds its use in predicting progression of symptoms, in predicting risk of development of dementia, and in therapeutic effectiveness monitoring.

BRIEF DESCRIPTION OF FIGURES

[0019] For a fuller understanding of the nature of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings in which:

Figure 1A to 1C: show boxplots of plasma biomarker levels stratified for amyloid PET status. Figure 1A to 1C X-axis from left to right: Normal amyloid PET, Abnormal amyloid PET. Figure 1A Y-axis: Plasma Ab42/Ab40 ratio; Figure IB Y-axis: Plasma GFAP in pg/mL. Figure 1C Y-axis: Plasma NfL in pg/mL. Figure ID to IF show boxplots of plasma biomarker levels stratified for syndrome diagnosis. X-axis from left to right: SCD, MCI, AD. Figure ID Y-axis: Plasma Ab42/Ab40 ratio. Figure IE Y-axis: Plasma GFAP in pg/mL.; Figure IF Y-axis: Plasma NfL in pg/mL. The p-values were calculated using univariate analysis of variance adjusted for age and gender and in case of more than two groups bonferroni-corrections for multiple comparisons was applied.

Figure 2A to 2C: show boxplots of plasma biomarker levels for amyloid PET status-stratified syndrome diagnosis. Figure 2A to 2C X-axis from left to right: Amyloid normal SCD; Amyloid abnormal SCD; Amyloid normal MCI; Amyloid abnormal MCI; Amyloid abnormal AD. Figure 2A Y-axis: Plasma Ab42/Ab40 ratio; Figure 2B Y-axis: Plasma GFAP in pg/mL.; Figure 2C Y-axis: Plasma NfL in pg/mL. The p-values were calculated using univariate analysis of variance adjusted for age and gender with post-hoc bonferroni- correction. For plasma GFAP and plasma NfL, levels were natural log transformed for normal distribution of the data prior to age and gender adjusted group comparisons.

Figure 3: shows ROCs for prediction of an abnormal amyloid PET scan by the plasma biomarkers or a combined model in the total study population in Figure 3A; and in the non-demented population only in Figure 3B. X-axis: Specificity; Y-axis: Sensitivity. Individual biomarkers plasma GFAP (— line), Abeta 1-42/1-40 ratio (-o- line) and NfL (-+- line) are plotted and the best-fitting model best predicting an abnormal amyloid PET status is plotted

Figure 4: shows Pearson's correlations of plasma with CSF biomarkers, stratified for syndrome diagnosis in Fig.4A=SCD , Fig.4B=MCI or Fig.4C=AD population. X-axis: CSF markers from left to right: Ab42, tTau, pTau, pTau/ Ab42 ratio; Y-axis: plasma markers from bottom to top: GFAP, NfL, Abb42/ Abb40 ratio.

DEATAILED DESCRIPTION OF THE INVENTION

[0020] Large quantity of amyloid is deposited before exteriorization of the cognitive function decline in AD patients. It has been demonstrated that the accuracies of the Ab42 and Ab40 assays are not sufficient to be used on their own as a clinical test of Ab positivity, particularly in blood-based settings. Therefore, Ab42 and Ab40 assays need to be improved before they can be helpful in screening diagnostic and clinical capabilities. Studies have demonstrated that Ab42/Ab40 ratio correlates with brain Ab and can differentiate patients with AD from healthy control participants.

[0021] We hypothised that the marker Ab42/Ab40 ratio complemented with the additional analyte GFAP might improve the accuracy for detecting amyloid accumulation, even before any clinical symptoms of cognitive function decline are observed. Although GFAP plasma immunoassays are available, GFAP has to the best of our knowledge never been used in combination with plasma Ab42/Ab40 ratio to demonstrate clinical and screening diagnostic capabilities of an Area under ROC curve greater than 0,85 as shown in the example section.

[0022] In one aspect, the present invention provides a marker for determining a cerebral Ab accumulation state in a living body-derived sample from a test subject, the marker comprising a combination of the analytes Ab42/Ab40 ratio and GFAP. By combining GFAP to Ab40/Ab42 ratio, it is possible to estimate a cerebral Ab accumulation state with higher accuracy, as compared with the case where the Ab40/Ab42 ratio is used singly.

[0023] The term "Ab" is used as an abbreviation of beta-amyloid. Ab is a cleavage product of APP, through sequential proteolytic processing by b- and y-secretases The y-secretase, which cuts at the C- terminal end of the Ab peptide, cleaves within the transmembrane region of APP to generate a number of Ab isoforms of 36-43 amino acid residues in length. Three major forms of Ab detected in the CSF are Ab40, Ab38, and Ab42 (respectively 40, 38 and 42 in length) with other minor abundance forms 15, 16, 17, 34, 37, and 39 amino acids in length. Abc, Ab1-c^qΐ3c and Abetal-x are synonymously used herein and designate beta-amyloid peptide with 1-x amino acid residues in length.

[0024] "Glial fibrillary acidic protein" (GFAP) is a 55 kDa cytosolic protein that is a major structural component of astroglial filaments and is the major intermediate filament protein in astrocytes. The term "GFAP" as used herein must not be understood as implying only the full length molecule. It rather also refers to any physiological fragment or modification thereof, in particular, to immunologically detectable fragments.

[0025] In another embodiment of the present invention, the marker for determining a cerebral Ab accumulation state in a living body-derived sample from a test subject comprises, in addition to Ab40/Ab42 ratio and GFAP, one or more additional analytes, such as one or more known CSF biomarkers of AD pathology, brain atrophy and brain metabolism. The one or more additional analytes are preferably chosen from the group consisting of NfL, APOE s4 carriership and tTau. tTau measured in plasma is referred to as plasma tau.

[0026] The term "body-derived sample or test sample" as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, or evaluation of a test subject of interest, such as a patient. Living body-derived samples or test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva and body secreting fluid. Preferred test samples are blood, serum and plasma, with plasma representing the most preferred sample. Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot and removal of the clot. Plasma is produced when whole blood is treated with an anticoagulant, such as e.g. EDTA, and cells are removed.

[0027] In a preferred embodiment of the present invention, the test subject is a human being. Preferably the test subject does not show any clinical symptoms of cognitive function decline or the test subject is at the very early stages of a neurodegenerative disease showing only clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). Particularly, the test subject is a non- demented individual having a cerebral abnormal amyloid status. In one preferred embodiment, the test subject does not show any clinical symptoms of cognitive decline or has clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). In another preferred embodiment, the test subject does not show any clinical symptoms of cognitive function decline or has clinical symptoms of SCD only. In still further preferred embodiment, the test subject does not show any clinical symptoms of cognitive function decline. In another embodiment, the subject is at the very early stages of a neurodegenerative disease; preferably AD.

[0028] The term "neurodegenerative disease", as used herein, refers to a disease that is associated with an altered, preferably decreasing degree of neuronal viability and/or activity. Non-limiting examples of neurodegenerative disease include, but are not limited to Alzheimer's disease, Parkinson's disease, Huntington disease, Lewy body dementia and Prion diseases, and their corresponding stages with baseline syndrome diagnosis.

[0029] The term "marker" as used herein, refers to any kind of molecule that is, directly or indirectly, for example by itself or through precursors, derivatives or products thereof, or interactions with other molecules, organelles, cells, tissues or the like, associated with and, preferably, indicative of a certain physiological state of test subject. In a preferred embodiment the physiological state of the test subject is a disease involving neuronal damages. In one embodiment, the physiological state of the test subject is at the very early stage of disease, preferably before any clinical symptoms of cognitive function decline are observed, such that it is a potential indicator of SCD or MCI and eventual progression to dementia. The marker, as used herein, is composed of one or more analytes, the analyte most frequently being a single kind of biomolecule. Typically, the analyte is a protein or peptide that is differentially expressed in a disease or a diseased test subject compared to the healthy test subject, and the disease can be diagnosed by way of analysis of the protein level present in a body-derived or test samples obtained from the test subject. Alternatively, the analyte is an allele-carrier that is differentially present in a disease or a diseased test subject compared to the healthy test subject. In certain settings of the present invention, the marker is a combination of analytes, and may include the ratio of one analyte level to another analyte level. In the present invention, the marker comprises a combination of the analytes Ab42/Ab40 ratio and GFAP. In a preferred embodiment, the marker consists of a combination of the analytes Ab42/Ab40 ratio and GFAP, and one or more further analytes. In particular settings, the marker consists of a combination of the analytes Ab42/Ab40 ratio, GFAP and NfL. As shown in the example section, this marker setting turns out to accurately identify an abnormal cerebral amyloid status. In another setting, the marker for determining a cerebral Ab accumulation state in a test subject consists of a combination of the analytes Ab42/Ab40 ratio, GFAP and APOE s4 carriership. This marker setting turns out to accurately identify an abnormal amyloid status.

[0030] APOE polymorphisms have been associated with changes in brain function. APOE encodes apolipoprotein E. There are three human variants of the gene: APOE e2, APOE e3, and APOE e4. APOE e4 increases the lifetime risk of AD by twofold to fourfold (Nussbaum et al, 2015; Nussbaum and Ellis, 2003). As used herein, "AROE-eA" codes for the apolipoprotein-e4 (apoE4), which compared to other isoforms, is less effective in maintaining cerebral lipid homeostasis and in breaking down Ab peptide, thus facilitating the formation of extracellular insoluble oligomers. APOE s4 carriership can be determined using techniques for genotyping, PCR, sequencing,..., or commercial kits developed for that purpose (e.g. EUROArray APOE Direct).

[0031] Cerebral Ab accumulation precedes cognitive function decline. Using patient test performance on a large battery of neuropsychological tests as outcome, we show that the marker for determining a cerebral Ab accumulation state in the patient is highly associated with performance on various cognitive tests. Tests to measure cognitive impairment include Mini-Mental State Examination (MMSE) covering global cognition, but also various tests in major cognitive domain such as domain of attention, memory, executive functioning, etc... Accordingly, the marker as defined in the present invention can be used for syndrome diagnosis and/or differential diagnosis and/or early detection of vascular problems. The marker may be helpful in the differentiation of different psychological problems (depression, schizophrenie, bipolar disorder,...)

[0032] Thus, in an alternative embodiment, the invention provides a syndrome diagnosis marker for determining cognitive impairment in a test subject, the marker comprising a combination of the analytes Ab42/Ab40 ratio and GFAP. In a preferred embodiment, the marker for syndrome diagnosis comprises, in addition to Ab40/Ab42 ratio and GFAP, one or more additional analytes, such as one or more known CSF biomarkers of AD pathology, brain atrophy and brain metabolism. Most preferably, the syndrome diagnosis marker for determining cognitive impairment in test subject, comprises a combination of the analytes Ab42/Ab40 ratio and GFAP and NfL. Typically, such marker will be tested in a living body- derived sample from a test subject. In a preferred embodiment, the marker for syndrome diagnosis is evaluated in a test subject before the test subject shows any clinical symptoms of cognitive function decline, or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of SCD or MCI.

[0033] As shown in the example section, the invention relates to a marker according to any of the described embodiments used in a method for determining a cerebral Ab accumulation state in a living body-derived sample wherein a decreased Ab42/Ab40 ratio and increased GFAP identifies/predicts neural damage due to an abnormal amyloid status. Thus, the marker as described finds its application in an analytic method for determining a cerebral Ab accumulation state in a living body-derived sample. Accordingly, the present invention relates to an analytic method for determining a cerebral Ab accumulation state in a test subject, the method comprising:

o subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40 and GFAP, to obtain measurement levels of Ab42, Ab40 and GFAP;

o a calculation step of calculating the ratio of Ab42 level to Ab40 level: Ab42/ Ab40; and o an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for Ab accumulation, and the GFAP level of the test object is higher than the standard level which is the GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

In a preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD or MCI. In an even more preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decliner or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD. In a still a further and preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline.

The example section shows that plasma GFAP combined with plasma Ab42/Ab40 ratio demonstrate clinical and screening diagnostic capabilities of an Area under ROC curve greater than 0,85. Preferably, the marker of the method comprises one or more analytes additional to Ab42, Ab40 and GFAP.

[0034] In one embodiment, the measuring step applied in the present invention measures the levels of each of the analytes Ab42, Ab40 and GFAP, and optionally one or more analytes, in a qualitative or analytical manner using a immmuno-assay (IA). Sandwich lA's to that purpose are well known in the art, the most common one being an enzyme-linked immunosorbent assay (ELISA). Several types of ELISA exist and include i.e. direct ELISA, sandwich ELISA, competitive ELISA, reverse ELISA, etc... (Crowther RJ, 1995; O'Kennedy & Murphy, 2017), all of which are applicable in the measuring step applied in the present invention. ELISA's rely on specific antibodies to bind the target antigen (peptide or protein), and a detection system to indicate the presence and quantity of antigen binding. Different biochemical techniques may be used for detecting the binding of the de antibody and the analyte molecule in an ELISA set-up, nonlimiting examples of ELISA's using different biochemical techniques include colorimetric lAs, fluorescent lAs, chemiluminescence lAs, etc...

[0035] In one embodiment, the measuring step applied in the present invention, measures the levels of each of the analytes Ab42, Ab40 and GFAP using a fully-automated immunoassay. Preferably, the fully- automated immunoassay reaches a sensitivity at femtomolar. In a preferred embodiment, the measuring step applied in the present invention measures the levels of each of the analytes Ab42, Ab40 and GFAP using a chemiluminescence ELISA. In a further preferred embodiment, the measuring step applied in the present invention measures the levels of each of the analytes Ab42, Ab40 and GFAP using Elecsys technology.

[0036] New high-sensitive analytical platforms that apply single-molecule analysis have more recently been developed. Single molecule measurements are digital in nature and each molecule generates a signal that can be counted. In such systems, the presence or absence of signal is measured rather the absolute amount of signal. The systems apply the same reagents as in a conventional ELISA and reaches a sensitivity at femtomolar (fg/mL). One such system, Simoa (Quanterix), is described in W02012142300. Simoa is a digital form of ELISA in which individual immunocomplexes are trapped and sealed on paramagnetic beads in thousands femtoliter-sized reaction chambers in arrays found on the Simoa discs with the aid of enzyme-labeled capture antibodies. Typically 500 000 beads are added to a IOOmί sample. Similar to the traditional ELISA, non-specifically bound proteins are washed away and substrate is added before the sealing step. The protein concentration in the test sample is determined by counting the number of wells containing both a bead and fluorescent product relative to the total number of wells containing beads. Because Simoa enables concentration to be determined digitally rather than by measurement of the total analog signal, this approach to detecting single immunocomplexes has been termed digital ELISA.

[0037] Accordingly, in a preferred embodiment, the measuring step applied in the methods of the present invention measures the levels of each of the analytes Ab42, Ab40, GFAP and optionally NfL using a digital ELISA. In a more preferred embodiment, the measuring step applied in the methods of the present invention measures the levels of each of the analytes Ab42, Ab40, GFAP and optionally NfL using Simoa technology. Simoa technology has been described and is known in the art (Rissin et al., 2010; Wilson et al., 2016).

[0038] The analytical method of detecting a cerebral Ab accumulation state according to the present invention may be carried out in as singleplex or multiplex. In a singleplex set up, single protein molecules are counted. A "multiplex" experiment is defined herein as one which allows detection of susceptibility to, or the incidence of a cerebral Ab accumulation state by analysis of the Ab42, Ab40 and GFAP level linked to susceptibility to, or the incidence of, neural damage using a single sample. Multiplexing provides technical advantages because neural damage may be accurately diagnosed from a single sample by identifying the level of all analytes present in the marker. If many different samples are required for analyte of the panel to be analyzed, this may lead to problems of variability between samples, possibly leading to less consistent and accurate detection of cerebral Ab accumulation Furthermore, it is preferable for patients if a minimum sample and minimum number of samples are required in order to achieve an accurate diagnosis. In an alternative embodiment a single sample may be divided into several samples, each of which is used in an individual experiment to detect the level of at least one but not all of the panel of analytes whose analyte level is linked to the susceptibility to, or the incidence of, cerebral Ab accumulation. Therefore, a single sample or portion of a sample may provide the analyte level for analyte which is then used collectively in order to diagnose susceptibility to, or the incidence of, cerebral Ab accumulation.

[0039] In a preferred embodiment, the levels of each of the analytes Ab42, Ab40, GFAP and/or one or more other analytes are measured using digital ELISA technology, preferably Simoa technology or other high-sensitive immuno-assay technology, such as Erenna, to be able to measure with reasonable accuracy in these low analyte concentrations. In a preferred embodiment, measurement of the Ab42 and Ab40 analyte on the one hand, and measurement of GAFP and/or NfL analyte on the other hand are done in different freeze-thaw cycles. For the Ab42 and Ab40 analyte, measurement in the measuring step occurs in sequential order within the same run. The obtained Ab42 measure is normalized by using the Ab40 measure in the body-derived sample. This is done in the calculation step of calculating the ratio of Ab42 level to Ab40 level: Ab42/ Ab40.

[0040] As described in the example section, the marker for use in the method of the invention employs a marker comprising a combination of the analytes Ab42/Ab40 ratio, GFAP and NfL, or, alternatively the analytes Ab42/Ab40 ratio, GFAP and APOE s4 carriership.

[0041] In one embodiment, the analytic method for determining a cerebral Ab accumulation state of a test subject, the method comprises:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40, GFAP and one or more additional analytes, to obtain measurement levels of Ab42, Ab40 GFAP and one or more additional analytes;

• a calculation step of calculating the ratio of Ab42 level to Ab40 level: Ab42/ Ab40; and

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, when, the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level and one or more additional analytes of the test subject is higher than the standard level which is the GFAP and one or more additional analytes of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

[0042] Thus, in a further embodiment, the present invention relates to an analytic method for determining a cerebral Ab accumulation state of a test subject, the method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40, GFAP and NFL to obtain measurement levels of Ab42, Ab40 GFAP and NfL;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, when, the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and that both the GFAP level and the NfL level of the test object is higher than the standard level which is the GFAP level and the NfL level of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

[0043] In still another embodiment, the present invention relates to an analytic method for determining a cerebral Ab accumulation state of a test subject, the method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40, GFAP and APOE s4 carriership, to obtain measurement levels of Ab42, Ab40 GFAP and information on APOE s4 carriership;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, the GFAP level of the test object is higher than the standard level which is the GFAP level of the subject having normal cognitive function, and APOE s4 carriership of the test object is higher than the standard which is APOE s4 carriership of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

In a preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD or MCI. In an even more preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decliner or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD. In a still a further and preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline. [0044] In an alternative embodiment, the present invention relates to a method for early AD syndrome diagnosis of a test subject, the method comprising performing the analytic method as for determining a cerebral Ab accumulation state of a test subject on a living body-derived sample derived from the test subject to determine that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, and based thereon, diagnose that the test subject is at early stage of neurodegenerative disease when cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function and being negative for cerebral Ab accumulation. In such diagnostic method, the diagnosis of early stage of neurodegenerative disease is preferably a diagnosis of early stage of AD, preferably at the stage wherein no clinical symptoms of cognitive function decline are observed or wherein only subjective or mild cognitive decline is observed. More preferably the early stage of neurodegenerative disease is at the stage of clinical symptoms showing subjective cognitive decline (SCD) or mild cognitive decline (MCI).

[0045] In an alternative embodiment, the present invention relates to a method for identifying a test subject as having or being at risk of development of dementia, the method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42, Ab40 and GFAP, to obtain measurement levels of Ab42, Ab40 and GFAP;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for Ab accumulation, and the GFAP level of the test object is higher than the standard level which is the GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation;

and, based thereon, indicate whether the test subject has or is at risk of development of dementia if the cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function and being negative for cerebral Ab accumulation.

[0046] In order to find the best fitting model for cerebral amyloid status and/or syndrome diagnosis, apart from the variables used in the methods of the present invention, other variables such as age and gender may be taken into account when determining a cerebral Ab accumulation state of a test subject.

[0047] In a further aspect, the methods as described are conducted once or a plurality of times over time allowing predicting susceptibility to, or the incidence of, future progression of cerebral Ab accumulation, or predicting potential risk of developing dementia. Susceptibility to, or the incidence of developing dementia will be higher if higher cerebral Ab accumulation over time is determined.

[0048] The markers and methods for determining a cerebral Ab accumulation state of a test subject also find their application in triage settings. From a cost-effective and time-wise point of view, patient stratification for selecting a patient based on its larger cerebral Ab accumulation state for further examination such as PET or invasive techniques is particularly suitable. Patient stratification also helps in selecting the drug's potential patient to participate in a clinical trial. As disclosed, the markers and methods of the present invention allow for sensitive cerebral Ab accumulation measurement in test subjects at early-stage AD. Test subjects having larger cerebral Ab accumulation state compared to normals may benefit from further examination and/or early enrollment in clinical trials. Further, the methods as presented here can be applied before and after a therapeutic intervention, such as drug administration, and the cerebral Ab accumulation state before and after such medical intervention will determine efficacy of the medical intervention.

[0049] Thus, in another aspect, the present invention relates to a method for test subject stratification, the method comprising performing the analytic method according to any of the embodiments of the present invention for determining a cerebral Ab accumulation state of a test subject on a living body- derived sample derived from the test subject to determine that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, and based thereon, indicate the test subject as being suitable for

further examination: or

treatment with a product acting against cerebral Ab accumulation; if cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function. In a preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD or MCI. In an even more preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decliner or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD. In a still a further and preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline.

[0050] Alternatively, the present invention relates to a method for test subject stratification, the method comprising performing the analytic method for determining a cerebral Ab accumulation state of a test subject on a living body-derived sample derived from the test subject to determine that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, and based thereon, indicate the test subject as being suitable for

further examination: or

treatment with a product acting against cerebral Ab accumulation;

if cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function.

[0051] Depending on product type and development stage of the product, investigators may use the method for stratification of the present invention to enroll test subject into observational studies or clinical trials. Suitable treatments include vaccines, drugs, dietary choices, dietary supplements and medical devices.

In another aspect, the present invention relates to a method for determining efficacy of a therapeutic intervention, the method comprising performing before and after a therapeutic intervention the analytic method according to any of the embodiments of the present invention for determining a cerebral Ab accumulation state of a test subject on a living body-derived sample derived from the test subject to determine that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, and based thereon, determine efficacy of the therapeutic intervention. Typically, the therapeutic intervention in the test subject is efficient if cerebral Ab accumulation of the test object before therapeutic intervention is higher than after therapeutic intervention. Most suitable therapeutic intervention involves administration of a product acting against neurodegeneration, or, a product potentially acting against neurodegeneration. In a preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD or MCI. In an even more preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decliner or wherein the test subject is at the very early stages of a neurodegenerative disease showing SCD. In a still a further and preferred embodiment, said analytic method is typically characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline.

[0052] In all methods of the present invention, the marker comprises the combination of the analytes Ab42/ Ab40 ratio, and GFAP. In a further embodiment, the marker comprises one or more additional analytes; preferably said one or more additional analytes are selected from NfL and APOE s4 carriership. Hence, in any of the disclosed methods, the measurement levels of NfL can be obtained, whether or not in combination with evaluation of APOE s4 carriership.

[0053] In a further aspect, the present invention provides the use of Ab42, Ab40, and GFAP as a marker for determining a cerebral Ab accumulation state of a test subject wherein said test subject does not show any clinical symptom of cognitive decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). In particular, said use is characterized in that a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject.

[0054] In the context of the present invention, in the subject having normal cognitive function and being negative for cerebral Ab accumulation, the cerebral Ab accumulation can be measured using conventional methods, such for example PET imaging. Even further, the marker panel of the present invention shows that they are even more sensitive than the conventional methods for determination of cerebral Ab accumulation.

[0055] In another aspect, the present invention provides the use of b42, Ab40, and GFAP as a marker for identifying a test subject as having or being at risk of development of dementia, wherein said test subject does not show any clinical symptom of cognitive decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). In particular, said use is characterized in that a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation in the test subject has or is at increased risk of development of dementia if the cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function and being negative for cerebral Ab accumulation.

[0056] In a further aspect, the present invention provides the use of b42, Ab40, and GFAP as a marker for test subject stratification wherein said test subject does not show any clinical symptom of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subject cognitive function decline (SCD) or mild cognitive decline (MCI). In particular, said use is characterized in that a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation indicates that the test subject is being suitable for further examination and/or being suitable for treatment with a therapeutic product acting against cerebral Ab accumulation.

[0057] In still another aspect, the present invention provides the use of b42, Ab40, and GFAP as a marker for determining the efficacy of a therapeutic intervention against cerebral Ab accumulation in a test subject wherein said test subject does not show any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI). In particular, said use is characterized in that a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation indicates that the test subject is being suitable to undergo the therapeutic intervention against the cerebral Ab accumulation.

[0058] In a further embodiment, in the uses of the different embodiments of the present invention one or more additional analytes can be analyzed. Preferably, said one or more additional analytes are chosen from the group consisting of NfL and APOE s4 carriership. In a further embodiment, an increased level of NfL in the test subject is additionally indicative for cerebral Ab accumulation.

[0059] In still a further embodiment, in the uses of the different embodiment disclosed herein the levels of each of the analytes Ab42, Ab40, GFAP, and, optionally NfL, are determined using a digital ELISA.

[0060] In still another further embodiment, in the uses of the different embodiments disclosed herein the levels of each of the analytes Ab42, Ab40, GFAP, and, optionally NfL, are determined in a living body- derived sample of the test subject; preferably a living body-derived sample selected from a blood, plasma or serum sample. Even further, the living body-derived sample is obtained from the test subject at the very early stages of a neurodegenerative disease; preferably Alzheimer's disease.

[0061] The methods and use described may be implemented using various kits of the invention. Accordingly, in a further aspect the invention provides a kit for use in a method for determining a cerebral Ab accumulation state of a test subject, or, in a method for early AD syndrome diagnosis, comprising:

a) monoclonal antibodies against Ab42, Ab40, GFAP and, optionally NfL.

b) instructions for use

For a fuller understanding of the nature of the present invention, reference is made to the examples taken in conjunction with the accompanying drawings.

EXAMPLES

[0062] Study population. This study included subjects from the Amsterdam Dementia Cohort (n=252) (van der Flier et al., 2014; van der Flier et al., 201418) with baseline syndrome diagnosis SCD (n=70), MCI (n=50) or AD (n=132) who have undergone an amyloid PET scan within one year from baseline diagnosis. Additionally, a plasma EDTA sample had to be available in the Amsterdam UMC, VUmc biobank, which was collected within one year from the clinical diagnosis and the amyloid PET scan. Research received ethical consent of the VU University and was in accordance with the Helsinki Declaration act of 1975. All subjects provided written consent to use their medical data and biomaterials for scientific research. Between November 2008 and October 2018, all subjects visited the memory clinic of the Amsterdam UMC, VUmc for extensive dementia screening consisting of neurological, physical and neuropsychological evaluation as well as biomarker analysis in CSF and brain magnetic resonance imaging (MRI) (Oeckl et al., 2019; van der Flier et al., 2014). The screening day was followed by a multidisciplinary consensus meeting wherein diagnosis was established according to the then applicable guidelines. SCD was assigned when no abnormalities were observed on clinical or cognitive testing and thus criteria for MCI, dementia, but also for other medical conditions potentially causing cognitive decline were not met (van der Flier et al., 2018). AD diagnosis was, by definition, accompanied by an abnormal amyloid PET scan.

[0063] Amyloid PET. All subjects underwent an amyloid PET scan, which was visually read and scored by an experienced nuclear medicine physician as amyloid abnormal or amyloid normal. Subjects were scanned with [18F]florbetaben (n=133), [18F]flutemetamol (n=37), [18F]florbetapir (n=33) or [llC] Pittsburgh compound-B (PiB; n=49) radiotracers, infused trough a venous cannula. For [18F]florbetaben and [18F]flutemetamol, 20-minute static PET scans were acquired starting 90 minutes after tracer injection (approximately 250MBq [18F]florbetaben, 180MBq [18F]flutemetamol) on respectively the PET/MR and Gemini TF-64 PET/CT scanner (Philips Medical Systems, The Netherlands). A 90 minutes dynamic scan starting simultaneously with tracer infusion (approximately 370MBq [18F]florbetapir, 351MBq [llCJPiB) was acquired on the PET/CT Ingenuity TF or Gemini TF (Philips Medical Systems, The Netherlands) for [18F]florbetapir and on ECAT EXACT FIR+ scanner (Siemens/CTI, Tennessee, United States) for [11CJPIB.

[0064] Plasma analyses. EDTA plasma was sampled through venipuncture, centrifuged within one hour at 1800 x g for 10 minutes, and stored at -80 °C in aliquots of 500mI in polypropylene tubes in the Amsterdam UMC, VUmc Biobank. Prior to analysis, samples were shortly thawed at room temperature using a cold-air fan, centrifuged at 10.000 x g and subsequently kept on ice. All samples were measured in duplicates onboard of the automated Simoa FID-1 analyzer by trained personnel. Research prototype blood amyloid beta (Amyblood) Simoa assays were developed that specifically detect Abetal-42 and Abetal-40. In short, the automated two-step analytical Simoa procedure of the Amyblood singleplex assays were as follows. In step one for 120 minutes, 25pL of 250K helper beads (Quanterix) and 250K paramagnetic carboxylated beads that were activated with 0.1 mg/mL EDC and coated with 0.2 mg/mL of either monoclonal antibody 21F12 or 2G3 (ADx Neurosciences, Belgium) were incubated with 100pL of 4-fold (for Abetal-42) or 10-fold (for Abetal-40) pre-diluted plasma EDTA in PBS-based buffer with 0.1% casein, 0.1% Tween20, 200pg/mL FIBR-1 (Scantibodies Laboratory Inc., United States) and 20 pL of 0.1 pg/mL of 8x sNFIS-LC-biotinylated detector 3D6 monocolonal antibody (ADx Neurosciences). After a wash cycle, in step two a 5 minute 15 second incubation followed with 50 pM streptavidin-conjugated b-galactosidase (Quanterix). After a next wash, 25pL Resorufin b-D-galactopyranoside (Quanterix) was added and beads were pulled onto the imaging disc, followed by time-lapsed fluorescent imaging. The standard curves were constructed in a range from 0 to 20 pg/mL using Abetal-40 and Abetal-42 recombinant peptides (ADx neurosciences). For Amyblood analysis, samples were randomly divided over seven independent runs in which Abetal-40 and Abtal-42 measurement occurred in sequential order within the same run. All Amyblood analyses were performed on one single Simoa instrument. Abetal-42 was normalized by using Abetal-40 in the plasma Abeta 1-42/1-40 ratio. A good average intra-assay coefficient of variation was obtained. In the next freeze-thaw cycle, plasma NfL and plasma GFAP were measured in three independent runs per marker. The commercially available Simoa™ NF-Light Advantage Kit (Quanterix) and Simoa™ GFAP Discovery Kit (Quanterix) were used according to manufacturer's instructions and with on-board automated sample dilution. Good average intra-assay %CV of 5%CV for plasma NfL and 4%CV for GFAP were obtained. Inter-assay %CV of EDTA plasma pool quality controls was on average for three independent quality control pools 2 %CV for NfL (range: 1.6 - 2.3%CV) and 8%CV (range: 3 - 15%CV) for GFAP.

[0065] CSF analysis. Of a large subset (n=239), CSF obtained through lumbar puncture was available. Biomarkers Abetal-42, tTau and tau phosphorylated at threonine 181 (pTaul81) were quantified in CSF using Innotest ELISAs (Fuijirebio, Ghent, Belgium). Over the years, a drift in CSF Abetal-42 levels occurred that was explained by the analytical procedure. CSF Abetal-42 levels were corrected for this drift. CSF biomarker concentrations were used as a continuous measure, and CSF Abetal-42 was dichotomized as CSF amyloid abnormal <813 pg/mL.

[0066] APOE genotyping. For n=244, Apolipoprotein E (APOE) genotyping was available. Sequencing was performed in EDTA plasma using Sanger sequencing on ABI130XL, after DNA amplification by PCR technique and analysis for size and quantity by QIAxcel DNA Fast Analysis kit. APOE e4 carriers had one or two APOE e4 copies, whereas non-carriers only had APOE e2 or APOE e3 alleles.

[0067] Neuropsychological assessment. Cognition was assessed by trained neuropsychologists, using a large neuropsychological testing battery covering the main cognitive domains global cognition, attention, memory, language and executive functioning. Tests included were the Mini-Mental State Examination (MMSE), Dutch version of the Rey Auditory Verbal Learning Test (RAVLT) immediate recall, delayed recall and recognition, the Visual Association Test A trial (VAT A; sum of trial 1 and 2) and VAT naming, category fluency animals, letter fluency test, Digit Span Forward and backward, Trail Making Test (TMT) A and B, Stroop word naming, Stroop color naming and stroop color word naming. TMTA, TMT B and stroop scores were natural log transformed for normality of data, and subsequently inverted so that for all neuropsychological tests a lower score means worse performance. Missing TMT B values were imputed by multiplying an individual's TMT A score by the average TMT B to TMT A ratio. All neuropsychological test scores were transformed into Z-scores for comparability of effect sizes. The number of missing baseline neuropsychological test performance ranged from 87% to 98%

[0068] Statistical analysis. Using SPSS for Windows version 22 (IBM) statistical analysis was conducted, and using R version 3.4.2 graphs were constructed. An alpha of p<0.05 was considered statistically significant. Plasma biomarkers Abetal-40, NfL and GFAP and CSF biomarkers Abetal-42, tTau and pTaul81 were right-skewed, thus natural log (Ln) transformation was performed prior to statistical analyses that require normally distributed data. Z-transformation was performed on inverted Abetal- 42/1-40 ratio, on the log-transformed GFAP and on log-transformed NfL when comparability of effect sizes was required. Baseline demographics were performed using Chi square tests, T-tests or non- parametric equivalents as appropriate for two groups, and univariate analyses of variance for more than two groups. CSF and plasma biomarker levels were additionally compared using univariate analysis of variance with adjustment for possible covariates age and gender. Cross-sectional relationships between cognitive performance (dependent variables; Z-transformed neuropsychological test scores) and plasma biomarkers (independent variables; Z-transformed plasma markers) were assessed using linear regression analysis (all separate models). The analysis was performed adjusted for age, gender and education (model 1) and with an additional correction for diagnosis (model 2). P-values were corrected for multiple testing using the 10% false discovery rate (10%FDR) procedure (Olsson et al., 2016). Receiver operating characteristic (ROC) curves of plasma markers predicting an abnormal amyloid PET scan were constructed and youden's cut-offs were calculated as the maximum sum of sensitivity and specificity. A best fitting logistic regression model that optimally predicts an abnormal amyloid PET status was determined using the backward elimination regression procedure based on Wald's p statistics. Variables included in this model were age, gender, diagnosis, APOE e4 carriership and all plasma markers. Predicted values of the optimal panel were plotted as ROC curve and Youden's cut-off was established. Wald's elimination regression procedure with subsequent ROC analysis was repeated for the non-demented group only (i.e. SCD + MCI). Global associations between plasma biomarker levels and CSF biomarker levels were assessed using Pearson's correlation analysis, both for total group and after stratification for syndrome diagnosis SCD, MCI or AD.

RESULTS

[0069] Cohort characteristics. Baseline demographic features of the total study population and stratified for amyloid PET status are listed in table 1. In table 2, data is presented as mean ± SD, or % (n). Education scoring is according to the Verhage (1965) system with a scale ranging from 1 to 7. Group differences were calculated using independent t-tests, chi-square tests or Mann Whitney U test as appropriate. All CSF biomarkers and plasma biomarkers Abetal-40, NfL and GFAP were natural log- transformed prior to statistical analysis. CSF and plasma biomarkers were additionally compared using univariate analysis of variance adjusted for age and gender. APOE status was available for n=244, CSF biomarkers for n=239, plasma Abeta-42 and Abetal-42/1-40 ratio for n=238, plasma Abetal-40 for n=240, plasma NfL for n=251 and plasma GFAP for n=247 individuals. SCD = subjective cognitive decline, MCI = mild cognitive impairment, AD = Alzheimer's dementia, MMSE = mini mental state examination, CSF=cerebrospinal fluid, Abeta = amyloid beta, tTau = total tau, pTaul81 = tau phosphorylated at threonine 181, GFAP = Glial fibrillary acidic protein, NfL = Neurofilament light. * = upon re-analysis adjusted for age and gender, p-value remained p<0.001. t = upon re-analysis adjusted for age and gender, p=0.036.

[0070] The study cohort comprised n=252 individuals of whom 176 (66%) were PET amyloid abnormal (average ± SD age of 63 ± 7, 87 females, MMSE of 23 ± 4) and 76 (34%) were PET amyloid normal (average age of 61 ± 9, 27 females, MMSE of 27 ± 2). Syndrome diagnosis within the PET amyloid abnormal group was for 18 individuals SCD, for 26 MCI and for 132 AD. Within the PET amyloid normal group this was 52 SCD and 24 MCI. There were less males in the amyloid abnormal group as compared to the amyloid normal group (c2=4.14, p=0.042). Following expectations, MMSE (Mann Whitney U test: p<0.001) and APOE e4 carriership (c2=40.15, p<0.001) were distributed differently between the amyloid PET abnormal and normal groups, as were the CSF biomarkers (all: p<0.001).

[0071] Plasma biomarkers for PET amyloid status and syndrome diagnosis. Age and gender adjusted group comparisons revealed that plasma Abeta 1-42/1-40 was decreased in the PET amyloid abnormal group compared to the amyloid normal group (figure 1A; F=26.79, p<0.001). Plasma GFAP was higher in the PET amyloid abnormal group compared to the amyloid normal group (figure IB; F=82.51, p<0.001), as was plasma NfL (figure 1C; F=34.99, p<0.001).

[0072] When stratifying according to syndrome diagnosis following our hypothesis that plasma markers GFAP and NfL might better reflect the degree of neuronal damage, age and gender adjusted group comparisons revealed that plasma Abeta 1-42/1-40 (figure ID; F=11.17, p<0.001) was decreased in AD compared to MCI (p=0.033) and SCD (p<0.001). Plasma GFAP (figure IE; F=32.07, p<0.001) was increased in MCI compared to SCD (p=0.012) and further increased in MCI compared to AD (p<0.001), as was plasma NfL (figure IF; F=25.30, p<0.001; post-hoc: SCD vs MCI: p=0.013, MCI vs AD: p=0.011). Comparison of plasma biomarker levels after stratification according to both amyloid PET status and syndrome diagnosis, is presented in figure 2.

[0073] Using test performance on a large battery of neuropsychological tests as outcome instead of syndrome diagnosis, it was shown that all plasma markers were associated with performance on various cognitive tests (table 2). In table 2, data is presented as standardized Beta (sBeta) with 95% Cl, of cross- sectional linear regression analysis between plasma biomarker levels and performance on neuropsychological tests. Plasma biomarker levels as well as neuropsychological test scores were Z- transformed for comparability of effect sizes. Additionally, plasma Abetal-42/1-40 levels as well as natural log-transformed TMT and natural log-transformed Stroop scores were inverted, so that for all tests and markers lower beta's represent worse performance. Model 1 = age + gender + education adjusted, model 2 = age + gender + education + diagnosis. Z-scores of plasma markers. Z-scores of neuropsychological tests. In bold p<0.05^FDR; model 2: no significant associations. Abeta= amyloid beta, GFAP = Glial fibrillary acidic protein, NfL = Neurofilament light , MMSE = Mini-Mental State Examination TMT = Trail Making Test, RAVLT = Dutch version of the Rey Auditory Verbal Learning Test, VAT = Visual Association Task.

[0074] When adjusting for age, gender and education, baseline plasma Abetal-42/1-40 ratio was found associated to baseline test performance on the MMSE (global cognition), and to various tests in the domains of attention, memory and executive functioning (all: p<0.05FDR) but not language. Plasma GFAP levels were related to performance on all tests covering global cognition and all major cognitive domains (all: p<0.05FDR). Plasma NfL levels were related to performance on MMSE and to most tests covering all major cognitive domains. After adjusting for age, gender, education and diagnosis, none of the plasma markers were significantly related to baseline performance on any of the neuropsychological tests.

[0075] Prediction of amyloid-PET status using plasma biomarkers. To evaluate the potential of the plasma biomarkers to accurately identify an abnormal amyloid PET scan, ROC curve analysis was performed. Plasma Abetal-42/1-40, plasma GFAP as well as plasma NfL could predict an abnormal amyloid status (all: AUC>71%). AUC with sensitivity and specificity at Youden's cut-off of the individual biomarkers is presented in table 3. To evaluate what would be an optimal panel that could best identify an abnormal amyloid PET scan, Wald's backward elimination logistic regression analysis was conducted including plasma markers Abetal-42/1-40 ratio, GFAP and NfL and possible covariates and/or AD risk factors age, gender, and APOE e4 carriership. The best fitting model included the variables plasma Abetal-42/1-40 ratio, plasma GFAP, age and APOE e4 carriership. This combined model reached an accuracy of AUC=88% (95%CI: 83 - 93%) in predicting an abnormal amyloid PET status (table 3; figure 3A). At youden's cut-off, the sensitivity of the model was 82% and specificity was 86%.

[0076] Focusing on non-demented individuals only (SCD and MCI) as non-demented amyloid abnormal individuals are mostly included in clinical trials, Wald's backward elimination procedure was repeated. Including the same variables plasma Abetal-42/1-40, GFAP and NfL, and age and APOE e4 carriership, identified plasma Abetal-42/1-40, plasma GFAP and APOE e4 carriership as the best fitting model. This panel resulted in AUC=84% (95%CI: 76 - 92%) to predict an abnormal amyloid status in the non- demented study population. At youden's cut-off, sensitivity was 70% and specificity was 86% (figure 3B).

[0077] Relationships between plasma and CSF biomarkers. To further investigate if changes in plasma biomarkers relate to AD biological processes, Pearson's correlation analysis was conducted. In the total population, all plasma markers were significantly correlated with all CSF biomarkers Abetal-42, tTau and pTaul81 (table 4). When stratifying for syndrome diagnosis to investigate which syndrome diagnoses drive the observed relationships (figure 4), it was observed that plasma Abetal-42/1-40 was no longer related to CSF biomarker levels in any of the diagnostic groups. Plasma GFAP was associated with all CSF biomarker levels within the SCD group (Abetal-42: r=-0.47, p<0.001, tTau: r=0.35, p=0.008, pTau: r=0.29, p=0.031), and with CSF tTau (r=0.31, p=0.031) and ptau (r=0.29, p=0.041) but not Abetal-42 in the MCI group. Plasma NfL was associated with CSF Abetal-42 only in only the SCD group (r=-0.38, p=0.003).

[0078] Relationships between plasma biomarkers and neuropsychological test performance. Using test performance on a large battery of neuropsychological tests as outcome , all plasma markers were associated with performance on various cognitive tests (table 4). More specifically, when adjusting for age, gender and education, baseline plasma Abetal-42/1-40 ratio associated with baseline test performance on the MMSE (global cognition), and to various tests in the domains of attention, memory and executive functioning (all: p<0.05^FDR) but not language. Plasma GFAP levels related to a larger number of tests, i.e. all tests covering global cognition and all major cognitive domains (all: p<0.05^FDR). Plasma NfL levels were related to performance on MMSE and to several tests covering all major cognitive domains. After additionally adjusting for diagnosis, none of the plasma markers were significantly related to baseline performance on any of the neuropsychological tests (data not shown).

TABLES

Table 1. Demographics, clinical characteristics and biomarkers of the total study population and stratified for amyloid PET-status.

Stratified for amyloid PET status

Total Amyloid normal Amyloid abnormal p-value n=252 34% (n=76) 66% (n=176)

Age, y 63 ±8 61 ±9 63 ±7 0.957

Female gender 45% (114) 36% (27) 49% (87) 0.042 Education 5.3 ± 1.2 5.5 ± 1.3 5.2 ± 1.2 0.072

Diagnosis (SCD/MCI/AD) 70/50/132 52/24/0 18/26/132 <0.001 MMSE 24 ±4 27 ±2 23 ±4 <0.001

APOE e4 carrier 53% (134) 24% (18) 66% (116) <0.001

CSF Abetal-42, pg/mL 756 ± 279 1105 ± 246 617 ± 129 <0.001 * CSF tTau, pg/mL 567 ± 342 306 ± 168 671 ± 339 <0.001 * CSF pTaul81, pg/mL 74 ±35 48 ± 18 85 ±35 <0.001 * CSF pTaul81/Abetal-42 ratio 0.12 ±0.08 0.04 ± 0.02 0.14 ±0.07 <0.001 *

Plasma Abeta 1-42/1-40 ratio 0.15 ±0.03 0.17 ±0.03 0.14 ±0.03 <0.001 * Plasma Abetal-42, pg/mL 24 ±6 27 ±6 23 ±6 <0.001 * Plasma Abetal-40, pg/mL 160 ± 29 165 ± 30 157 ±28 0.053 f Plasma GFAP, pg/mL 146 ± 78 96 ±53 168 ±77 <0.001 * Plasma NfL, pg/mL 14 ±9 11 ±6 15 ± 10 <0.001 *

28 able 2. Associations of plasma biomarkers with neuropsychological test performanc

Plasma Abeta 1-42/1-40 Plasma GFAP Plasma NfL

Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 sBeta (95% Cl) sBeta (95% Cl) sBeta (95% Cl) sBeta (95% Cl) sBeta (95% Cl) sBeta (95% Cl)bal cognition

MSE -0.22 (-0.34 - -0.09) -0.03 (-0.13 -0.07) -0.40 (-0.51 - -0.28) -0.12 (-0.23- -0.02) -0.35 (-0.48 - -0.23) -0.09 (-0.19-0.02)ntion

igit span forward -0.02 (-0.15-0.11) 0.06 (-0.07-0.18) -0.13 (-0.26 - 0.00) -0.03 (-0.17-0.11) -0.10 (-0.23-0.04) 0.00 (-0.14-0.14) MT A -0.21 (-0.34 - -0.08) -0.07 (-0.19-0.05) -0.36 (-0.48 - -0.23) -0.15 (-0.28 - -0.02) -0.31 (-0.44 - -0.17) -0.11 (-0.24-0.02)troop word naming -0.16 (-0.29 - -0.03) -0.03 (-0.14-0.09) -0.28 (-0.41 - -0.14) -0.07 (-0.20 - 0.07) -0.23 (-0.37 - -0.09) -0.03 (-0.17-0.10)troop color naming -0.08 (-0.21-0.06) 0.07 (-0.05-0.18) -0.30 (-0.43 - -0.16) -0.07 (-0.21-0.06) -0.21 (-0.35 - -0.07) 0.01 (-0.13-0.14) ory

AVLT immediate recall -0.12 (-0.25-0.01) 0.07 (-0.03-0.17) -0.28 (-0.41 - -0.16) 0.03 (-0.08-0.13) -0.30 (-0.43 - -0.18) -0.02 (-0.13-0.09) AVLT delayed recall -0.15 (-0.28 - -0.02) 0.04 (-0.06-0.14) -0.29 (-0.42 - -0.17) 0.02 (-0.09-0.13) -0.25 (-0.38 - -0.12) 0.05 (-0.06-0.16) AVLT recognition -0.15 (-0.28 - -0.02) 0.00 (-0.11-0.12) -0.24 (-0.37- -0.11) 0.02 (-0.10-0.15) -0.20 (-0.33 - -0.06) 0.05 (-0.07-0.18) AT A -0.19 (-0.32 - -0.06) -0.04 (-0.15-0.07) -0.28 (-0.42 - -0.15) -0.03 (-0.16-0.10) -0.19 (-0.33 - -0.06) 0.05 (-0.08-0.18)guage

AT naming -0.02 (-0.16-0.11) 0.06 (-0.07-0.20) -0.20 (-0.33 - -0.06) -0.09 (-0.24 - 0.06) -0.13 (-0.27-0.01) -0.02 (-0.17-0.13) ategory fluency animals -0.05 (-0.18-0.08) 0.13 (0.02-0.24) -0.31 (-0.44 - -0.18) -0.07 (-0.19-0.05) -0.19 (-0.33 - -0.06) 0.05 (-0.07-0.18)cutive functioning

igit span backward -0.09 (-0.22-0.03) 0.07 (-0.04-0.18) -0.18 (-0.31 - -0.04) 0.08 (-0.04-0.20) -0.15 (-0.29 - -0.02) 0.08 (-0.04-0.20) MT B -0.18 (-0.31- -0.05) 0.00 (-0.10-0.10) -0.36 (-0.49 - -0.24) -0.09 (-0.20-0.03) -0.34 (-0.47 - -0.21) -0.08 (-0.20 - 0.04)troop color word naming -0.07 (-0.20-0.06) 0.09 (-0.02-0.20) -0.33 (-0.46 - -0.19) -0.10 (-0.23 -0.03) -0.17 (-0.32 - -0.03) 0.05 (-0.08-0.18) etter fluency -0.01 (-0.14-0.11) 0.09 (-0.03-0.21) -0.24 (-0.37- -0.11) -0.11 (-0.25-0.03) -0.21 (-0.34 - -0.08) -0.09 (-0.22-0.05)

Table 3. AUC and sensitivity and specificity at Youden's cut-off to identify an abnormal amyloid PET scan in the total study population.

AUC (95%CI) Youden’s cut-off Sensitivity Specificity

Plasma Abeta 1-42/ 1-40 73% (66 - 81%) 0.16 70% 76%

Plasma GFAP 81 % (75 - 87%) 125 pg/mL 73% 79%

Plasma NfL 71 % (64 - 79%) 1 1.5 pg/mL 73% 64%

Model 88% (83 - 93%) 82% 86%

AUC with 95% confidence interval was calculated using receiver operator curve analysis. Youden's cut-off is specified as the cut-off at the maximum sum of sensitivity and specificity. Model includes the predicted values of the combined plasma Abetal-42/1-40 ratio, plasma GFAP, APOE e4 carriership and age panel, for which values were predicted using logistic regression analysis. Abeta = amyloid beta, GFAP = Glial fibrillary acidic protein, NfL = Neurofilament light, AUC = Area Under the Curve, 95%CI = 95% confidence interval.

Table 4. Pearson's correlations of plasma and CSF biomarkers for the total group.

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Claims

1. An analytic method for determining a cerebral Ab accumulation state in a test subject, the method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42/ Ab40 ratio and GFAP, to obtain measurement levels of Ab42, Ab40 and GFAP;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation, when, the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level of the test subject is higher than the standard level which is the GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation,

the method characterized in that the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI).

2. The method of claim 1 wherein the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subjective cognitive decline (SCD).

3. The method of claim 1 or 2 wherein the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline.

4. The method of any of the preceding claims wherein a decreased Ab42/ Ab40 ratio and an increased GFAP identifies/predicts neural damage and/or glial activation due to abnormal amyloid status.

5. A method for identifying a test subject as having or being at risk of development of dementia, the method comprising: • subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42/ Ab40 ratio and GFAP to obtain measurement levels of Ab42, Ab40 and GFAP;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level of the test object is higher than the standard level of GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation;

and, based thereon, indicate the test subject as having or being at risk of development of dementia if the cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function,

6. An analytical method for test subject stratification, the method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42/ Ab40 ratio and GFAP to obtain measurement levels of Ab42, Ab40 and GFAP;;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level of the test object is higher than the standard level of GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation; and based thereon, indicate the test subject as being:

• suitable for further examination; • suitable for treatment with a product acting against cerebral Ab accumulation; if the cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function,

7. An analytical method for determining efficacy of a medical intervention regarding a cerebral Ab accumulation state of a test subject, the method comprising conducting at least once before and at least once after the medical intervention an analytic method comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42/ Ab40 ratio and GFAP to obtain measurement levels of Ab42, Ab40 and GFAP;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level of the test object is higher than the standard level of GFAP level of the subject having normal cognitive function and being negative for cerebral Ab accumulation; and based thereon, indicate that the medical intervention in the test subject is efficient if cerebral Ab accumulation of the test object before therapeutic intervention is higher than after therapeutic intervention,

8. The method according to any one of claims 5 to 7 wherein the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurodegenerative disease showing clinical symptoms of subjective cognitive decline (SCD).

9. The method according to any one of claims 5 to 7 wherein the cerebral Ab accumulation state is determined in the test subject before the test subject shows any clinical symptoms of cognitive function decline.

10. The method according any of claims 1 to 9 wherein the marker comprises one or more additional analytes.

11. The method according to claim 10 wherein the one or more additional analytes are chosen from the group consisting of NfL and APOE s4 carriership.

12. The method according claim 11 comprising:

• subjecting a living body-derived sample derived from the test subject to a measuring step of a marker, said marker comprising the combination of the analytes Ab42/ Ab40 ratio, GFAP and NfL, to obtain measurement levels of Ab42, Ab40, GFAP and NfL;

• an evaluation step of determining that an amount of cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function who is negative for cerebral Ab accumulation when the ratio Ab42/ Ab40 of the test subject is lower than the standard levels which is the ratio Ab42/ Ab40 of the subject having normal cognitive function and being negative for cerebral Ab accumulation, and the GFAP level and NfL level of the test object is higher than the standard level of GFAP level and NfL level of the subject having normal cognitive function and being negative for cerebral Ab accumulation.

13. The method according to any of the preceding claims, wherein the measuring step measures the levels of each of the analytes Ab42, Ab40, GFAP, and, optionally NfL, using a digital ELISA.

14. The method according to any of the preceding claims , wherein the living body-derived sample is a blood, plasma or serum sample.

15. The method according to any of the preceding claims, wherein the living body-derived sample is obtained from a test subject at the very early stages of neurodegenerative disease; preferably Alzheimer's disease.

16. Use of Ab42, Ab40, and GFAP as a marker for determining a cerebral Ab accumulation state of a test subject wherein said test subject does not show any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI).

17. The use of claim 16 wherein a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject.

18. Use of Ab42, Ab40, and GFAP as a marker for identifying a test subject as having or being at risk of development of dementia, wherein said test subject does not show any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI).

19. The use of claim 18 wherein a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation in the test subject has or is at increased risk of development of dementia if the cerebral Ab accumulation of the test subject is larger than an amount of cerebral Ab accumulation of a subject having normal cognitive function.

20. Use of Ab42, Ab40, and GFAP as a marker for test subject stratification wherein said test subject does not show any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI).

21. The use of claim 20 wherein a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation indicates that the test subject is being suitable for further examination and/or being suitable for treatment with a product acting against cerebral Ab accumulation.

22. Use of Ab42, Ab40, and GFAP as a marker for determining the efficacy of a therapeutic intervention against cerebral Ab accumulation in a test subject wherein said test subject does not show any clinical symptoms of cognitive function decline or wherein the test subject is at the very early stages of a neurogenerative disease showing clinical symptoms of subjective cognitive decline (SCD) or mild cognitive decline (MCI).

23. The use according to claim 22 wherein a reduced ratio Ab42/ Ab40 of the test subject compared to the standard levels of the ratio Ab42/ Ab40 in a subject having normal cognitive function and being negative for cerebral Ab accumulation and an increased GFAP level in the test subject compared to the GFAP level of a subject having normal cognitive function and being negative for cerebral Ab accumulation are indicative for cerebral Ab accumulation in the test subject and wherein said increased cerebral Ab accumulation indicates that the test subject is being suitable to undergo the therapeutic intervention against the cerebral Ab accumulation.

24. The use according to any of the preceding claims 16 to 23 wherein one or more additional analytes are analyzed.

25. The use according to claim 24 wherein the one or more additional analytes are chosen from the group consisting of NfL and APOE s4 carriership.

26. The use according to claim 25 wherein an increased level of NfL in the test subject is additionally indicative for cerebral Ab accumulation.

27. The use according to any one of the claims 16 to 26 wherein the levels of each of the analytes Ab42, Ab40, GFAP, and, optionally NfL, are determined using a digital ELISA.

28. The use according to any one of the claims 16 to 27 , wherein the levels of each of the analytes Ab42, Ab40, GFAP, and, optionally NfL, are determined in a living body-derived sample of the test subject; preferably a living body-derive sample selected from a blood, plasma or serum sample.

29. The use according to claim 28, wherein the living body-derived sample is obtained from the test subject at the very early stages of neurodegenerative disease; preferably Alzheimer's disease.

30. A kit for use in a method for determining a cerebral Ab accumulation state of a test subject, or, in a method for early AD syndrome diagnosis, comprising:

a. monoclonal antibodies against Ab42, Ab40, GFAP and, optionally NfL;

b. instructions for use.