Biomarkers for diagnosing the subclinical phase of alzheimer's disease and uses thereof

Abstract

To provide molecular signatures of the asymptomatic phase of Alzheimer's disease, methods for diagnosing the asymptomatic phase of Alzheimer's disease in a subject, for predicting the progression of the asymptomatic phase of Alzheimer's disease and for determining a personalized course of treatment in a subject suffering from the asymptomatic phase of Alzheimer's disease, and a computer system comprising a machine learning algorithm trained to diagnose the asymptomatic phase.SOLUTION: The computer system comprises the steps of receiving inputs of the levels, amounts or concentrations of the at least five biomarkers as determined in a sample obtained from the subject, analyzing and transforming the inputs of the levels, amounts or concentrations of the at least five biomarkers by organizing and / or modifying each level, amount or concentration input via a machine learning algorithm to derive a probability score and / or a classification label, and generating an output which is a classification label or a probability score.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to molecular signatures of the subclinical stage of Alzheimer's disease and methods of using the same for diagnosing the subclinical stage of Alzheimer's disease in a subject, stratifying the subclinical stage of Alzheimer's disease in a subject into different subclinical stage grades, predicting the progression of the subclinical stage of Alzheimer's disease in a subject, and determining individualized courses of treatment in subjects affected by the subclinical stage of Alzheimer's disease. The present invention also relates to a computer system including a machine learning algorithm trained to diagnose the subclinical stage of Alzheimer's disease in a subject. [Background technology]

[0002] Alzheimer's disease (AD) is the most common cause of dementia in Western societies. In clinical terms, AD is characterized by progressive cognitive decline that usually begins with memory impairment. As the disease progresses, AD inevitably affects all intellectual functions, including executive function, leading to complete dependency for basic activities of daily living and premature death. Approximately 50 million people worldwide have AD, and the number of patients is estimated to skyrocket to 131.5 million by 2050 if no cure is found (Prince et al., 2015. World Alzheimer Report 2015. The global impact of dementia: An analysis of prevalence, incidence, cost and trends (Rep.). London: Alzheimer's disease international (ADI)).

[0003] The current cost of the disease is approximately US$1 trillion per year and is predicted to double by 2030. In the United States, out-of-pocket costs for families affected by AD average more than $8,000 per year, making AD the most expensive disease for families during the last 5 years of life (Kelley et al., 2013. J Gen Intern Med. 28(2):304-9). Unfortunately, although several drugs can alleviate the associated symptoms, there is no effective treatment for AD.

[0004] A century ago, Dr. Alois Alzheimer described the first patient with AD. Dr. Alois Alzheimer identified the cerebral lesions of this disease over a century ago (Shampo et al., 2013. Mayo Clin Proc. 88(12):e155). His patient, Auguste Deter, presented with progressive memory loss, thought disorder, disorientation, and personality changes. At the microscopic level, Dr. Alois Alzheimer identified two major cerebral aggregates associated with this disease: senile plaques and neurofibrillary tangles. However, it was not until 1984 that researchers discovered that the primary component of senile plaques was amyloid peptides derived from cleavage of the amyloid precursor protein (APP) (McKhann et al., 1984. Neurology. 34(7):939-44). Just a few years after these discoveries, neurofibrillary tangles were characterized as hyperphosphorylated Tau aggregates (Jellinger, 2006. J Neural Transm (Vienna). 113(11):1603-23). ​​These major discoveries marked the start of more than 30 years of intensive research.

[0005] Despite 30 years of intensive research, nearly 100% of clinical trials fail. To date, two key events in AD have been well established. AD is characterized by the progressive accumulation of beta-amyloid peptide (Aβ), which leads to gradual Tau hyperphosphorylation. As a result, patients present with a progressive cognitive decline followed by the deposition of senile plaques and the formation of fibrillary tangles. In the end-stage, dementia emerges in a sequence of events known as the "amyloid cascade" (Figure 1).

[0006] Neurological evaluation and concurrent diagnosis of patients are performed only after the first signs of dementia appear. Despite billions of dollars invested in R&D to find effective treatments, clinical trials for AD still have the lowest success rate of any disease area—less than 1% compared to 19% for cancer (Cummings et al., 2017. Alzheimer's Dement (NY). 3(3):367-384). This high failure rate is due to the fact that clinical trials target too late stages (i.e., dementia stages), a lack of basic knowledge of the disorder, and current animal models that do not fully replicate the human AD process. In particular, the pathophysiological link between APP processing (including soluble Aβ peptide production) and tau pathology remains challenging in AD animal models.

[0007] Thus, the lack of animal models that mimic key events observed in human AD raises the question of the validity of the modeling techniques used.

[0008] Without early diagnosis, there is no hope of salvation. Until recently, the diagnosis of AD was based exclusively on neuropsychological assessments. Despite recent advances in biomarkers, the sensitivity and specificity of these remains inadequate.

[0009] The first biological signs of the disease appear at least 20 years before clinical diagnosis (Figure 2). Therefore, diagnosis is established when most of the damage has occurred to the brain and when the patient already has severe dementia (Sperling et al., 2014. Neuron. 84(3):608-22), making the chances of successful treatment extremely low. However, it is impossible to identify biomarkers for asymptomatic AD from diagnosed AD patients. In fact, blood biomarkers evolve throughout the progression of the disease. Therefore, it is impossible to extrapolate variations during the presymptomatic phase based on variations from patients diagnosed with AD. This explains why identifying biomarkers from the asymptomatic phase is difficult and why scientists are unable to make early diagnoses.

[0010] Currently, most biomarkers under investigation are of three main types and are based on testing of patients diagnosed with AD: (1) Imaging of cerebral amyloid beta or blood Aβ 42 Measurement of; (2) cerebral tau imaging or blood tau measurement; or (3) Common biomarkers for all neurodegenerative disorders.

[0011] (1) Imaging of cerebral amyloid beta or blood Aβ 42 Measurement of For example, Dr. Koichi Tanaka and his group 42 Most amyloidogenic amyloid beta peptides (Aβ) in the blood are known to be present in very low concentrations. 42 They have developed a powerful technique to measure Aβ (a type of peptide) in the brain. This technique opens up a novel way to successfully identify people with cerebral amyloid-β plaque burden through a simple blood test. In the near future, they hope to overcome the costly and unsafe Aβ assay, which currently consists of in vivo imaging (PIB-PET) and cerebrospinal fluid biomarkers after lumbar puncture. 42 It is intended to replace peptide measurements.

[0012] However, this technique is severely limited with regard to its use as a suitable diagnostic tool for both subclinical and late-stage AD.

[0013] First, cerebral amyloid-β plaque burden is known to correlate poorly with AD status. In a paper (Nakamura et al., 2018. Nature. 554(7691):249-254), the authors acknowledged that "in the NCGG dataset, 9 of 29 patients (31%) were diagnosed with AD but were PIB-PET Aβ-negative" and "31 patients with AD (22 Aβ-negative) were PIB-PET Aβ-negative." + and 9 Aβ - , classified by PIB-PET) and 20 non-AD patients (8 Aβ + and 12 Aβ - ) cases.” In summary, approximately 30% of AD patients have PIB-PET Aβ - Approximately 40% of healthy individuals had PIB-PET Aβ + (Figure 3).

[0014] Nakamura et al. conclude that "these results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-β burden at the individual level," however, due to the lack of correlation between brain amyloid-β burden and AD status, this technique cannot accurately diagnose individuals with AD.

[0015] Second, this technique does not measure the outcome of the other major pathology involved in AD: tauopathy. For the same amount of amyloid-β in the brain, some individuals will develop AD (including the tauopathy portion) and some will not, depending on their individual susceptibility to amyloid-β toxicity. The more "responsive" an individual is to the toxicity of amyloid-β peptide, the higher their chance of developing AD, regardless of the amount of amyloid-β peptide (in the brain, cerebrospinal fluid, or blood).

[0016] (2) Imaging of cerebral tau or measurement of blood tau Cerebral tau burden is currently under investigation. However, due to the limited accuracy of tau imaging, aggregated tau is only visible in the later stages of progression, when the number of tangles is enormous. Tau imaging cannot be used as a biomarker in the asymptomatic stage.

[0017] Furthermore, due to their specific cellular localization, Tau and phospho-Tau can only be measured in the blood after neuronal death, thus constituting a late biomarker and cannot be used to detect patients during the asymptomatic phase of AD (long before atrophy appears).

[0018] (3) Common biomarkers for all neurodegenerative disorders All of these biomarkers have been identified primarily through a priori approaches. This approach limits the discovery of novel biomarkers unrelated to amyloid proteins, neurotrophic factors (NFTs), or neuroinflammatory biomarkers. It is important to note that blood levels of amyloid proteins correlate poorly with AD status (preventing their use as an AD diagnostic) and that both neurotrophic factors and neuroinflammatory processes are involved only in the clinical phase of AD. Again, these biomarkers are not relevant for detecting patients during the asymptomatic phase of AD. Furthermore, growth factor and neuroinflammatory biomarkers are insufficiently specific for AD and cannot be used as differential diagnostics for AD.

[0019] Therefore, to identify appropriate biomarkers for the asymptomatic phase, it is necessary to have a faithful model of AD that reproduces this asymptomatic phase. However, transgenic animal models do not match the human AD pathology.

[0020] The limitations of transgenic AD models reduce the specificity that would allow the development of diagnostics for the subclinical stages of AD. Most AD models used in laboratories are transgenic mice expressing human mutant genes associated with familial forms of AD (e.g., amyloid protein precursor [APP], presenilin-1 [PSEN1], and presenilin-2 [PSEN2]). Because each of these mutations results in increased Aβ production, these models are well-suited to rapidly mimic amyloid plaque deposition in a very short period of time. Furthermore, they are suitable models for developing appropriate positron emission tomography (PET) or magnetic resonance imaging (MRI) tracers to identify senile plaques or neurofibrillary tangles in patient brains.

[0021] However, these existing transgenic animal models have at least three major limitations.

[0022] First, several studies have shown that the development of AD features in transgenic mice is dependent on transgene expression. As a result, aging—the strongest risk factor for AD—is often ignored in AD studies because most mouse models exhibit AD-like phenotypes within just a few months. The first limitation is the fact that all of these mice develop accelerated aging, which is not similar to the human disease.

[0023] Second, genetic mutations in the MAPT gene (encoding the tau protein) have not been found in AD patients. Therefore, mouse models have been developed using MAPT mutations found in a subset of tauopathies to develop neurofibrillary tangles. Several cross-breeding studies have been performed to generate transgenic models that develop both amyloid and tau pathology, such as the 3xTg-AD mouse (Duyckaerts et al., 2008. Acta Neuropathol. 115(1):5-38). However, in human disease, both pathologies manifest independently: Aβ is the causative agent that triggers the amyloid cascade, which induces tau pathology. This amyloid cascade is not recapitulated in these mouse models, representing a second limitation.

[0024] Third, transgenes overexpressed in transgenic animals are not overexpressed in patients (except for the form of AD that occurs in patients with Down's syndrome), which explains why levels of neurotoxic peptides such as Aβ in these transgenic models are significantly higher than in the brains of AD patients (Audrain et al., 2016. Mol Neurodegener. 11:5). Thus, a final limitation is the supra-pathological concentrations of pathological metabolites expressed by transgenic AD models.

[0025] Additionally, other modeling strategies have been developed, such as injection-based animal models induced by intracerebral injection of amyloid or tau peptides directly into the brain (Puzzo et al., 2017. Elife. 6.pii:e2699). Again, similar limitations as transgenic models may be presented. Despite these limitations, existing AD animal models have provided a wealth of data leading to an understanding of neurological AD pathology and the evaluation of various potential therapeutic strategies. Overall, the research community is concerned about the lack of appropriate models. This lack of human-like AD models appears to be a limiting factor for the development of diagnostics (Lecanu & Papadopoulos, 2013. Alzheimers Res Ther. 5(3):17). In all cases, key factors, including aging, the impact of soluble Aβ peptides on tau pathology, and faithful clinical Aβ concentrations, remain challenges and should be addressed in the design of appropriate AD animal models.

[0026] Emergence of non-transgenic models that more closely resemble human pathology To mimic the progression of this disease in an in vivo model and in a manner that more closely replicates clinical observations, an innovative AD rat model, the AgenT rat, was recently developed by injecting adeno-associated viruses (AAVs) encoding human mutant APP protein and presenilin 1 (PS1) into the hippocampus of adult rodents (U.S. Pat. No. 10,159,227 and European Patent No. 3066203).

[0027] This model can be described as a disruptive technique and time course that more closely resembles the progression of AD in humans.

[0028] In fact, the technology used is not based on transgenic approaches. Because AD induction is performed only in adult animals, AgenT rats are not subject to developmental compensation or genetic drift. Furthermore, the pattern of APP expression in AgenT rats mimics the genetic mosaicism recently described in sporadic human AD, as increased copy number of the APP gene has been observed in limited neuronal subunits (Bushman et al., 2015. Elife. 4) and the occurrence of somatic mutations known to be associated with familial forms of Alzheimer's disease has been described (Lee et al., 2018. Nature. 563(7733):639-645). Therefore, AgenT rats may be considered a closer model of sporadic AD than transgenic animals.

[0029] Furthermore, the induced APP pathology was associated with amyloid peptide levels and Aβ 42 / 40The induced amyloid pathology leads to pathophysiological mechanisms including progressive Tau hyperphosphorylation. The slow progression of APP pathology allows for the progressive development of endogenous Tau pathology without the appearance of early interfering inflammation and plaque formation. These steps can be considered the asymptomatic phase of AD, which begins in patients at least 18 years before the current clinical diagnosis (Rajan et al., 2015. Neurology. 85(10):898-904). The next phase of AD disease progression consists of AD-related cerebral lesions, such as senile plaques, cerebral amyloid angiopathy, and tangle-like aggregates, which appear only in aged AgenT rats.

[0030] All these characteristics make the AgenT rat model a powerful tool for better predicting the behavior of blood biomarkers according to the stage of progression, and therefore constitute a suitable test system for characterizing novel biomarkers or panels of biomarkers for the development of early diagnostics.

[0031] In this sense, the inventors used artificial intelligence techniques to identify a panel of 119 best-in-class biomarkers suitable for predicting AD. Surprisingly, the inventors were able to demonstrate that an artificial neural network trained using data from AgenT rats (i.e., rats that have AD but are asymptomatic) and healthy rats was ultimately able to predict pre- or subclinical AD from a subset of approximately five or fewer biomarkers randomly taken from the complete list of 119 best-in-class biomarkers.

[0032] Furthermore, the inventors were surprisingly able to demonstrate that an artificial neural network trained using a random subset of these biomarkers, up to about five, can not only predict the asymptomatic phase of AD, but also further stratify asymptomatic AD into different grades. Summary of the Invention

[0033] The present invention relates to a molecular signature of the asymptomatic stage of Alzheimer's disease comprising at least five biomarkers selected from the group of biomarkers in Table 1A.

[0034] In one embodiment, the molecular signature of the asymptomatic stage of Alzheimer's disease comprises the biomarkers of Table 10A, Table 10B, Table 10C, or Table 10D.

[0035] The present invention further provides a method for diagnosing the asymptomatic stage of Alzheimer's disease in a subject, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers of Table 1A in a sample previously obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; c) diagnosing the subject as suffering from asymptomatic Alzheimer's disease based on a correlation between the reference signature and the molecular signature; The present invention relates to a method, including:

[0036] The present invention further provides a method for predicting the progression of subclinical phase of Alzheimer's disease in a subject, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers in Table 1A in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; c) predicting the progression of Alzheimer's disease based on the correlation between the reference signature and the molecular signature; The present invention relates to a method, including:

[0037] The present invention further provides a method for determining an individualized course of treatment for a subject suffering from the asymptomatic stage of Alzheimer's disease, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers in Table 1A in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; c) determining a personalized course of treatment for the subject based on the correlation between the reference signature and the molecular signature; The present invention relates to a method, including:

[0038] The present invention further provides a method for stratifying the subclinical stage of Alzheimer's disease in a subject into different subclinical stage grades, preferably S1, S2, or S3 grades, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers in Table 1A in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; c) stratifying the subject into subclinical grades of Alzheimer's disease based on the correlation between the reference signature and the molecular signature; The present invention relates to a method, including:

[0039] In certain embodiments of the method of stratifying a subject's subclinical stage of Alzheimer's disease into different subclinical stage grades, the molecular signature comprises at least 14 biomarkers selected from the group of biomarkers in Table 1A.

[0040] In one embodiment, the reference signature comprises the levels, amounts, or concentrations of the same at least five biomarkers measured in samples previously obtained from substantially healthy subjects, preferably measured in samples previously obtained from a population of substantially healthy subjects.

[0041] In one embodiment, the correlation in step c) is measured by comparing the variation in the level, amount, or concentration of at least five biomarkers in said molecular signature and said reference signature with the variation profile of biomarkers in Table 3.

[0042] In one embodiment, the molecular signature comprises the biomarkers of Table 10A, Table 10B, Table 10C, or Table 10D.

[0043] In one embodiment, the comparison in step b) is performed using at least one machine learning algorithm.

[0044] In one embodiment, the at least one machine learning algorithm is selected from the group comprising an artificial neural network (ANN), a perceptron algorithm, a deep neural network, a clustering algorithm, a k-nearest neighbors (k-NN), a decision tree algorithm, a random forest algorithm, a linear regression algorithm, a linear discriminant analysis (LDA) algorithm, a quadratic discriminant analysis (QDA) algorithm, a support vector machine (SVM), a Bayesian algorithm, a simple rule algorithm, a clustering algorithm, a meta-classifier algorithm, a Gaussian mixture model (GMM) algorithm, a nearest centroid algorithm, an extreme gradient boosting (XGBoost) algorithm, a linear mixed effects model algorithm, and combinations thereof.

[0045] In one embodiment, at least one machine learning algorithm is trained on a training dataset that includes information related to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A from samples previously obtained from substantially healthy subjects and subjects known to be suffering from the asymptomatic stage of Alzheimer's disease.

[0046] In one embodiment, at least one machine learning algorithm is trained with a training dataset comprising variation profiles of the biomarkers in Table 3.

[0047] The present invention further provides a computer system for diagnosing the asymptomatic stage of Alzheimer's disease in a subject, comprising: (i) at least one processor; (ii) when executed by said processor, a. receiving an input of levels, amounts, or concentrations of at least five biomarkers selected from the group of biomarkers in Table 1A determined in a sample previously obtained from said subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers by codifying and / or modifying each level, amount, or concentration input via a machine learning algorithm to derive a probability score and / or classification label; the machine learning algorithm is trained on a training dataset; the training dataset comprises information relating to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A from samples previously obtained from subjects with a known Alzheimer's disease status; Steps and c. Producing an output that is a classification label or a probability score; d. providing a diagnosis of the subject as having or not having asymptomatic Alzheimer's disease based on the output; and at least one storage medium storing at least one code readable by said processor that causes said processor to perform The present invention relates to a computer system including:

[0048] The present invention further provides a computer-implemented method for diagnosing a subclinical stage of Alzheimer's disease in a subject, comprising: a. receiving an input of levels, amounts, or concentrations of at least five biomarkers selected from the group of biomarkers in Table 1A determined in a sample previously obtained from said subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers by codifying and / or modifying each level, amount, or concentration input via a machine learning algorithm to derive a probability score and / or classification label; the machine learning algorithm is trained on a training dataset; the training dataset comprises information relating to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A from samples previously obtained from subjects with a known Alzheimer's disease status; Steps and c. Producing an output that is a classification label or a probability score; d. providing a diagnosis of the subject as having or not having asymptomatic Alzheimer's disease based on the output; and The present invention relates to a method, including:

[0049] In one embodiment, the training dataset includes information related to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A from samples previously obtained from substantially healthy subjects and subjects known to be suffering from the asymptomatic stage of Alzheimer's disease.

[0050] In one embodiment, providing a diagnosis in step d comprises providing a stratification of subjects suffering from asymptomatic Alzheimer's disease into a grade of said asymptomatic Alzheimer's disease, preferably into grades S1, S2, or S3.

[0051] In certain embodiments, where step d. comprises providing stratification, step a. comprises receiving an input of levels, amounts, or concentrations of at least 14 biomarkers selected from the group of biomarkers in Table 1A.

[0052] In one embodiment, the training dataset comprises variation profiles of the biomarkers in Table 3.

[0053] The present invention further relates to a computer program comprising software code readable by a processor adapted to perform the computer-implemented method according to the present invention when executed by said processor.

[0054] The present invention further relates to a non-transitory computer readable storage medium containing code which, when executed by a computer, causes a processor to perform the computer-implemented method of the present invention.

[0055] Detailed Description The present invention relates to a molecular signature or profile of the asymptomatic stage of Alzheimer's disease.

[0056] As used herein, the terms "silent phase / stage," "pre-dementia phase / stage," or "preclinical phase / stage" are used interchangeably when referring to Alzheimer's disease and refer to a preclinical state in a subject who is not cognitively impaired but who exhibits at least one of the following characteristics of Alzheimer's disease: dysregulation of soluble Aβ peptide, an increase in hyperphosphorylated Tau protein, and the appearance of senile plaques and fibrillary tangles. These terms encompass both the "silent phase" and the "prodromal phase" of Alzheimer's disease. The "silent phase" spans the period from the subject's first molecular event of Alzheimer's disease (i.e., dysregulation of Aβ peptide production or clearance) to the onset of the first clinical symptoms. For detailed definitions, see Dubois et al., 2016 (Alzheimers Dement. 12(3):292-323) or Sperling et al., 2011 (Alzheimers Dement. 7(3):280-292), the entire contents of which are incorporated herein by reference.

[0057] As used herein, the terms "asymptomatic" and "prodromal" are interchangeable when referring to Alzheimer's disease and refer to the preclinical state of subjects who are not cognitively impaired but who exhibit at least one of the following characteristics of Alzheimer's disease at the brain level: dysregulation of soluble Aβ peptides, an increase in hyperphosphorylated Tau protein, and possibly the appearance of senile plaques and fibrillary tangles. These subjects develop clinical symptoms of Alzheimer's disease after several years or decades (Hubbard et al., 1990. Neuropathol Appl Neurobiol. 16(2):111-21). At this stage of the pathology, cerebral changes are primarily molecular. Patients are effectively ill but do not exhibit any objective cognitive impairment. Biomarkers in cerebrospinal fluid (CSF) and PET imaging are usually negative.

[0058] As used herein, the terms "prodromal" and "mild cognitive impairment (MCI)" are used interchangeably to refer to Alzheimer's disease and describe the stage between the first cognitive abnormalities (associated with normal age-related cognitive decline) and the onset of dementia symptoms. This is characterized by problems with memory, language, thinking, or judgment, but without the symptoms of AD dementia. Cerebral concentrations of amyloid peptides tend to increase, while CSF concentrations tend to decrease. However, baseline levels vary from person to person. This explains why 32% of cognitively normal individuals test above the amyloid positivity threshold, while 35% to 52% of prodromal patients test negative (Landau, 2020 July 28. Imaging biomarkers and Alzheimer's disease prevention. Speech presented at the Alzheimer's Association International Conference (AAIC) 2020, online). Therefore, amyloid levels are not specific enough to identify Alzheimer's disease patients. At this stage, some patients begin to show an increased concentration of Tau protein both at the brain and peripheral level (CSF, blood), but this remains low and therefore does not allow the diagnosis of all prodromal Alzheimer's disease patients.

[0059] The terms "clinical signs," "clinical symptoms," "AD dementia," "Alzheimer's disease dementia," and "AD dementia symptoms," when referring to Alzheimer's disease, refer to symptoms ranging from, but not limited to, memory loss that disrupts daily life, difficulty posing or solving problems, difficulty completing familiar tasks at home, work, or leisure, confusion about time or place, difficulty understanding visual images and spatial relationships, novel problems with words in speech or writing, misplacing objects and losing the ability to retrace steps, diminished or poor judgment, withdrawal from work or social activities, or mood and personality changes. Such clinical signs are described, for example, on the Alzheimer's Association website: https: / / www.alz.org / alzheimers-dementia / 10_signs.

[0060] In one embodiment, a molecular signature or profile of the invention comprises biomarkers whose average profile of levels, amounts, or concentrations, when compared to a reference signature or profile, is characteristic of the asymptomatic stage of Alzheimer's disease.

[0061] "Characteristic of," when referring to a level, amount, or concentration of a biomarker, means that the level, amount, or concentration of a given biomarker—or the average profile of levels, amounts, or concentrations of the biomarker—is substantially different from or substantially similar to the level, amount, or concentration of the same biomarker—or the average profile of levels, amounts, or concentrations of the biomarker—from a reference subject. "Characteristic" should be understood as "substantially different from" or "substantially similar" depending on the reference subject and its disease state.

[0062] In one embodiment, the level, amount, or concentration of a given biomarker is "substantially different" if it is about more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more higher, or about more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more lower than the level, amount, or concentration of the same biomarker in a reference subject. In one embodiment, the level, amount, or concentration of a given biomarker is "substantially different" if it is more than about 5% higher or more than 5% lower than the level, amount, or concentration of the same biomarker in a reference subject.

[0063] In one embodiment, the level, amount, or concentration of a given biomarker is "substantially similar" if it is less than about 1% higher, 2% higher, 3% higher, 4% higher, 5% higher, 6% higher, 7% higher, 8% higher, 9% higher, 10% higher, 15% higher, 20% higher, or more higher than the level, amount, or concentration of the same biomarker in a reference subject; or less than about 1% lower, 2% lower, 3% lower, 4% lower, 5% lower, 6% lower, 7% lower, 8% lower, 9% lower, 10% lower, 15% lower, 20% lower, or more lower than the level, amount, or concentration of the same biomarker in a reference subject. In one embodiment, the level, amount, or concentration of a given biomarker is "substantially similar" if it is less than about 5% higher or less than 5% lower than the level, amount, or concentration of the same biomarker in a reference subject.

[0064] In one embodiment, the level, amount, or concentration of a biomarker can be measured by methods well known in the art, including, but not limited to, mass spectrometry (e.g., tandem mass spectrometry [MS / MS], chromatography-assisted mass spectrometry, and combinations thereof), immunohistochemistry, multiplex methods (Luminex), Western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), RT-PCR, RT-qPCR, Northern blot, hybridization techniques (e.g., using microarrays, and combinations thereof, including, but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing, e.g., next-generation DNA sequencing (NGS) or RNA-seq (also known as "whole transcriptome shotgun sequencing")), and the like.

[0065] In one embodiment, a molecular signature or profile of the invention comprises biomarkers whose level, amount, or concentration, when compared to a reference signature or profile, is characteristic of asymptomatic Alzheimer's disease grade S1, asymptomatic Alzheimer's disease grade S2, and / or asymptomatic Alzheimer's disease grade S3.

[0066] In one embodiment, the asymptomatic stage of Alzheimer's disease is defined as the asymptomatic stage of Alzheimer's disease grade S1. Thus, the present invention relates to a molecular signature or profile of the asymptomatic stage of Alzheimer's disease grade S1.

[0067] As used herein, the term "subclinical stage of grade S1 of Alzheimer's disease" or "grade S1" refers to a subclinical stage of Alzheimer's disease in which a subject does not exhibit clinical symptoms such as mild cognitive impairment (MCI) and dementia, but physiopathological characteristics are observable. Such physiopathological characteristics of grade S1 include the increase in cerebral soluble Aβ. 42Physiopathological characteristics of grade S1 do not include physiopathological characteristics of grade S2 and / or grade S3, as defined hereinbelow.

[0068] In one embodiment, the asymptomatic stage of Alzheimer's disease is defined as the asymptomatic stage of Alzheimer's disease grade S2. Thus, the present invention relates to a molecular signature or profile of the asymptomatic stage of Alzheimer's disease grade S2.

[0069] As used herein, the term "asymptomatic stage of grade S2 of Alzheimer's disease" or "grade S2" refers to a grade of asymptomatic stage of Alzheimer's disease in which a subject does not exhibit clinical symptoms such as mild cognitive impairment (MCI) and dementia, but physiopathological characteristics are observable. Such physiopathological characteristics of grade S2 include those of grade S1 plus soluble Aβ. 42 The physiopathological characteristics of grade S2 include at least one of peptide accumulation, Tau hyperphosphorylation, and accelerated forgetting. The physiopathological characteristics of grade S2 do not include the characteristics of grade S3, as defined herein below.

[0070] In one embodiment, the asymptomatic stage of Alzheimer's disease is defined as the asymptomatic stage of Alzheimer's disease grade S3. Thus, the present invention relates to a molecular signature or profile of the asymptomatic stage of Alzheimer's disease grade S3.

[0071] As used herein, the term "Alzheimer's disease grade S3 subclinical stage" or "grade S3" refers to a grade of Alzheimer's disease in which a subject does not exhibit clinical symptoms such as dementia, but physiopathological characteristics are observable. Such physiopathological characteristics of grade S3 include at least one of the characteristics of grade S1 and grade S2 plus increased hyperphosphorylated tau, senile plaques, fibrillary tangles, and mild or severe memory impairment. In some cases, mild cognitive impairment may be considered a symptom of grade S3.

[0072] Figure 1 summarizes these three subclinical grades of Alzheimer's disease.

[0073] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least one biomarker selected from the group of biomarkers in Table 1 A. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises one biomarker selected from the group of biomarkers in Table 1 A. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of one biomarker selected from the group of biomarkers in Table 1 A.

[0074] [Table 1] TIFF2026009931000003.tif132162

[0075] In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease does not include at least one biomarker, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 biomarkers selected from the group comprising or consisting of 1-methyladenosine, 3,4-dihydroxybutyrate, 3-amino-2-piperidone, 4-methyl-2-oxopentanoate, arabonate / xylonate, creatine, creatinine, cysteine-glutathione disulfide, dimethyl sulfone, erythronate, glucose, N-acetylalanine, sphingosine 1-phosphate, and tartronate (hydroxymalonate).

[0076] As used herein, the term "14-3-3 protein" refers to any one or more of the following proteins: 14-3-3 protein β / α, 14-3-3 protein γ, 14-3-3 protein ε, 14-3-3 protein ζ / δ, 14-3-3 protein η, and 14-3-3 protein θ.

[0077] As used herein, the term "apolipoprotein" refers to any one or more of the following proteins: apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein CI, apolipoprotein C-II (predicted), apolipoprotein C-III, apolipoprotein C-IV, apolipoprotein D, apolipoprotein E, rat apolipoprotein E protein, apolipoprotein H (beta-2-glycoprotein I), apolipoprotein M, and apolipoprotein N.

[0078] As used herein, the term "Arp2 / 3 complex protein" refers to any one or more of the following proteins: actin-related protein 2, actin-related protein 2 / 3 complex subunit 1B, actin-related protein 2 / 3 complex subunit 3, actin-related protein 2 / 3 complex subunit 4, actin-related protein 2 / 3 complex subunit 5, actin-related protein 3, and arp2 / 3 complex 34 kDa subunit.

[0079] As used herein, the term "carboxylesterase 1 family" refers to any one or more of the following proteins: carboxylesterase 1, carboxylesterase 1C, and carboxylesterase 1E.

[0080] As used herein, the term "carnitine and conjugates" refers to the following molecules: 2-methylbutyrylcarnitine (C5), acetylcarnitine (C2), arachidonoylcarnitine (C20:4), butyrylcarnitine (C4), carnitine, cis-4-decenoylcarnitine (C10:1), isobutyrylcarnitine (C4), isovalerylcarnitine (C5), laurylcarnitine (C12), linoleic acid, ... It represents any one or more of leoylcarnitine (C18:2), myristoylcarnitine (C14), octanoylcarnitine (C8), oleoylcarnitine (C18), palmitoleoylcarnitine (C16:1), palmitoylcarnitine (C16), propionylcarnitine (C3), stearoylcarnitine (C18), (S)-3-hydroxybutyrylcarnitine, and deoxycarnitine.

[0081] As used herein, the term "cholates and conjugates" refers to any one or more of the following molecules: chenodeoxycholate, cholate, deoxycholate, glycocholate, taurochenodeoxycholate, taurocholate, and taurodeoxycholate.

[0082] As used herein, the term "clotting factor family" refers to any one or more of the following proteins: clotting factor V, clotting factor IX, clotting factor VII, clotting factor X, clotting factor XI, clotting factor XII, clotting factor XIII A chain, and clotting factor XIII B chain.

[0083] As used herein, the term "complement system family" refers to any one or more of the following proteins: complement factor B, complement C1q small component subunit A, complement C1q small component subunit B, complement C1q small component subunit C, complement C1r small component, complement C1r small component-like protein, complement C1s small component, complement C1s small component, complement C2, complement C3, complement C4, complement C4A, complement C4B, C4B binding protein α chain, C4B binding protein β chain, complement C4-like, complement C5, complement C6, complement C7, complement C8 α chain, complement component C8 β chain, complement C8 γ chain, complement component C9, complement factor D, complement factor H, complement factor H-related protein, complement factor H-related protein 1, complement factor H-related protein 2, complement factor H-related protein 3, complement factor H-related protein 4, and complement factor I.

[0084] As used herein, the term "creatine kinase family" refers to any one or more of the following proteins: creatine kinase type B and creatine kinase type M.

[0085] As used herein, the term "globin family" refers to any one or more of the following proteins: globin a2, globin a4, globin c2, globin c3, globin d1, haptoglobin, haptoglobin-related protein, hemoglobin subunit alpha, hemoglobin subunit beta, hemoglobin subunit delta, and myoglobin.

[0086] As used herein, the term "globulin family" refers to the following proteins: alpha-2 antiplasmin, murinoglobulin-2, vitamin K-dependent protein C, serum albumin, angiotensinogen, murinoglobulin-1, Ig kappa chain C, Igh-6 protein, alpha-2-macroglobulin, murinoglobulin-1, complement factor properdin, haptoglobin, beta-2-microglobulin, ceruloplasmin, serotransferrin, and proteins similar to immunoglobulin kappa-chain Any one or more of VK-1, serine (or cysteine) proteinase inhibitor clade A member 4, alpha-2-macroglobulin, IgG-2a protein, prothrombin, alpha-1-macroglobulin, serum albumin, thyroxine-binding globulin, immunoglobulin heavy chain variable region, corticosteroid-binding globulin, Ig heavy chain V region IR2, murinoglobulin-2, Ig ​​gamma-2 B chain C region, Igh-6 protein, Ig lambda-2 chain C region, Ig delta chain C region, Ig gamma-2 C chain C region, Igh-6 protein, immunoglobulin J chain, Ig kappa chain V region S211, serum amyloid A-1 protein, serum amyloid A-2 protein, serum amyloid A-4 protein, and serum amyloid A protein are represented.

[0087] As used herein, the term "kininogen family" refers to any one or more of the following proteins: kininogen, kininogen 1, and T-kininogen 2.

[0088] As used herein, the term "lysine and conjugates" refers to the following molecules: 5-hydroxylysine, fructosyl lysine, γ-glutamyl-α-lysine, lysine, N 6 ,N 6 ,N 6 -trimethyllysine, N 6 -acetyllysine, N 6-methyllysine, N,N,N-trimethyl-5-aminovalerate, and pipecolate.

[0089] As used herein, the term "proteasome complex family" refers to any one or more of the following molecules: proteasome subunit alpha type, proteasome subunit alpha type-7, proteasome subunit alpha type-1, proteasome subunit alpha type-2, proteasome subunit alpha type-3, proteasome subunit alpha type-4, proteasome subunit alpha type-6, proteasome subunit beta, proteasome subunit beta type, proteasome subunit beta type-1, proteasome subunit beta type-10, and proteasome subunit beta type-3.

[0090] As used herein, the term "serpin superfamily member" refers to any one or more of the following proteins: alpha-1-antiproteinase, heparin cofactor II, plasma protease C1 inhibitor, protein Z-dependent protease inhibitor, serine (or cysteine) peptidase inhibitor clade B member 10, serine (or cysteine) peptidase inhibitor clade B member 6a, serine (or cysteine) peptidase inhibitor clade C member 1, serine protease inhibitor A3C, serine protease inhibitor A3F, serine protease inhibitor A3K, serine protease inhibitor A3L, serine protease inhibitor A3M, serine protease inhibitor A3N, serine protease inhibitor Kazal-3-like, serpin A11, serpin family F member 2, and thyroxine-binding globulin.

[0091] As used herein, the term "valerate and conjugates" refers to any one or more of the following molecules: 2,3-dihydroxyisovalerate, 2-hydroxy-3-methylvalerate, 3-methyl-2-oxovalerate, α-hydroxyisovalerate, β-hydroxyisovalerate, and N,N,N-trimethyl-5-aminovalerate.

[0092] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least one biomarker selected from the group of biomarkers in Table 1 B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises one biomarker selected from the group of biomarkers in Table 1 B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of one biomarker selected from the group of biomarkers in Table 1 B.

[0093] [Table 2] TIFF2026009931000005.tif241160TIFF2026009931000006.tif242161TIFF2026009931000007.tif237162

[0094] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least one biomarker selected from the group of biomarkers in Table 2A. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises one biomarker selected from the group of biomarkers in Table 2A. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of one biomarker selected from the group of biomarkers in Table 2A.

[0095] [Table 3] TIFF2026009931000009.tif27162

[0096] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least one biomarker selected from the group of biomarkers in Table 2B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises one biomarker selected from the group of biomarkers in Table 2B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of one biomarker selected from the group of biomarkers in Table 2B.

[0097] [Table 4] TIFF2026009931000011.tif33160

[0098] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least one biomarker selected from the group of biomarkers in Table 2C. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises one biomarker selected from the group of biomarkers in Table 2C. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of one biomarker selected from the group of biomarkers in Table 2C.

[0099] [Table 5] TIFF2026009931000013.tif33162

[0100] In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0101] In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0102] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least two biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises two biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of two biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0103] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least two biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises two biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of two biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0104] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least three biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises three biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of three biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0105] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least three biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises three biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of three biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0106] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least four biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises four biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of four biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0107] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least four biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises four biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of four biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0108] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least five biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises five biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of five biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0109] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least five biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises five biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of five biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0110] In one exemplary and non-limiting embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises or consists of at least five biomarkers selected from the following five biomarkers: fructosyl lysine, integrin beta, isobutyrylcarnitine (C4), myosin regulatory light chain RLC-A, and talin 2.

[0111] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least six biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises six biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of six biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0112] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least six biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises six biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of six biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0113] In one exemplary and non-limiting embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease includes or consists of at least six biomarkers selected from the following six biomarkers: fructosyl lysine, Igh-6 protein, myosin regulatory light chain RLC-A, octadecandioate (C18), ribonate (ribonolactone), and talin 2.

[0114] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least seven biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises seven biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of seven biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0115] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least seven biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises seven biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of seven biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0116] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least eight biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises eight biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of eight biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0117] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least eight biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises eight biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of eight biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0118] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least nine biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises nine biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of nine biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0119] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least nine biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises nine biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of nine biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0120] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 10 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 10 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 10 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0121] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 10 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 10 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 10 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0122] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 11 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 11 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 11 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0123] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 11 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 11 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 11 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0124] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 12 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 12 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 12 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0125] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 12 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 12 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 12 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0126] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 13 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 13 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 13 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0127] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 13 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 13 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 13 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0128] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 14 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 14 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 14 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0129] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 14 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 14 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 14 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0130] In one exemplary and non-limiting embodiment, the molecular signature or profile of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease comprises or consists of at least 14 biomarkers selected from the following 14 biomarkers: 10 kDa heat shock protein, mitochondrial; 5-hydroxylysine; adenylate kinase 4, mitochondrial; calreticulin; creatine kinase type B; ergothioneine; peptidyl-prolyl cis-trans isomerase FKBP1A; fructosyl lysine; globin c2; integrin subunit αV; myoglobin; retinoic acid receptor responder 2; Tmprss13 protein, and transferrin receptor protein 1.

[0131] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 15 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 15 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 15 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0132] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 15 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 15 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 15 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0133] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 16 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 16 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 16 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0134] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 16 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 16 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 16 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0135] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 17 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 17 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 17 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0136] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 17 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 17 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 17 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0137] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 18 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 18 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 18 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0138] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 18 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 18 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 18 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0139] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 19 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 19 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 19 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0140] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 19 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 19 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 19 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0141] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 20 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 20 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 20 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0142] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 20 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 20 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 20 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0143] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 21 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 21 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 21 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0144] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 21 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 21 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 21 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0145] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 22 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 22 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 22 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0146] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 22 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 22 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 22 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0147] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 23 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 23 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 23 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0148] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 23 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 23 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 23 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0149] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 24 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 24 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 24 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0150] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 24 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 24 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 24 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0151] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 25 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 25 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 25 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0152] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 25 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 25 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 25 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0153] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 26 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 26 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 26 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0154] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 26 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 26 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 26 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0155] In one exemplary and non-limiting embodiment, the molecular signature or profile of the subclinical stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease includes the following 26 biomarkers: rat apolipoprotein E protein; Arp2 / 3 complex 34 kDa subunit; carnitine; isobutyrylcarnitine (C4); isovalerylcarnitine (C5); coagulation factor VII; serine (or cysteine) proteinase inhibitor clade A member 4; Igh-6 protein; serum amyloid P component; allantoic acid; carpa The present invention relates to a method for treating inflammatory bowel disease, comprising administering to a subject a therapeutic target for ...

[0156] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 27 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 27 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 27 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0157] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 27 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 27 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 27 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0158] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 28 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 28 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 28 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0159] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 28 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 28 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 28 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0160] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 29 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 29 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 29 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0161] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 29 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 29 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 29 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0162] In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 30 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 30 biomarkers selected from the group of biomarkers in Table 1A or Table 1B. In one embodiment, the molecular signature or profile of the asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 30 biomarkers selected from the group of biomarkers in Table 1A or Table 1B.

[0163] In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises at least 30 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 comprises 30 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. In one embodiment, a molecular signature or profile of an asymptomatic stage of Alzheimer's disease of grade S1, grade S2, and / or grade S3 consists of 30 biomarkers selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C.

[0164] In one embodiment, a determination as to whether a given biomarker level, amount, or concentration, or an average profile of biomarker levels, amounts, or concentrations, is characteristic of the asymptomatic stages of Alzheimer's disease at grades S1, S2, and / or S3, is made by comparison to a reference signature or profile, which may be implemented in software, or an overall median or other arithmetic mean across measurements may be constructed.

[0165] In one embodiment, a reference signature or profile may be compared to a signature or profile derived from a study of a population including, but not limited to, subjects having a similar age range, subjects of the same or similar ethnic group, similar cancer history, etc.

[0166] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 1A or Table 1B in reference samples derived from or obtained from one or more reference subjects.

[0167] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 2A, Table 2B, or Table 2C in reference samples derived from or obtained from one or more reference subjects.

[0168] In one embodiment, the reference subject is an animal, preferably a mammal.

[0169] Examples of mammals include, but are not limited to, humans, non-human primates (such as chimpanzees and other ape and monkey species), farm animals (such as cows, horses, sheep, goats, and pigs), domestic animals (such as rabbits, dogs, and cats), laboratory animals (such as rats, mice, and guinea pigs), etc. The term does not denote a particular age or sex unless otherwise specified.

[0170] In one embodiment, the reference subject is a primate, including humans and non-human primates, hi one embodiment, the reference subject is a human.

[0171] In one embodiment, the reference subject is a substantially healthy subject.

[0172] As used herein, a "substantially healthy subject" refers to a subject that has not been previously diagnosed or identified as having or suffering from Alzheimer's disease. Preferably, a "substantially healthy subject" refers to a subject that has not been previously diagnosed or identified as having or suffering from an asymptomatic stage of Alzheimer's disease. Preferably, a "substantially healthy subject" refers to a subject that has not been previously diagnosed or identified as having or suffering from any of Alzheimer's disease-related mild cognitive impairment (MCI), Alzheimer's type dementia, grade S1 physiopathological characteristics, grade S2 physiopathological characteristics, and grade S3 physiopathological characteristics, as defined hereinabove.

[0173] In one embodiment, the reference subject is a subject who has never been diagnosed or identified as having or suffering from Alzheimer's disease, either before or after death.In one embodiment, the reference subject is a subject who has never been diagnosed or identified as having or suffering from an asymptomatic stage of Alzheimer's disease, either before or after death.Preferably, the reference subject is a subject who has never been diagnosed or identified as having any of Alzheimer's disease-related mild cognitive impairment (MCI), Alzheimer's dementia, physiopathological characteristics of grade S1, physiopathological characteristics of grade S2, and physiopathological characteristics of grade S3, as defined herein above, either before or after death.

[0174] In one embodiment, the reference signature or profile is derived from measurements of the levels, amounts, or concentrations of the biomarkers of Table 1A or Table 1B in reference samples derived from or obtained from reference subjects of a reference population.

[0175] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 2A, Table 2B, or Table 2C in reference samples derived from or obtained from reference subjects of a reference population.

[0176] In one embodiment, the reference population comprises substantially healthy subjects as defined herein above, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 substantially healthy subjects.

[0177] In one embodiment, the reference population includes subjects who have never been diagnosed or identified as having or suffering from Alzheimer's disease, either pre- or post-mortem, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 subjects who have never been diagnosed or identified as having or suffering from Alzheimer's disease, either pre- or post-mortem. In one embodiment, the reference population includes subjects who have never been diagnosed or identified as having an asymptomatic phase of Alzheimer's disease, either pre- or post-mortem, preferably at least 50, more preferably at least 100, more preferably at least 200, and even more preferably at least 500 subjects who have never been diagnosed or identified as having an asymptomatic phase of Alzheimer's disease, either pre- or post-mortem. In one embodiment, the reference population comprises subjects who have never been diagnosed or identified, either before or after death, as having mild cognitive impairment (MCI), dementia, physiopathological characteristics of grade S1, physiopathological characteristics of grade S2, and physiopathological characteristics of grade S3 as defined herein above, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 subjects who have never been diagnosed or identified, either before or after death, as having mild cognitive impairment (MCI), dementia, physiopathological characteristics of grade S1, physiopathological characteristics of grade S2, and physiopathological characteristics of grade S3 as defined herein above.

[0178] In one embodiment, the reference subject is a subject of grade S1.

[0179] As used herein, a "subject of grade S1" has been previously diagnosed or identified as having or suffering from a subclinical stage of grade S1 of Alzheimer's disease. Preferably, a "subject of grade S1" has not been previously diagnosed or identified as having or suffering from a subclinical stage of grade S2 or S3 of Alzheimer's disease, or has not been diagnosed or identified. Preferably, a "subject of grade S1" has been previously diagnosed or identified as having physiopathological characteristics of grade S1, but has not been previously diagnosed or identified as having physiopathological characteristics of grade S2 and physiopathological characteristics of grade S3 as defined herein above, and has not been previously diagnosed or identified as having mild cognitive impairment (MCI) and dementia.

[0180] In one embodiment, the subject with grade S1 is an animal, preferably a mammal.

[0181] Examples of mammals include, but are not limited to, humans, non-human primates (such as chimpanzees and other ape and monkey species), farm animals (such as cows, horses, sheep, goats, and pigs), domestic animals (such as rabbits, dogs, and cats), laboratory animals (such as rats, mice, and guinea pigs), etc. The term does not denote a particular age or sex.

[0182] In one embodiment, the reference subject is a subject who has been previously diagnosed or identified, ante- or post-mortem, as having or suffering from a subclinical stage of Alzheimer's disease grade S1.

[0183] In one embodiment, the reference signature or profile is derived from measurements of the levels, amounts, or concentrations of the biomarkers of Table 1A or Table 1B in reference samples derived from or obtained from reference subjects of a reference population.

[0184] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 2A, Table 2B, or Table 2C in reference samples derived from or obtained from reference subjects of a reference population.

[0185] In one embodiment, the reference population comprises at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 subjects of grade S1 as defined herein above.

[0186] In one embodiment, the reference population includes at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 deceased subjects who have been previously diagnosed or identified as having or suffering from a subclinical phase of Alzheimer's disease grade S1 before or after death, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 deceased subjects who have been previously diagnosed or identified as having or suffering from a subclinical phase of Alzheimer's disease grade S1 before or after death.

[0187] In one embodiment, the reference subject is a subject of grade S2.

[0188] As used herein, a "subject of grade S2" refers to a subject who has previously been diagnosed or identified as having or suffering from an asymptomatic stage of grade S2 of Alzheimer's disease. Preferably, a "subject of grade S2" has not been previously diagnosed or identified as having or suffering from an asymptomatic stage of grade S3 of Alzheimer's disease, or has not been diagnosed or identified. Preferably, a "subject of grade S2" refers to a subject who has previously been diagnosed or identified as having physiopathological characteristics of grade S2, as defined hereinabove, but has not previously been diagnosed or identified as having physiopathological characteristics of grade S3, and has not previously been diagnosed or identified as having Alzheimer's disease-related mild cognitive impairment (MCI) or Alzheimer's type dementia.

[0189] In one embodiment, the subject with grade S2 is an animal, preferably a mammal.

[0190] Examples of mammals include, but are not limited to, humans, non-human primates (such as chimpanzees and other ape and monkey species), farm animals (such as cows, horses, sheep, goats, and pigs), domestic animals (such as rabbits, dogs, and cats), laboratory animals (such as rats, mice, and guinea pigs), etc. The term does not denote a particular age or sex.

[0191] In one embodiment, the reference subject is a subject who has been previously diagnosed or identified as having or suffering from a subclinical phase of Alzheimer's disease grade S2, either antemortem or postmortem.

[0192] In one embodiment, the reference signature or profile is derived from measurements of the levels, amounts, or concentrations of the biomarkers of Table 1A or Table 1B in reference samples derived from or obtained from reference subjects of a reference population.

[0193] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 2A, Table 2B, or Table 2C in reference samples derived from or obtained from reference subjects of a reference population.

[0194] In one embodiment, the reference population comprises subjects of grade S2 as defined herein above, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 subjects of grade S2.

[0195] In one embodiment, the reference population includes at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 deceased subjects who have been previously diagnosed or identified as having or suffering from an asymptomatic phase of Alzheimer's disease grade S2, either before or after death.

[0196] In one embodiment, the referent subject is a subject of grade S3.

[0197] As used herein, a "subject of grade S3" refers to a subject who has previously been diagnosed or identified as having or suffering from an asymptomatic stage of grade S3 of Alzheimer's disease. Preferably, a "subject of grade S3" refers to a subject who has previously been diagnosed or identified as having physiopathological characteristics of grade S3, but has not previously been diagnosed or identified as having Alzheimer's dementia. In some cases, a subject with Alzheimer's disease-related MCI may be considered a subject of grade S3.

[0198] In one embodiment, the subject with grade S3 is an animal, preferably a mammal.

[0199] Examples of mammals include, but are not limited to, humans, non-human primates (such as chimpanzees and other ape and monkey species), farm animals (such as cows, horses, sheep, goats, and pigs), domestic animals (such as rabbits, dogs, and cats), laboratory animals (such as rats, mice, and guinea pigs), etc. The term does not denote a particular age or sex.

[0200] In one embodiment, the reference subject is a subject who has been previously diagnosed or identified as having or suffering from a subclinical phase of Alzheimer's disease grade S3, either antemortem or postmortem.

[0201] In one embodiment, the reference signature or profile is derived from measurements of the levels, amounts, or concentrations of the biomarkers of Table 1A or Table 1B in reference samples derived from or obtained from reference subjects of a reference population.

[0202] In one embodiment, the reference signature or profile is derived from measuring the levels, amounts, or concentrations of the biomarkers of Table 2A, Table 2B, or Table 2C in reference samples derived from or obtained from reference subjects of a reference population.

[0203] In one embodiment, the reference population comprises subjects of grade S3 as defined herein above, preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 subjects of grade S3.

[0204] In one embodiment, the reference population includes at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, and even more preferably at least 500 deceased subjects who have been previously diagnosed or identified as having a subclinical phase of or suffering from Alzheimer's disease grade S3, either pre- or post-mortem.

[0205] By implication of a large number of samples from the reference population, it is possible to envision calculating the median and / or mean level, amount, or concentration of each biomarker in Table 1A or Table IB (or Table 2A, Table 2B, or Table 2C), or alternatively constructing a reference signature or profile of the biomarkers at the levels, amounts, or concentrations in Table 1A or Table IB (or at the levels, amounts, or concentrations in Table 2A, Table 2B, or Table 2C). In connection with these results, each level, amount, or concentration of a given biomarker, or alternatively each reference signature or profile of the biomarker levels, amounts, or concentrations, can be monitored if they are substantially different (such as substantially higher or lower) or substantially similar.

[0206] In one embodiment, reference signature or profile is constructed using statistical and structural classification algorithms and other methods.The samples from reference population are used to calculate the average profile of at least one biomarker selected from the group of biomarkers in Table 1A or Table 1B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more biomarkers.In one embodiment, reference signature or profile is constructed using statistical and structural classification algorithms and other methods. Samples from the reference population are used to compute average profiles for at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C. These reference signatures or profiles are computed for four reference groups: (1) healthy subjects, (2) subjects with grade S1, (3) subjects with grade S2, and (4) subjects with grade S4, and are subsequently referred to as "group centroids."

[0207] In one embodiment, the centroid is centered. In one embodiment, the centroid is scaled by the biomarker. In one embodiment, the centroid is centered and scaled by the biomarker.

[0208] Cancer class prediction from gene expression profiling based on centroid classification is a technique well known to those skilled in the art.For example, see Tibshirani et al., 2002. Proc Natl Acad Sci US A. 99(10):6567-72; Dabney, 2005. Bioinformatics. 21(22):4148-54; and Shen et al., 2009. J Biomed Inform. 42(1):59-65.

[0209] In one embodiment, a molecular signature or profile of the invention is characteristic of the asymptomatic stage of grade S1, grade S2, and / or grade S3 Alzheimer's disease when the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 1A or Table IB (or Table 2A, Table 2B, or Table 2C), varies as set forth in Table 3 when compared to a reference signature or profile derived from or obtained from a substantially healthy subject.

[0210] [Table 6] TIFF2026009931000015.tif241162TIFF2026009931000016.tif241160TIFF2026009931 000017.tif241160TIFF2026009931000018.tif241161TIFF2026009931000019.tif24116 2TIFF2026009931000020.tif241160TIFF2026009931000021.tif241161TIFF2026009931 000022.tif240162TIFF2026009931000023.tif241161TIFF2026009931000024.tif87162

[0211] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic grade S1 Alzheimer's disease if it has a substantially lower (i.e., more than 5% lower) level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 4A when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0212] [Table 7] TIFF2026009931000026.tif206162

[0213] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic grade S1 Alzheimer's disease if the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 4B when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0214] [Table 8] TIFF2026009931000028.tif57162

[0215] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S1 if the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 biomarkers selected from the group of biomarkers in Table 4C is substantially similar (i.e., no more than 5% lower or higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0216] [Table 9]

[0217] In one embodiment, the molecular signature or profile of the present invention comprises: the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 4A, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects, the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 4B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially higher (i.e., more than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 4C, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 biomarkers, when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects, is substantially similar (i.e., no more than 5% higher or lower); This is specific to grade S1, the asymptomatic stage of Alzheimer's disease.

[0218] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S2 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5A, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0219] [Table 10] TIFF2026009931000031.tif132162

[0220] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S2 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially elevated (i.e., more than 5% elevated) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0221] [Table 11] TIFF2026009931000033.tif120162

[0222] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S2 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5C, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 biomarkers, when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects, is substantially similar (i.e., no more than 5% higher or lower).

[0223] [Table 12]

[0224] In one embodiment, the molecular signature or profile of the present invention comprises: the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5A, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially higher (i.e., more than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 5C, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 biomarkers, when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects, is substantially similar (i.e., no more than 5% higher or lower); It is specific to grade S2, the asymptomatic stage of Alzheimer's disease.

[0225] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S3 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 6A, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0226] [Table 13] TIFF2026009931000036.tif166162

[0227] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S3 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 6B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially elevated (i.e., more than 5% elevated) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0228] [Table 14] TIFF2026009931000038.tif79162

[0229] In one embodiment, a molecular signature or profile of the invention is characteristic of asymptomatic Alzheimer's disease grade S3 if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 6C, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 biomarkers, is substantially similar (i.e., no more than 5% higher or lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects.

[0230] [Table 15]

[0231] In one embodiment, the molecular signature or profile of the present invention comprises: if the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 6A, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; the level, amount, or concentration of at least one biomarker selected from the group of biomarkers in Table 6B, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers, is substantially higher (i.e., more than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 biomarkers selected from the group of biomarkers in Table 6C, is substantially similar (i.e., no more than 5% higher or lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects; It is specific to the asymptomatic stage of Alzheimer's disease, grade S3.

[0232] The present invention also relates to a method of diagnosing the asymptomatic stage of Alzheimer's disease in a subject in need thereof using the molecular signature or profile of the present invention.

[0233] The present invention also relates to a method for stratifying the subclinical stage of Alzheimer's disease in a subject into grades, preferably S1, S2, or S3, using the molecular signatures or profiles of the present invention.

[0234] The present invention also relates to methods of predicting the progression of subclinical stages of Alzheimer's disease in a subject using the molecular signatures or profiles of the present invention.

[0235] In one embodiment, the method of the present invention comprises the step of providing a sample from a subject.

[0236] The term "sample," as used herein, generally refers to any sample that can be tested for the expression level of a biomarker, preferably a biomarker selected from the group of biomarkers in Table 1A or Table 1B (or Table 2A, Table 2B, or Table 2C).

[0237] In one embodiment, the method of the present invention comprises the step of providing a sample from a subject.

[0238] In one embodiment, the sample is a body tissue or fluid sample.

[0239] In one embodiment, the sample is a body tissue sample. Examples of body tissues include, but are not limited to, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testes, prostate, ovaries, hair, skin, bone, breast, uterus, bladder, and spinal cord.

[0240] In a preferred embodiment, the sample is not a body tissue sample.

[0241] In a preferred embodiment, the sample is a bodily fluid, including, but not limited to, blood, plasma, serum, lymph, ascetic fluid, cyst fluid, urine, bile, nipple exudate, synovial fluid, bronchoalveolar lavage fluid, sputum, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, semen, saliva, sweat, feces, stool, and alveolar macrophages.

[0242] In a preferred embodiment, the sample is a bodily fluid selected from the group comprising or consisting of blood, plasma, and serum.

[0243] In a preferred embodiment, the sample is not a sample of cerebrospinal fluid.

[0244] In a preferred embodiment, the sample is not feces or stool.

[0245] In one embodiment, the sample has been previously taken from the subject, i.e. the method of the invention does not include a step of retrieving the sample from the subject; consequently, in this embodiment the method of the invention is a non-invasive method or "in vitro method".

[0246] In one embodiment, the method of the invention comprises determining a molecular signature or profile according to the invention of a subject in a sample from said subject.

[0247] In one embodiment, the step of determining the subject's molecular signature or profile comprises the substep of measuring the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, selected from the group of biomarkers in Table 1A or Table 1B described herein above.

[0248] In one embodiment, the step of determining the subject's molecular signature or profile comprises the substep of measuring the level, amount, or concentration of at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more, selected from the group of biomarkers in Table 2A, Table 2B, or Table 2C described herein above.

[0249] In one embodiment, the level, amount, or concentration of a biomarker can be measured by methods well known in the art, including, but not limited to, mass spectrometry (e.g., tandem mass spectrometry [MS / MS], chromatography-assisted mass spectrometry, and combinations thereof), immunohistochemistry, multiplex methods (Luminex), Western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), RT-PCR, RT-qPCR, Northern blot, hybridization techniques (e.g., using microarrays, and combinations thereof, including, but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing, e.g., next-generation DNA sequencing (NGS) or RNA-seq (also known as "whole transcriptome shotgun sequencing")), and the like.

[0250] In one embodiment, the method of the present invention comprises the step of correlating the subject's molecular signature or profile with at least one reference signature or profile as described herein above.

[0251] The reference signature or profile may be implemented in software, or an overall median or other arithmetic mean across measurements may be constructed.

[0252] In one embodiment, correlating the subject's molecular signature or profile with at least one reference signature or profile may be performed by entering the subject's molecular signature or profile into an algorithm that has been previously trained with the levels, amounts, or concentrations of biomarkers determined in the reference subjects to decipher each of the reference signatures or profiles. The trained algorithm compares the subject's molecular signature or profile to the reference signatures or profiles.

[0253] In one embodiment, the algorithm returns the percentage of match of the subject's molecular signature or profile with each of at least one reference signature or profile, preferably with each of four reference signatures or profiles: healthy, grade S1, grade S2, and grade S3.

[0254] In one embodiment, if the subject's molecular signature or profile matches a healthy reference signature or profile, the subject is assigned as not suffering from the asymptomatic stage of Alzheimer's disease.

[0255] In one embodiment, if the subject's molecular signature or profile matches any of the reference signatures or profiles for grade S1, grade S2, or grade S3, the subject is assigned to have asymptomatic Alzheimer's disease, preferably asymptomatic Alzheimer's disease grade S1, grade S2, or grade S3.

[0256] In one embodiment, if the subject's molecular signature or profile matches any of the biomarker variation profiles for grade S1, grade S2, or grade S3 in Table 3, the subject is assigned to have asymptomatic Alzheimer's disease, preferably asymptomatic grade S1, grade S2, or grade S3 of Alzheimer's disease.

[0257] In one embodiment, the subject's molecular signature or profile comprises: at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 4A, whose level, amount, or concentration is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 4B, whose level, amount, or concentration is substantially higher (i.e., greater than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or At least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 biomarkers selected from the group of biomarkers in Table 4C, whose level, amount, or concentration is substantially similar (i.e., no more than 5% lower or higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects. If it contains Subjects are assigned to have asymptomatic grade S1 Alzheimer's disease.

[0258] In one embodiment, the subject's molecular signature or profile comprises: at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 5A, whose level, amount, or concentration is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 5B, whose level, amount, or concentration is substantially higher (i.e., greater than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or At least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 biomarkers selected from the group of biomarkers in Table 5C, whose level, amount, or concentration is substantially similar (i.e., no more than 5% lower or higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects. If it contains Subjects are assigned to have asymptomatic grade S2 Alzheimer's disease.

[0259] In one embodiment, the subject's molecular signature or profile comprises: at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 6A, whose level, amount, or concentration is substantially lower (i.e., more than 5% lower) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; at least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers selected from the group of biomarkers in Table 6B, whose level, amount, or concentration is substantially higher (i.e., greater than 5% higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or a population of substantially healthy subjects; and / or At least one biomarker, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 biomarkers selected from the group of biomarkers in Table 6C, whose level, amount, or concentration is substantially similar (i.e., no more than 5% lower or higher) when compared to the level, amount, or concentration of the same biomarker in a substantially healthy subject or population of substantially healthy subjects. If it contains Subjects are assigned to have asymptomatic grade S3 Alzheimer's disease.

[0260] In one embodiment, correlations are made to the match scores for each of four reference signatures or profiles: healthy, grade S1, grade S2, and grade S3, thereby allowing for secondary stratification.

[0261] In one embodiment, the methods of the invention comprise diagnosing a subject as suffering from the asymptomatic stage of Alzheimer's disease based on correlation of the subject's individual signature or profile with a reference signature or profile.

[0262] In one embodiment, the methods of the invention comprise stratifying the subject's asymptomatic stage of Alzheimer's disease into a grade, preferably S1, S2, or S3, based on correlation of the subject's individual signature or profile with a reference signature or profile, e.g., based on correlation with variations in the level, amount, or concentration of at least one biomarker set forth in Table 3, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more biomarkers.

[0263] In one embodiment, the method of the invention comprises predicting the progression of the subclinical stage of Alzheimer's disease in a subject based on correlation of the subject's individual signature or profile with a reference signature or profile.

[0264] In one embodiment, the methods of the present invention comprise determining an individualized course of treatment for a subject based on correlation of the subject's individualized signature or profile with a reference signature or profile.

[0265] The present invention also relates to a method for treating a subject suffering from an asymptomatic stage of Alzheimer's disease using the molecular signature or profile of the present invention, or to a method for treating a subject suffering from an asymptomatic stage of Alzheimer's disease, for example, an S1, S2, or S3 grade of asymptomatic stage of Alzheimer's disease, using the molecular signature or profile of the present invention.

[0266] The present invention also relates to a method for determining an individualized course of treatment for a subject suffering from an asymptomatic stage of Alzheimer's disease, using the molecular signature or profile of the present invention.The present invention also relates to a method for determining an individualized course of treatment for a subject suffering from an asymptomatic stage of Alzheimer's disease, for example, an asymptomatic stage of Alzheimer's disease, grade S1, S2, or S3, using the molecular signature or profile of the present invention.

[0267] The present invention also relates to a method for defining the clinical management of a subject suffering from the asymptomatic stage of Alzheimer's disease using the molecular signature or profile of the present invention.The present invention also relates to a method for defining the clinical management of a subject suffering from the asymptomatic stage of Alzheimer's disease, for example, grades S1, S2, or S3 of the asymptomatic stage of Alzheimer's disease, using the molecular signature or profile of the present invention.

[0268] In one embodiment, the method of treating or determining an individual's course of treatment or defining clinical management comprises diagnosing the subclinical stage of Alzheimer's disease in a subject as described herein above.

[0269] In one embodiment, the method of treating or determining an individual course of treatment or defining clinical management comprises stratifying the subject's asymptomatic stage of Alzheimer's disease into a grade, preferably an S1, S2, or S3 grade as described herein above.

[0270] In one embodiment, the method of treating or determining an individual's course of treatment or defining clinical management comprises the further step of treating the subject.

[0271] In one embodiment, treating the subject is for the purpose of preventing or reducing or alleviating the risk of developing clinical symptoms of Alzheimer's disease or dementia due to Alzheimer's disease in the subject.

[0272] Examples of treatments for the asymptomatic stage of Alzheimer's disease include, but are not limited to, beta-secretase 1 (Bace1) inhibitors, anti-amyloid antibodies, anti-inflammatory agents, anti-Tau antibodies, memory enhancers, synaptic plasticity enhancers, neuroprotective enhancers, microbiota modifiers, inhibitors of Tau or Aβ aggregation and dissemination, and anti-anxiety agents.

[0273] Examples of treatments for the asymptomatic stage of Alzheimer's disease, grade S1, include, but are not limited to, beta-secretase 1 (Base1) inhibitors, anti-amyloid antibodies, inhibitors of Aβ dissemination, anti-inflammatory agents, and anti-anxiety agents.

[0274] Examples of treatments for the asymptomatic stage of Alzheimer's disease, grade S2, include, but are not limited to, beta-secretase 1 (Bace1) inhibitors, anti-amyloid antibodies, anti-inflammatory agents, anti-tau antibodies, synaptic plasticity enhancers, neuroprotective enhancers, inhibitors of tau or Aβ aggregation and dissemination, memory enhancers, microbiota modifiers, and anti-anxiety medications.

[0275] Examples of treatments for the asymptomatic stage of Alzheimer's disease, grade S3, include, but are not limited to, beta-secretase 1 (Bace1) inhibitors, anti-amyloid antibodies, anti-inflammatory agents, anti-tau antibodies, memory enhancers, synaptic plasticity enhancers, neuroprotective enhancers, inhibitors of tau or Aβ aggregation and dissemination, and anti-anxiety medications.

[0276] Examples of Base1 inhibitors include, but are not limited to, CTS-21166 (CoMentis Inc.), verubecestat (MK-8931; Merck & Co., Inc.), solanezumab (Eli Lilly & Co.), lanabecestat (AZD3293; AstraZeneca and Eli Lilly & Co.), elenbecestat (Biogen), and LY2886721 (Eli Lilly & Co.).

[0277] Examples of anti-amyloid antibodies include, but are not limited to, bapineuzumab (Janssen / Pfizer), solanezumab (Eli Lilly), crenezumab (Genentech), gantenerumab (Hoffman-La Roche), BAN2401 (Biogen / Eisai Inc.), GSK 933776 (GlaxoSmithKline), AAB-003 (Janssen / Pfizer), SAR228810 (Sanofi), BIIB037 / BART (Biogen), ACI-24 (AC Immune), and aducanumab (Biogen / Eisai Inc.).

[0278] Examples of anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), beta agonists, anticholinergics, and methylxanthines.

[0279] Examples of anti-Tau antibodies include, but are not limited to, ABBV-8E12 (Abbvie), ACI-35 (AC Immune), BIIB092 (Biogen), and gosuranemab (Biogen).

[0280] Examples of memory enhancers include, but are not limited to, metabolic substances (e.g., glucose, ketones, oxygen supplements), alkaloids (e.g., theobromine, caffeine), vitamins, amino acids, minerals, micronutrients, plant extracts and derivatives, herbs or herbal dietary supplements (e.g., ginkgo biloba, ginseng root).

[0281] Examples of inhibitors of Tau aggregation and seeding include, but are not limited to, TRx0237 (TauRx) and Morphomer Tau (AC Immune).

[0282] Examples of synaptic plasticity enhancers include, but are not limited to, blarcamesine (Anavex Life Sciences), CT1812 (Cognition Therapeutics), GRF6019 (Alkahest), and LM11A-31-BHS (Pharmatrophix).

[0283] Examples of microbiota-modifying drugs include, but are not limited to, sodium oligomannate (Green Valley Pharmaceuticals), SLAB51, ProBiotic-4, and fecal transplant therapy (FMT) from substantially healthy subjects. For a review of microbiota-modifying drugs for the prevention or treatment of Alzheimer's disease, see Bonfili et al., 2020 (FEBS J. Epub ahead of print).

[0284] Examples of neuroprotective enhancers include, but are not limited to, huperzine A, nefiracetam, propentofylline, rivastigmine, and SGS-742.

[0285] Examples of anti-anxiety agents include, but are not limited to, 5-HT1AR agonists (e.g., buspirone, gepirone, and tandospirone), GABA A Receptor positive allosteric modulator (GABA A R PAMs) (e.g., adinazolam, alprazolam, bromazepam, camazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clotiazepam, cloxazolam, diazepam, ethyl loflazepate, etizolam, fludiazepam, halazepam, ketazolam, lorazepam, medazepam, nordazepam, oxazepam, pinazepam, prazepam, alpidem, phenobarbital, carisoprodol, meprobamate, chlormezanone, ethanol (alcohol), etifoxine, imepitoin, cava, skullcap, and valerian), α2δ voltage-dependent calcium channel (VDCC) blockers (e.g., gabapentin, gabapentin enacarbil, phenibut, and pregabalin). antidepressants (e.g., escitalopram, duloxetine, trazodone, clomipramine, mirtazapine, phenelzine, agomelatine, bupropion, tianeptine, vilazodone, and vortioxetine), sympatholytics (e.g., prazosin, clonidine, dexmedetomidine, guanfacine, and propranolol), benzoctamine, cannabidiol, cycloserine, favomotizole, hydroxyzine, kanna, lavender, lorpiprazole, mebicar, mepiprazole, nicotine, opipramol, oxaflozane, phenaglycodol, phenibut, picamilon, selank, tiagabine, tofisopam, and validolum.

[0286] For a review of the (2020) pipeline of drugs and biopharmaceuticals in clinical trials for the treatment of Alzheimer's disease, see Cummings et al., 2020 (Alzheimers Dement (NY). 6(1):e12050), which lists 121 agents currently in clinical trials. The contents of Cummings et al., 2020, particularly the drugs and biopharmaceuticals listed in Figure 1 and Tables 1, 2, 3, and 4, are incorporated by reference.

[0287] In one particular embodiment, the method of treating or determining an individual course of treatment or defining clinical management comprises administering to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease, at least one beta-secretase 1 (Bace1) inhibitor, anti-amyloid antibody, anti-inflammatory agent, anti-Tau antibody, memory enhancer, synaptic plasticity enhancer, neuroprotective enhancer, microbiota modifier, inhibitor of Tau or Aβ aggregation and dissemination, or anti-anxiety agent, as defined herein above.

[0288] In one particular embodiment, the method of treating or determining an individual course of treatment or defining clinical management comprises administering at least one anti-amyloid antibody as defined herein above to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease.

[0289] In one particular embodiment, a method of treating or determining an individual course of treatment or defining clinical management comprises administering to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease, at least one anti-amyloid antibody selected from the group comprising or consisting of bapineuzumab, solanezumab, crenezumab, gantenerumab, BAN2401, GSK 933776, AAB-003, SAR228810, BIIB037 / BART, ACI-24, and aducanumab.

[0290] In one particular embodiment, the method of treating or determining an individual's course of treatment or defining clinical management includes administering aducanumab to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease.

[0291] In one particular embodiment, the method of treating or determining an individual's course of treatment or defining clinical management includes administering gantenerumab to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease.

[0292] In one particular embodiment, the method of treating or determining an individual's course of treatment or defining clinical management comprises administering an oligomannate to a subject diagnosed with an asymptomatic stage of Alzheimer's disease, e.g., an asymptomatic or prodromal stage of Alzheimer's disease.

[0293] In one embodiment, treating the subject is for the purpose of preventing or reducing or mitigating the risk of cardiovascular disease associated with Alzheimer's disease.

[0294] Cardiovascular disease is known to be a contributing factor to the onset of Alzheimer's disease or an increased risk of developing Alzheimer's disease. Therefore, preventing, reducing, or alleviating the risk of cardiovascular disease can be a secondary preventive measure for preventing, reducing, or alleviating the risk of developing clinical symptoms of Alzheimer's disease or dementia due to Alzheimer's disease in a subject.

[0295] Means and methods for preventing or reducing or mitigating the risk of cardiovascular disease are known in the art and include, but are not limited to, quitting smoking, minimizing alcohol consumption, consuming a healthy and balanced diet, exercising at least 150 minutes per week, controlling blood pressure, undergoing regular health checkups, treating diabetes if applicable, etc.

[0296] In one embodiment, treating the subject is for the purpose of delaying the risk of cognitive decline associated with Alzheimer's disease.

[0297] Cognitive decline is known to be a factor that contributes to the onset or increased risk of developing clinical symptoms of Alzheimer's disease or Alzheimer's disease dementia in a subject.

[0298] Means and methods for delaying the risk of cognitive decline are well known in the art and include, but are not limited to, reading, learning a foreign language, playing a musical instrument, and maintaining an active social life (e.g., by volunteering in the local community, participating in group sports, or trying a new activity or hobby).

[0299] In one embodiment, the step of treating the subject is for the purpose of treating or alleviating factors associated with Alzheimer's disease.

[0300] Factors associated with Alzheimer's disease are known in the art and include, but are not limited to, hearing loss, depression, loneliness or social isolation, and a sedentary lifestyle.

[0301] The present invention also relates to methods for recruiting subjects with asymptomatic stages of Alzheimer's disease in clinical trials using the molecular signatures or profiles of the present invention.The present invention also relates to methods for selecting subjects with asymptomatic stages of Alzheimer's disease for enrollment in clinical trials using the molecular signatures or profiles of the present invention.

[0302] The present invention also relates to methods for recruiting subjects with asymptomatic stages of Alzheimer's disease, e.g., asymptomatic Alzheimer's disease grades S1, S2, or S3, in clinical trials using the molecular signatures or profiles of the invention.The present invention also relates to methods for selecting subjects with asymptomatic stages of Alzheimer's disease, e.g., asymptomatic Alzheimer's disease grades S1, S2, or S3, for enrollment in clinical trials using the molecular signatures or profiles of the invention.

[0303] In one embodiment, the method of recruiting a subject in a clinical trial or selecting a subject for enrollment in a clinical trial comprises diagnosing the subclinical stage of Alzheimer's disease in the subject as described herein above.

[0304] In one embodiment, the method of recruiting a subject for a clinical trial or selecting a subject for enrollment in a clinical trial comprises stratifying the subject's asymptomatic stage of Alzheimer's disease into grades as described herein above, preferably into grades S1, S2, or S3.

[0305] In one embodiment, the method of recruiting a subject in a clinical trial or selecting a subject for enrollment in a clinical trial comprises the further step of recruiting a subject in a clinical trial or selecting a subject for enrollment in a clinical trial.

[0306] In one embodiment, the clinical trial involves treatment of the asymptomatic phase of Alzheimer's disease. In one embodiment, the clinical trial involves investigating the safety and / or efficacy of treatment of the asymptomatic phase of Alzheimer's disease.

[0307] In one embodiment, optionally during a clinical trial, the methods of the invention may be performed at least once or twice, e.g., during and / or at the end of the clinical trial, to monitor the molecular signature or profile of the invention while the subject is treated with the test compound.

[0308] Alternatively, the methods of the present invention may be used at the end of a clinical trial as a measure of a primary or secondary endpoint: change from baseline in one or more biomarkers of a molecular signature or profile of the present invention.

[0309] In one embodiment, the subject is an animal, preferably a mammal.

[0310] Examples of mammals include, but are not limited to, humans, non-human primates (such as chimpanzees and other ape and monkey species), farm animals (such as cows, horses, sheep, goats, and pigs), domestic animals (such as rabbits, dogs, and cats), laboratory animals (such as rats, mice, and guinea pigs), etc. The term does not denote a particular age or sex unless otherwise specified.

[0311] In one embodiment, the subject is a primate, including human and non-human primates.

[0312] In one embodiment, the subject is a human.

[0313] In one embodiment, the subject is male or female.

[0314] In one embodiment, the subject is a child. In one embodiment, the subject is an adult.

[0315] In one embodiment, the subject is at risk of developing Alzheimer's disease. Risk factors for Alzheimer's disease include, but are not limited to, age, family history, genetics, and others.

[0316] Age is the largest known factor in Alzheimer's disease. Most subjects with symptomatic Alzheimer's disease are over the age of 65. After age 65, the risk of Alzheimer's disease doubles every five years. After age 85, the risk factor reaches nearly one-third.

[0317] Thus, in one embodiment, the subject is over 20 years old. In one embodiment, the subject is over 30 years old. In one embodiment, the subject is over 40 years old. In one embodiment, the subject is over 50 years old. In one embodiment, the subject is over 60 years old. In one embodiment, the subject is over 70 years old. In one embodiment, the subject is over 80 years old.

[0318] In one embodiment, the subject is 0-20 years old. In one embodiment, the subject is 20-40 years old. In one embodiment, the subject is 40-50 years old. In one embodiment, the subject is 50-55 years old. In one embodiment, the subject is 55-60 years old. In one embodiment, the subject is 60-65 years old. In one embodiment, the subject is 65-70 years old. In one embodiment, the subject is 70-75 years old. In one embodiment, the subject is 75-80 years old. In one embodiment, the subject is 80-85 years old. In one embodiment, the subject is 85-90 years old.

[0319] Family history is another risk factor for Alzheimer's disease.

[0320] Thus, in one embodiment, the subject has a relative, preferably a parent, grandparent, great-grandparent, brother, sister, aunt, uncle, niece, nephew, or cousin, who has been diagnosed or identified as having Alzheimer's disease.

[0321] Heredity is another risk factor for Alzheimer's disease. Several studies have shown that single nucleotide polymorphisms (SNPs) at some loci can affect the risk of Alzheimer's disease. See, for example, Jansen et al., 2019. Nat Genet. 51(3):404-413.

[0322] Thus, in one embodiment, the subject has a single nucleotide polymorphism (SNP) at at least one locus selected from those defined in Table 1 of Jansen et al., 2019, which is incorporated by reference.

[0323] Other risk factors for Alzheimer's disease are known, including, but not limited to, Down's syndrome, sleep deprivation, head trauma, heart disease, diabetes, stroke, high blood pressure, and hypercholesterolemia.

[0324] The present invention also relates to a computer system for diagnosing the asymptomatic stage of Alzheimer's disease in a subject in need thereof using the molecular signature of the present invention. The present invention also relates to a computer-implemented method for diagnosing the asymptomatic stage of Alzheimer's disease in a subject using the molecular signature of the present invention.

[0325] The present invention also relates to a computer system for stratifying a subject's asymptomatic stage of Alzheimer's disease into grades, preferably S1, S2, or S3, using the molecular signatures of the present invention. The present invention also relates to a computer-implemented method for stratifying a subject's asymptomatic stage of Alzheimer's disease into grades, preferably S1, S2, or S3, using the molecular signatures of the present invention.

[0326] The present invention also relates to a computer system for predicting the progression of asymptomatic phase of Alzheimer's disease in a subject using the molecular signature of the present invention. The present invention also relates to a computer-implemented method for predicting the progression of asymptomatic phase of Alzheimer's disease in a subject using the molecular signature of the present invention.

[0327] The present invention also relates to a computer system for determining an individualized course of treatment for a subject suffering from the asymptomatic stage of Alzheimer's disease using the molecular signature of the present invention. The present invention also relates to a computer-implemented method for determining an individualized course of treatment for a subject suffering from the asymptomatic stage of Alzheimer's disease using the molecular signature of the present invention.

[0328] As used herein, the term "computer system" refers to any device that can store and process information and / or use the stored information to control the behavior or implementation of the device itself, regardless of whether the device is electronic, mechanical, logical, or virtual in nature. The term "computer system" can refer to a single computer as well as multiple computers working together to perform a function described as being performed in or by the computer system. A method performed using a computer system is called a "computer-implemented method."

[0329] In one embodiment, a computer system according to the present invention comprises: (i) at least one processor; (ii) at least one computer-readable storage medium storing processor-readable code; Includes:

[0330] As used herein, the term "processor" is meant to include any integrated circuit or other electronic device capable of performing operations on at least one instruction word, such as executing instructions, code, computer programs, and scripts accessed from a storage medium. However, the term "processor" should not be construed as being limited to hardware capable of executing software, but rather refers to a processing device in a general sense and may include, for example, a computer, a microprocessor, an integrated circuit, or a programmable logic device (PLD). A processor may also include one or more graphics processing units (GPUs), whether utilized for computer graphics and image processing or other functions. Furthermore, instructions and / or data capable of performing associated and / or resulting functions may be stored on any processor-readable medium, including, but not limited to, an integrated circuit, a hard disk, a magnetic tape (including floppy disks and zip diskettes), an optical disk (including Blu-ray, compact disks, and digital versatile disks), flash memory (including memory cards and USB flash drives), random access memory (RAM) (including dynamic RAM and static RAM), read-only memory (ROM), or a cache. The instructions may be stored in particular hardware, software, firmware, or any combination thereof.

[0331] Examples of processors include, but are not limited to, central processing units (CPUs), microprocessors, digital signal processors (DSPs), general-purpose microprocessors, application-specific integrated circuits (ASICs), field programmable logic arrays (FPGAs), and other equivalent integrated circuits or other logic circuits.

[0332] The present invention also relates to a computer program comprising software code readable by a processor which, when executed by said processor, is adapted to perform the computer-implemented methods described herein.

[0333] The present invention also relates to a computer readable storage medium containing processor readable code that, when executed by a processor, causes the processor to perform the steps of the computer-implemented methods described herein.

[0334] Examples of computer-readable storage media include, but are not limited to, integrated circuits, hard disks, magnetic tapes (including floppy disks and zip diskettes), and optical disks (including Blu-ray, compact disks, and digital versatile disks), flash memory (including memory cards and USB flash drives), random access memory (RAM) (including dynamic RAM and static RAM), read-only memory (ROM) or cache.

[0335] In one embodiment, the computer readable storage medium is a non-transitory computer readable storage medium.

[0336] In one embodiment, the code stored on the computer readable storage medium, when executed by a processor of a computer system, causes the processor to: a. receiving an input of levels, amounts, or concentrations of at least five biomarkers selected from Table 1A or Table 1B determined in a sample previously obtained from the subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers via at least one machine learning algorithm by codifying and / or modifying the inputs for each level to derive a probability score and / or classification label; c. Producing outputs that are classification labels and / or probability scores; and d. providing a diagnosis of the subject as having or not having asymptomatic Alzheimer's disease based on the output; or providing a stratification of the subject's Alzheimer's disease into a subclinical grade, preferably S1, S2, or S3 grade, based on the output; or providing a prognosis for the progression of subclinical stages of Alzheimer's disease based on the output; or The output is used to provide a specific course of treatment or information for determining a specific course of treatment for the subject.

[0337] In one embodiment, the code stored on the computer readable storage medium, when executed by a processor of a computer system, causes the processor to: a. receiving an input of levels, amounts, or concentrations of at least five biomarkers selected from Table 2A, Table 2B, or Table 2C determined in a sample previously obtained from the subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers via at least one machine learning algorithm by codifying and / or modifying the inputs for each level to derive a probability score and / or classification label; c. Producing outputs that are classification labels and / or probability scores; and d. providing a diagnosis of the subject as having or not having asymptomatic Alzheimer's disease based on the output; or providing a stratification of the subject's Alzheimer's disease into a subclinical grade, preferably S1, S2, or S3 grade, based on the output; or providing a prognosis for the progression of subclinical stages of Alzheimer's disease based on the output; or The output is used to provide a specific course of treatment or information for determining a specific course of treatment for the subject.

[0338] As used herein, the term "learning algorithm" or "machine learning algorithm" refers to a computer-implemented algorithm that automates the construction of analytical models, e.g., for clustering, classification, or profile recognition. A learning algorithm performs analysis on a training data set that is provided to the algorithm. The learning algorithm outputs a "model," also called a "classifier," "classification algorithm," or "diagnostic algorithm." A model receives test data as input and makes an inference or classification of the input data as belonging to one or another class, cluster group, or location on a measure, e.g., diagnosis, stage, prognosis, disease progression, response to a drug, etc.

[0339] A "dataset" is a collection of data used to build a machine learning mathematical model to make data-driven predictions or decisions. In "supervised learning" (i.e., inferring a function from known input-output examples in the form of labeled training data), three types of machine learning datasets are typically dedicated to each of three types of tasks: "training," i.e., fitting parameters; "validation," i.e., adjusting machine learning hyperparameters (which are parameters used to control the learning process); and "testing," i.e., confirming, independent of the training dataset, that the mathematical model is used to build and subsequently provides satisfactory results.

[0340] Various learning algorithms can be used to infer the pathology or condition of a subject. Machine learning algorithms can be supervised or unsupervised. Learning algorithms include, but are not limited to, artificial neural networks (e.g., backpropagation networks), discriminant analysis (e.g., Bayesian classifiers, Fisher analysis), support vector machines, decision trees (e.g., recursive partitioning processes, e.g., CART (classification and regression trees)), random forests, linear classifiers (e.g., multiple linear regression [MLR], partial least squares [PLS] regression, principal component regression [PCR]), hierarchical clustering, and cluster analysis. Learning algorithms create models or classifiers that can be used to make inferences, such as inferences about the disease state of a subject.

[0341] In one embodiment, the at least one machine learning algorithm has been previously trained on at least one training dataset.

[0342] In one embodiment, at least one training dataset includes information related to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A (as the at least five biomarkers in step a. of the computer-implemented method) from samples previously obtained from reference subjects (i.e., subjects with a known state of Alzheimer's disease).

[0343] In one embodiment, at least one training data set comprises information relating to the levels, amounts, or concentrations of the same at least five biomarkers of Table 1A from samples previously obtained from substantially healthy subjects and subjects known to be suffering from the asymptomatic stage of Alzheimer's disease.

[0344] In one embodiment, the training dataset comprises variation profiles of the biomarkers in Table 3.

[0345] In one embodiment, the at least one machine learning algorithm is selected from the group including an artificial neural network (ANN), a perceptron algorithm, a deep neural network, a clustering algorithm, a k-nearest neighbor (k-NN), a decision tree algorithm, a random forest algorithm, a linear regression algorithm, a logistic regression algorithm, a linear discriminant analysis (LDA) algorithm, a quadratic discriminant analysis (QDA) algorithm, a support vector machine (SVM), a Bayesian algorithm, a simple rule algorithm, a clustering algorithm, a meta-classifier algorithm, a Gaussian mixture model (GMM) algorithm, a nearest centroid algorithm, a gradient boosting algorithm (e.g., an extreme gradient boosting [XG Boost] algorithm or an AdaBoost (adaptive boosting) algorithm), a linear mixed-effects model algorithm, and combinations thereof. [Brief explanation of the drawings]

[0346] [Figure 1] Stratification of the subclinical stage of Alzheimer's disease into grades S1, S2, and S3 in function of key cerebral events: production of soluble Aβ peptides, hyperphosphorylation of Tau, and the appearance of aggregated lesions (senile plaques and tangles). The onset of dementia in a subject marks the beginning of the so-called clinical phase. [Figure 2] Comparison of Alzheimer's disease progression in humans, transgenic mice, and AgenT rats. As shown, transgenic mice are not suitable for recapitulating the progression of AD observed in humans, especially its asymptomatic stage. In contrast, the characteristics of the AgenT rat model make it a more faithful model of the asymptomatic stage of AD than transgenic animals. All these characteristics make the AgenT rat model a powerful tool for better predicting the behavior of blood biomarkers according to the stage of progression. Therefore, this model constitutes a suitable test system for characterizing new biomarkers or panels of biomarkers for the development of early diagnosis. [Figure 3] Cerebral imaging of amyloid does not constitute an effective method for detecting subjects with Alzheimer's disease. Indeed, 30% of AD patients are PIB-PET (positron emission tomography (PET) using PIB (Pittsburgh compound B)) Aβ-, while 40% of healthy individuals are PIB-PET Aβ+. This strongly reduces its validity as a diagnostic. [Figure 4] Clinical validation of AgenT grade S3 plasma variations. To decipher the clinical relevance of the plasma variations we observed in AgenT rats, we compared the variations observed in diagnosed patients (Doecke et al., 2012. Arch Neurol. 69(10):1318-25; Mapstone et al., 2014. Nat Med. 20(4):415-8; Olazaran et al., 2015. J Alzheimers Dis. 45(4):1157-73; Kim et al., 2017. J Alzheimers Dis. 60(3):809-817, meta-analysis) with the variations observed in grade S3 rats. We observed that 75% of the previously described variations were also present in AgenT rats (***p<0.0001; ***r2=0.71). This result strongly supports the clinical relevance of the high level of plasma variation observed in AgenT grade S3 rats. [Figure 5]Clinical Validation of Agent T Grade S1 Plasma Variations. To decipher the clinical relevance of the plasma variations we observed in Agent T rats, we cross-compared the variations observed in young Down syndrome individuals (Caracausi et al., 2018. Sci Rep. 8(1):2977) with the variations observed in Agent T Grade S1 rats. We observed that 74% of the previously described variations were also present in Agent T rats (***p<0.0001; ***r2=0.76). This result strongly supports the high level of clinical relevance of the plasma variations observed in Agent T Grade S1 rats. [Figure 6] Example of blood biomarker variation during the progression of Alzheimer's disease. Blood biomarkers evolve throughout the progression of this condition in a nonlinear fashion. Therefore, it is not possible to estimate variation during the asymptomatic phase based solely on variation from patients diagnosed with AD. Three representative examples are shown in this figure (α-2-macroglobulin, 5-hydroxylysine, and ethyl malonate). The points on the curves marked (1) represent variation observed in the plasma of AgenT rats assessed by mass spectrometry. [Figure 7] The identification process of blood biomarkers. The identification of 119 "best-in-class" blood biomarkers suitable for detecting AD in asymptomatic subjects appears to be an innovative strategy combining neuroscience and artificial intelligence. [Figure 8]The scientific literature has questioned the diagnostic validity of some of the identified blood biomarkers. However, their subclinical AD profile, especially their specific nonlinear variations throughout the asymptomatic phase, remains unknown and cannot be deciphered from available preclinical or clinical data. Therefore, our approach provides a high level of confidence in understanding the evolution of biomarkers over time in the asymptomatic phase of AD (ApoE, serpin A1, and complement C3). The points on the curve represented by (1) represent the variation observed in the plasma of AgenT rats as assessed by mass spectrometry; the points on the curve represented by (2) represent the variation observed in the plasma of patients diagnosed with Alzheimer's disease (adapted from Thambisetty et al., 2011. PLoS One. 6(12):e28527); the points on the curve represented by (3) represent the variation observed in the plasma of patients diagnosed with Alzheimer's disease (adapted from Wang et al., 2014. PLoS One. 9(2):e89041); and the points on the curve represented by (4) represent the variation observed in the plasma of patients diagnosed with Alzheimer's disease (adapted from Liao et al., 2007. Proteomics Clin Appl. 1(5):506-12). [Figure 9] Comparison between biomarkers released in the brain and those produced by peripheral organs. Measuring biomarkers released from peripheral organs in an "amyloid stress state" significantly increases the specificity (i.e., true positive rate) and sensitivity (i.e., true negative rate) of the test. [Figure 10]Figure 10A-B: Example of a neural network based on 14 randomly selected biomarkers from Table 1A for diagnosing the asymptomatic stage of Alzheimer's disease in a subject. The list of biomarkers is as follows: 10 kDa heat shock protein, mitochondrial; 5-hydroxylysine (from the biomarker family "Lysine and Conjugates"); adenylate kinase 4, mitochondrial; calreticulin; creatine kinase type B (from the biomarker family "Creatine Kinase Family"); ergothioneine; fructosyl lysine (from the biomarker family "Lysine and Conjugates"); globin c2 (from the biomarker family "Globin Family"); integrin subunit αV; myoglobin (from the biomarker family "Globin Family"); peptidyl-prolyl cis-trans isomerase FKBP1A; retinoic acid receptor responder 2; Tmprss13 protein; and transferrin receptor protein 1. Figure 10A. Structure of the neural network trained to identify AD status and stratification. In this illustrative example, the neural network includes 14 inputs on the left (i.e., 14 randomly selected biomarkers from Table 1A) and 4 outputs on the right (i.e., 4 profiles: healthy, grade S1, grade S2, and grade S3). Figure 10B. Accuracy of the trained neural network for detecting subclinical AD across 5 cross-validations. [Figure 11]Figures 11A-B. Example of a neural network based on 14 randomly selected biomarkers from Table 1A for stratifying a subject's subclinical stage of Alzheimer's disease into different subclinical grades. The 14 biomarkers are the same as those listed in Figure 10. Figure 11A. A stratification method illustrated with two samples (A and B). The method involves measuring the level, amount, or concentration of biomarkers, processing the raw data with a trained neural network to compare the subject's signature or profile with each of the reference signatures or profiles (healthy, grade S1, grade S2, and grade S3), calculating a fit score, and stratifying the subject by the profile. Figure 11B. Ad hoc confusion matrix of a neural network trained for stratifying subclinical AD across five cross-validations. [Figure 12] Experimental design used to validate 119 best-in-class biomarkers in humans via transfer learning. [Figure 13] Mean accuracy for 2, 5, 15, and 25 randomly selected biomarkers of Table 1A or non-Table 1A components. Analysis was realized with 250 random selections using two-way ANOVA. [Figure 14] Mean accuracy for 2, 5, 15, and 25 randomly selected biomarkers of Table 1A or non-Table 1A components. Analysis was realized with 1000 random selections using the Mann Whitney non-parametric test. [Figure 15] Figures 15A-C. Performance obtained with 1000 random selections using two-way ANOVA. Figure 15A. Percentage of accuracy for 2, 5, 15, and 25 randomly selected biomarkers from Table 1A or non-Table 1A components. Figure 15B. Percentage of biomarker combinations with accuracy greater than 70%. Figure 15C. Mean accuracy for 2, 5, 15, and 25 randomly selected biomarkers from Table 1A or non-Table 1A components. DETAILED DESCRIPTION OF THE INVENTION

[0347] Example The invention is further illustrated by the following examples.

[0348] Example 1 material and method animal The AgenT rat model (U.S. Pat. No. 10,159,227; European Patent No. 3066203) was induced by injecting adeno-associated viruses (AAVs) encoding human mutant APP (double mutant APP751 cDNA containing the Swedish and London mutations) and presenilin 1 (PS1) (cDNA containing the M146L mutation (pENTR4-PS1-S182M146L)) genes into the hippocampus of adult rodents (8-week-old male Wistar rats).

[0349] Control rats were injected with an AAV encoding presenilin 1 (PS1) alone.

[0350] This disruptive technique allowed for the localized production of exogenous mutant APP and PS1 proteins in a small number of neurons, which then counteracted the progressive spread of Aβ throughout the hippocampal tissue. 42 Thus, the majority of hippocampal cells do not carry the genetic modification, making this a relevant model for the non-genetic form of the disease, which represents over 92% of cases (Prince et al., 2015. World Alzheimer Report 2015. The global impact of dementia: An analysis of prevalence, incidence, cost and trends (Rep.). London: Alzheimer's disease international (ADI)).

[0351] The pathophysiological relevance of this model is being validated by comparing it with postmortem samples from AD patients. 42The peptide concentration gradually increases, reaching concentrations comparable to those measured in the hippocampus of AD patients in later stages. Gradual hyperphosphorylation of endogenous tau protein coincides with a decline in memory capacity, recapitulating the sequence of events observed in the clinic. Amyloid plaques and cerebral amyloid angiopathy develop only in aged AgenT rats. Intraneuronal aggregates of hyperphosphorylated tau protein confirm the full involvement of tau pathology (Audrain et al., 2018. Cereb Cortex. 28(11):3976-3993).

[0352] plasma extraction To identify plasma biomarkers, blood was sampled from 33 control rats and 33 AgenT rats.

[0353] The sampling age is 16 control rats (grade S1) aged 1–3 months after injection; Sixteen AgenT rats (grade S1), aged 1–3 months after injection; 10 control rats (grade S2) aged 8–10 months after injection; Ten AgenT rats (grade S2) aged 8–10 months after injection; seven control rats (grade S3) aged 15–30 months after injection, and Seven AgenT rats (grade S3) aged 15 to 30 months after injection We are working to achieve this.

[0354] To avoid batch effects, these experiments were based on six unrelated cohorts of rats.

[0355] Each blood sample was associated with a specific grade of progression (S1, S2, S3) corresponding to a different neurological disorder. This stratification allows characterizing the evolution of molecules that are deregulated as the disease progresses.

[0356] EDTA plasma was obtained via cardiac puncture after centrifugation at 2,000 g for 10 min, aliquoted into 0.5 mL polypropylene tubes, and stored at −80°C.

[0357] Quantification of plasma components by mass spectrometry Proteomics Mass Spectrometry Plasma samples were shipped frozen on dry ice. Five microliters of the sample were denatured, reduced, and alkylated using Biognosys Denaturation and Reduction / Alkylation Buffer for 30 minutes at 37°C.

[0358] 80 μg of protein was then digested overnight at 37° C. with 1.6 μg of trypsin (Promega) per sample. Peptides were desalted using C18 MacroSpin columns (The Nest Group) according to the manufacturer's instructions and dried using a SpeedVac system.

[0359] Peptides were resuspended in 22 μL of LC solvent A (1% aqueous acetonitrile containing 0.1% formic acid) and spiked with calibration peptides from the Biognosys iRT kit before analysis by mass spectrometry.

[0360] The concentration of the peptides was determined using a microBCA (Thermo Fisher) and a UV / Vis spectrometer (SPECTROstar Nano, BMG Labtech).

[0361] For data-independent acquisition (DIA) liquid chromatography tandem mass spectrometry (LC-MS / MS) measurements, 5 μg of peptides per sample were injected onto a C18 column (CSH-C18 1.7 μm, 300 μm internal diameter, 150 mm length) on a Waters M-Class LC connected to a Thermo Scientific Fusion Lumos Tribrid mass spectrometer equipped with a next-generation nanoFlex electrospray source.

[0362] LC solvents are: LC solvent A: 1% aqueous acetonitrile containing 0.1% formic acid; LC solvent B: 15% water in acetonitrile with 0.1% formic acid It was.

[0363] The nonlinear LC gradient was 1 to 49% solvent B in 40 min, followed by steps of 90% B for 1 min and 1% B for 4 min.

[0364] The DIA method with one full-coverage survey scan and 29 DIA windows was used.

[0365] HRM mass spectrometry data were analyzed using Spectronaut Pulsar X software (Biognosys). The false positive rate for proteins and peptides was set at 1%, and data were filtered using row-based extraction. The assay library (protein catalog) created in this project was used in this analysis. HRM measurements analyzed by Spectronaut were normalized using local regression normalization (Callister et al., 2006. J Proteome Res. 5(2):277-86).

[0366] Heatmap distances were calculated using the "Manhattan" method and clustering was calculated using "ward.D" on both axes.

[0367] Principal component analysis was performed in R using prcomp and the ggbiplot function modified for plotting, and partial least squares discriminant analysis was performed using the mixOMICS package.

[0368] General plotting was performed in R using the ggplot2 package.

[0369] Metabolomic Mass Spectrometry Samples were prepared using an automated MicroLab STAR® system manufactured by Hamilton Company. Several recovery standards were added before the first step of the extraction process for quality control.

[0370] Samples were extracted with methanol (Glen Mills GenoGrinder 2000) with vigorous agitation for 2 min to protein precipitate and release small molecules bound to proteins or trapped in the precipitated protein matrix, followed by centrifugation to recover chemically diverse metabolites.

[0371] The resulting extract was divided into five fractions: two for analysis by two separate reversed-phase (RP) / ultra-performance liquid chromatography (UPLC)-MS / MS methods using positive-ion mode electrospray ionization (ESI); one for analysis by RP / UPLC-MS / MS using negative ion mode ESI; one for analysis by hydrophilic interaction liquid chromatography (HILIC) / UPLC-MS / MS using negative ion mode ESI, and One saved for backup purposes.

[0372] Samples were briefly placed on a TurboVap® (Zymark) to remove the organic solvent. Sample extracts were stored under nitrogen overnight before being prepared for analysis.

[0373] All methods utilize a Waters ACQUITY UPLC and Thermo Scientific Q-Exactive high resolution / accurate mass spectrometer fitted with a HESI-II (heated electrospray ionization) source and an Orbitrap mass spectrometer operating at 35,000 mass resolution.

[0374] Sample extracts were dried and then reconstituted in solvents compatible with each of the four methods, each of which contained a series of standards at consistent concentrations to ensure injection and chromatographic consistency.

[0375] One aliquot was analyzed using acidic cation conditions, which were chromatographically optimized for more hydrophilic compounds, in which the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 μm) using water and methanol containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA).

[0376] A second aliquot was also analyzed using acidic cation conditions, which were chromatographically optimized for more hydrophobic compounds, in which the extract was gradient eluted from the C18 column described above using methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA, operating at a higher overall organic content.

[0377] A third aliquot was analyzed using a separate dedicated C18 column using basic anion optimized conditions. The basic extract was gradient eluted from the column using methanol and water but with 6.5 mM ammonium bicarbonate (pH 8).

[0378] The fourth aliquot was analyzed via negative ionization by elution from a HILIC column (Waters UPLC BEH Amide 2.1 x 150 mm, 1.7 μm) using a gradient containing water and acetonitrile with 10 mM ammonium formate (pH 10.8).

[0379] MS analysis was performed by alternating MS and data-dependent MS scans using dynamic exclusion. The scan range varied slightly between these methods but covered approximately 70–1000 m / z. Hardware and software were used to extract raw data, identify peaks, and process quality control data. Compounds were identified by comparing them to library entries of purified standards or recurrent unknown entities. The mass spectrometry facility maintains libraries based on certified standards, including retention time / index (RI), mass-to-charge ratio (m / z), and chromatographic data (including MS / MS spectral data) of all molecules present in the library. Furthermore, biochemical identification was based on three criteria: retention index within a narrow RI window of the proposed identification, an accurate mass match with the library at ±10 ppm, and MS / MS forward and reverse scores. MS / MS scores were based on a comparison of ions present in the experimental spectrum to those present in the library entry spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and differentiate biochemicals. Over 4,500 commercially available purified standard compounds have been obtained and registered in the LIMS for analysis on all platforms to determine their analytical properties. Various curation methods are used to ensure high-quality data sets are available for statistical analysis and data interpretation. The quality control and curation processes are designed to ensure accurate and consistent identification of true chemical entities and to remove system artifacts, misassignments, redundancies, and equivalents of background noise. Data analysis uses visualization and interpretation software to confirm the consistency of peak identifications between various samples. Library matches for each compound are confirmed for each sample and corrected as necessary. Peaks are quantified as area-under-the-curve detector ion counts.In studies spanning multiple days, a data adjustment step is performed to correct for block variations resulting from differences in instrument day-to-day adjustments while preserving the variance that occurs within a day. Essentially, each compound is corrected for balanced blocks of run days by registering a daily median value equal to 1 (1.00) and adjusting each data point proportionally (called "block correction"). For studies that do not require analysis of more than one day, no adjustment of the raw data is necessary, other than scaling for data visualization.

[0380] Identification of plasma biomarkers The starting point of this analysis was to exclude irrelevant biomarkers. To do this, we performed the following three steps progressively: (1) We excluded all biomarkers whose variance did not match some threshold, i.e., biomarkers that had approximately the same values ​​in all samples; (2) We performed several linear clustering algorithms (such as linear SVM, gradient tree boosting, random forest, and logistic regression) to provide the relative importance of biomarkers, and we filtered out those with negligible importance. At the end of this step, we could consider that all significantly irrelevant biomarkers had been eliminated. (3) Next, we performed different recursive feature elimination (RFE) in cross-validation using several algorithms to assign weights to the features relative to the remaining biomarkers, and finally we selected these biomarkers. More specifically, RFE is a feature selection method that fits a model and recursively eliminates the weakest biomarkers until a relevant number of features is achieved.

[0381] After identifying the most relevant biomarkers, we selected the most useful ones for the asymptomatic phase of AD as follows: (1) We recursively tested all potential combinations of these biomarkers by cross-validating two different machine learning algorithms (multilayer perceptron and support vector machine with a cubic polynomial kernel). Thus, we successfully found the best combination of n biomarkers (n ranges from 1 to 250). (2) Among the biomarker combinations for which we were able to obtain the best average score for cross-validation prediction, we selected the one with the fewest number of biomarkers to avoid overfitting as much as possible.

[0382] In this last analysis, we obtained a list of 119 biomarkers (or biomarker families) that could be considered the most characteristic of the different grades of AD.

[0383] result Stratification of subclinical AD By combining longitudinal behavioral and brain biochemical analyses in AgenT rats, we stratified the subclinical stage of AD according to three grades (Fig. 1 and Table 7).

[0384] [Table 16]

[0385] Grade S1 indicates the presence of soluble Aβ in cerebral tissue at concentrations sufficient to induce anxiety-like symptoms. 42 It is defined by the production of

[0386] Second, grade S2 induces Aβ in cerebral tissue at concentrations sufficient to induce pathological hyperphosphorylation of tau epitopes and promote accelerated long-term amnesia. 42 It is defined as the accumulation of

[0387] Finally, grade S3 is defined by aggregation of both amyloid peptides (senile plaques) and phospho-Tau (fibrillary tangles).

[0388] Stratification of the asymptomatic phase appears to be key to successful biomarker identification, and this method may enable the development of a diagnostic for the asymptomatic phase of AD.

[0389] Determination of the overall plasma constituent profile We performed a comprehensive analysis by mass spectrometry, and proteomic, lipidomic, and metabolomic approaches were implemented to identify specific profiles in the plasma samples of each rat according to their progression grade.

[0390] Thus, 2400 components have been measured. We generated a complete dataset by correlating the plasma profile and the actual stage of progression (grade S1, S2, or S3) to begin identifying suitable blood biomarkers.

[0391] AgenT rats demonstrated high clinical relevance. A meta-analysis of four published articles (Doecke et al., 2012. Arch Neurol. 69(10):1318-25; Mapstone et al., 2014. Nat Med. 20(4):415-8; Olazaran et al., 2015. J Alzheimers Dis. 45(4):1157-73; Kim et al., 2017. J Alzheimers Dis. 60(3):809-817) identified 90 deregulated molecules in the plasma of diagnosed AD patients.

[0392] Of these 90 molecules, 45 were expected to be measured during the mass spectrometry assay in grade S3 AgenT rats.

[0393] To decipher the clinical relevance of the plasma variations we observed in AgenT rats, we cross-compared the variations observed in diagnosed patients with those observed in grade S3 rats. We observed that 75% of the previously described variations were also present in AgenT rats (***p<0.0001; ***r 2 = 0.71). This result strongly supports the clinical relevance of the high level of plasma variation observed in grade S3 AgenT rats (Figure 4 and Table 8).

[0394] [Table 17] TIFF2026009931000042.tif215162

[0395] Epidemiological evidence suggests that by the age of 40, all individuals with Down syndrome (DS) have the neuropathology of AD (Lott & Head, 2005. Neurobiol Aging. 26(3):383-9). The complete penetrance of AD in individuals with DS is due to an additional duplication of the amyloid precursor protein (APP) gene caused by trisomy 21 (Rovelet-Lecrux et al., 2006. Nat Genet. 38(1):24-6; Sleegers et al., 2006. Brain. 129(Pt 11):2977-83). This genetic predisposition results in the onset of an asymptomatic phase of AD from birth in individuals with DS. Therefore, we believe that individuals with DS rapidly deteriorate to AD grade S1 in the earliest stages.

[0396] To the best of our knowledge, analysis of the only published paper on plasma biomarker variations in young DS individuals (Caracausi et al., 2018. Sci Rep. 8(1):2977) led to the identification of 46 deregulated molecules.

[0397] Of these molecules, 23 appeared to be measured during the mass spectrometry assay in grade S1 AgenT rats.

[0398] To decipher the clinical relevance of the plasma variations we observed in AgenT rats, we cross-compared the variations observed in young DS individuals with those observed in grade S1 rats. We observed that 74% of the variations previously described were also present in AgenT rats (***p<0.0001; ***r 2 =0.76). This result strongly supports the clinical relevance of the high level of plasma variation observed in grade S1 AgenT rats (Figure 5 and Table 9).

[0399] [Table 18] TIFF2026009931000044.tif44162

[0400] Interestingly, blood biomarkers evolve throughout the progression of the disease, making it impossible to extrapolate variations during the asymptomatic phase based solely on variations from patients diagnosed with AD. Three typical examples (α-2-macroglobulin, 5-hydroxylysine, and ethylmalonate) are shown in Figure 6.

[0401] Identification of plasma biomarkers suitable for the asymptomatic stage of Alzheimer's disease After confirming the clinical validity of the AgenT rats, we used artificial intelligence techniques to identify best-in-class biomarkers suitable for detecting subclinical AD (Figure 7).

[0402] We identified 119 best-in-class plasma biomarkers (biomarker families) suitable for detecting the subclinical phase of AD (Table 1A).

[0403] Interestingly, some of the identified biomarkers have already been suspected as promising AD biomarkers, however, their subclinical AD profiles, especially their specific nonlinear variations across the asymptomatic phase (Figure 8), were still unknown and could not be deciphered from available preclinical or clinical data.

[0404] Our approach therefore provides an understanding of the evolution of biomarkers over time during the asymptomatic phase of AD with a high level of confidence.

[0405] Within the "biomarker families" in Table 1A, the following summarize proteins: 14-3-3 family: 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. 14-3-3 proteins have the property of binding to a large number of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. More than 200 signaling proteins have been reported as 14-3-3 ligands. The major 14-3-3 family members are 14-3-3 protein β / α, 14-3-3 protein ε, 14-3-3 protein η, 14-3-3 protein γ, 14-3-3 protein θ, and 14-3-3 protein ζ / δ. Arp2 / 3 complex proteins: The Arp2 / 3 complex is a seven-subunit protein complex that plays a major role in regulating the actin cytoskeleton. It is a major component of the actin cytoskeleton and is found in most eukaryotic cells, including the actin cytoskeleton. The major Arp2 / 3 complex proteins are actin-related protein 2, actin-related protein 2 / 3 complex subunit 1B, actin-related protein 2 / 3 complex subunit 3, actin-related protein 2 / 3 complex subunit 4, actin-related protein 2 / 3 complex subunit 5, actin-related protein 3, and Arp2 / 3 complex 34 kDa subunit. Apolipoproteins: Apolipoproteins are proteins that bind lipids (oil-soluble substances, such as fats and cholesterol) to form lipoproteins. They transport lipids (and fat-soluble vitamins) in the blood, cerebrospinal fluid, and lymph. The major apolipoproteins are apolipoprotein AI, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein B-100, apolipoprotein CI, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein C-IV, apolipoprotein D, apolipoprotein E, apolipoprotein H, apolipoprotein M, and apolipoprotein N. Clotting Factor Family: Clotting factors are proteins in the blood that help control bleeding. Complement System Family: The complement system is part of the immune system that clears microorganisms and damaged cells from organs, promotes inflammation, and enhances the ability of antibodies and phagocytes to attack pathogenic cell membranes. It is part of the innate immune system, is not adaptive, and remains unchanged throughout an individual's life. However, the complement system can be recruited and acted upon by antibodies produced by the adaptive immune system. Globin family: Globins are a superfamily of heme-containing globular proteins involved in binding and / or transporting oxygen. Globulin family: Globulins are a family of globular proteins that have a higher molecular weight than albumin and are insoluble in pure water but soluble in dilute salt solutions. Some globulins are produced in the liver, others by the immune system. Globulins, albumin, and fibrinogen are the major blood proteins. Kininogen family: Kininogens are proteins defined by their role as precursors of kinins, but may have additional roles as well. Kinins are biologically active peptides, the parent form of which is bradykinin. The major kininogens are kininogen, kininogen 1, and T-kininogen 2. Proteasome complex family: Proteasomes are cylindrical complexes containing a "core" of four stacked rings that form a central pore. Each ring is composed of seven individual proteins. The two inner rings are made up of seven β subunits, and the two outer rings each contain seven α subunits. Serpin Superfamily: Serpins are a superfamily of proteins with similar structures that have been identified for their protease inhibitory activity.

[0406] Other "biomarker families" in Table 1A summarize metabolites: Lysine and Derivatives: Lysine plays several roles in humans, most importantly in proteinogenesis, but also in the cross-linking of collagen polypeptides, the uptake of essential mineral nutrients, and the production of carnitine, which is important in the metabolism of fatty acids. Carnitine and Derivatives: Carnitine is a conditionally essential nutrient that plays a key role in energy production and fatty acid metabolism. Carnitine, which cannot be obtained from food, is endogenously synthesized from two essential amino acids, lysine and methionine. Dysregulation of carnitine has been implicated in complications of diabetes mellitus, dialysis, trauma, malnutrition, cardiomyopathy, obesity, starvation, drug interactions, endocrine imbalances, and other disorders (Flanagan et al., 2010. Role of carnitine in disease). Cholic acid, also known as 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid, is a water-insoluble primary bile acid. Salts of cholic acid are called cholates. Cholic acid, along with chenodeoxycholic acid, is one of the two major bile acids produced by the liver and is synthesized from cholesterol. These two major bile acids are roughly equal in concentration in humans. Derivatives are made from cholyl-CoA by exchanging the CoA with either glycine or taurine, resulting in glycocholate and taurocholate, respectively. Valerate and derivatives: Valerate compounds are salts or esters of valeric acid. They are also known as pentanoates. Many steroid-based medicines, such as those based on betamethasone or hydrocortisone, contain the steroid as a valerate ester.

[0407] Peripheral biomarkers are more relevant than those released in the brain for predicting an individual's AD status. All blood biomarkers currently under development are brain-released biomarkers, particularly Aβ 42 Based on peptides, Tau or phospho-Tau, growth factors, neuroinflammatory players, or neuronal cell death markers (e.g., neurofilament light chain (NfL)). This type of biomarker has many limitations, significantly reducing their specificity for detecting asymptomatic AD patients.

[0408] Aβ 42 Peptides are poorly correlated with AD status. Indeed, soluble Aβ in the brain 42 With the same peptide concentration, an individual may or may not develop AD. This is a result of the individual's sensitivity to "amyloid stress." Without considering this individual sensitivity, it is impossible to accurately detect subclinical AD.

[0409] Circulating Tau, phospho-Tau, growth factors, neuroinflammatory players, or neuronal cell death markers may be of interest to improve the current clinical diagnosis of AD, but are less useful for detecting asymptomatic patients due to slow deregulation.

[0410] To address these issues, the use of peripheral blood biomarkers appears to be the best solution: measuring biomarkers released from peripheral organs in an "amyloid stress state" significantly increases the specificity (i.e., true positive rate) and sensitivity (i.e., true negative rate) of the test.

[0411] Indeed, only individuals under and responsive to "amyloid stress" develop AD and exhibit deregulated blood peripheral biomarkers, the majority of which are peripheral biomarkers (Figure 9).

[0412] High-level accuracy based biomarker prediction neural network By using a few randomly selected biomarkers from the list of 119 best-in-class plasma biomarkers identified, neural networks can be trained with reference subjects (training set) to define the four reference profiles described above.Using this trained neural network, it can be calculated its accuracy by using a new batch of subjects (a test set that has not been used to train the algorithm) or by cross-validation techniques.The performance obtained using artificial neural networks is greater than 75%.These performances are calculated with respect to the trained algorithm's ability to separate healthy subjects from asymptomatic Alzheimer's subjects.

[0413] By using a randomly selected subset of 5 (Table 10A), 6 (Table 10B), 14 (Table 10C) and 26 (Table 10D) biomarkers from Table 1A and a feed-forward neural network, more precisely a multilayer perceptron, with a logistic activation function, we were able to obtain an accuracy of detection of subclinical AD over five cross-validations of more than 75%.

[0414] [Table 19]

[0415] [Table 20]

[0416] [Table 21]

[0417] [Table 22]

[0418] Using a subset of 14 biomarkers (Tables 10C and 11) and a feed-forward neural network with a logistic activation function, more precisely a multilayer perceptron (Figure 10A), we were able to obtain an accuracy for detecting asymptomatic AD over a five-fold cross-validation of 84%, a specificity (true negatives, i.e., healthy subjects identified as such) of 84.9%, and a sensitivity (true positives, i.e., asymptomatic AD subjects identified as such) of 81% (Figure 10B).

[0419] [Table 23]

[0420] Stratification of the asymptomatic stage of Alzheimer's disease Furthermore, it is possible to detect the AD stratification shown in the confusion matrix of FIG. 11B, still performed using five-fold cross-validation, using the same 14 randomly selected biomarkers.

[0421] The stratification method is illustrated in Figure 11A. In summary, the blood biomarker profile of the test subject is compared with each of the reference signatures or profiles. A "match" score is calculated by a trained algorithm based on the percentage of match between the molecular signature or profile of the test individual and the reference signature or profile. Subjects with high match scores are assigned to stratification (healthy, grade S1, grade S2, or grade S3).

[0422] Example 2: Validation of 119 best-in-class plasma biomarkers in humans material and method By sampling the plasma of a non-transgenic animal model that successfully recapitulates the continuum of Alzheimer's disease progression at the brain level (Audrain et al., 2018. Cereb Cortex. 28(11):3976-3993), we used artificial intelligence to identify 119 best-in-class plasma biomarkers.

[0423] Next, we analyzed the behavior of these biomarkers in 232 human plasma samples collected up to 13 years before the onset of dementia (Figure 12). Three unrelated cohorts were used: two cohorts with sporadic AD (one from France and one from Spain) and one with Down syndrome (from Spain). Table 12 shows the typology of the patients tested: Alzheimer's disease patients (including asymptomatic, prodromal, and dementia patients) and non-Alzheimer's disease individuals as negative controls (healthy controls and patients with neurodegenerative diseases other than AD, such as frontotemporal dementia (FTD), dementia with Lewy bodies, vascular dementia, psychological disorders, SNAP (suspected non-Alzheimer's disease pathophysiology), focal amyloidosis, primary progressive aphasia, multiple system atrophy, corticobasal degeneration, or mixed dementia). Tables 13A-C show disease characteristics from the three cohorts.

[0424] [Table 24]

[0425] [Table 25]

[0426] [Table 26]

[0427] [Table 27]

[0428] To confirm the informativeness of the biomarkers in Table 1A, we compared them with the rest of the plasma components (i.e., plasma components not identified in Table 1A, hereafter referred to as "non-Table 1A components") as follows: 1) We randomly selected a set of "n" biomarkers (n = 2, 5, 15, or 25 biomarkers) from Table 1A and evaluated the performance for detecting Alzheimer's disease patients using logistic regression based only on these n biomarkers in a 5-fold cross-validation. Here, we purposely used logistic regression because it is a fundamental classifier that allows us to construct the informative nature of the randomly selected biomarkers by linearly combining them, thereby reducing the risk of overfitting compared to other algorithms that perform nonlinear combinations, such as neural networks. 2) This approach was performed 250 and 1000 times, and we obtained the accuracy of detecting the asymptomatic stage of AD for each of 5000 randomly selected biomarker sets. We chose to perform two independent experimental runs (250 and 1000 combinations) to demonstrate the robustness of the average performance obtained with 2, 5, 15, and 25 random biomarkers. 3) The same method was also performed considering non-Table 1A components, thus making it possible to compare the performance of biomarkers for Table 1A and non-Table 1A components. 4) We tested for differences in distribution using the Mann-Whitney nonparametric test or two-way ANOVA. We also set a 70% threshold for correct diagnosis as the performance threshold for diagnostic tests available in clinics. We compared the percentage of randomly selected combinations of Table 1A biomarkers and non-Table 1A components that reached this threshold.

[0429] result 250 random selections The average performance obtained for combinations of 2, 5, 15, and 25 plasma components is shown in Table 14.

[0430] [Table 28]

[0431] Bold values ​​represent significant differences between Table 1A biomarkers and non-Table 1A constructs (p<0.0001, Mann Whitney non-parametric test).

[0432] It is important to note that the performance obtained with the five biomarkers from Table 1A is comparable to that obtained with the 15 and 25 non-Table 1A components (Figure 13).

[0433] 1000 random selections Two random biomarkers In 1000 random selections, the performance of using the two biomarkers in correctly identifying AD patients is, on average, 56.92% ± 0.002% for Table 1A biomarkers and 53.28% ± 0.002% for non-Table 1A constructs. This difference is significantly different at a p-value < 0.0001 (Mann Whitney nonparametric test).

[0434] This confirms that whatever the two random biomarkers taken in Table 1A, the performance obtained will statistically outperform that obtained with two random non-Table 1A components (Figure 14).

[0435] Thus, having at least two biomarkers from Table 1A increases the detection of asymptomatic Alzheimer's disease, demonstrating the superiority of all 119 biomarkers from Table 1A (when at least two of them are used) for asymptomatic detection of patients with AD over all other plasma components.

[0436] Five random biomarkers The performance of using the five biomarkers in correctly identifying patients with Alzheimer's disease is, on average, 61.65% ± 0.002% for Table 1A biomarkers and 56.17% ± 0.002% for non-Table 1A constructs. This difference is significantly different at a p-value < 0.0001 (Mann Whitney nonparametric test).

[0437] This confirms that whatever the five random biomarkers taken in Table 1A, the performance obtained will statistically outperform that obtained with five random non-Table 1A components (Figure 14).

[0438] Thus, having at least five biomarkers from Table 1A increases the detection of asymptomatic Alzheimer's disease, demonstrating the superiority of all 119 biomarkers from Table 1A (when at least five of them are used) for asymptomatic detection of patients with AD over all other plasma components.

[0439] 15 random biomarkers The performance of using the 15 biomarkers in correctly identifying patients with Alzheimer's disease is, on average, 68.27% ± 0.002% for Table 1A biomarkers and 61.56% ± 0.002% for non-Table 1A constructs. This difference is significantly different at a p-value < 0.0001 (Mann Whitney nonparametric test).

[0440] This confirms that whatever the 15 random biomarkers taken from Table 1A, the performance obtained will statistically exceed that obtained with 15 random non-Table 1A components (Figure 14). It is also interesting to note that the accuracy obtained with the five random biomarkers from Table 1A is comparable to that obtained with the 15 non-Table 1A components.

[0441] Thus, having at least 15 biomarkers from Table 1A increases the detection of asymptomatic Alzheimer's disease, demonstrating the superiority of all 119 biomarkers from Table 1A (when at least 15 of them are used) for asymptomatic detection of patients with AD over all other plasma components.

[0442] 25 random biomarkers The performance of using the 25 biomarkers in correctly identifying patients with Alzheimer's disease is, on average, 71.47% ± 0.001% for Table 1A biomarkers and 64.08% ± 0.002% for non-Table 1A constructs. This difference is significantly different at a p-value < 0.0001 (Mann Whitney nonparametric test).

[0443] This confirms that whatever the 25 random biomarkers taken in Table 1A, the performance obtained will statistically outperform that obtained with 25 random non-Table 1A components (Figure 14).

[0444] Thus, having at least 25 biomarkers from Table 1A increases the detection of asymptomatic Alzheimer's disease, demonstrating the superiority of all 119 biomarkers from Table 1A (when at least 25 of them are used) for asymptomatic detection of patients with AD over all other plasma components.

[0445] As can be seen in Figures 15A-B, there is a 65% chance of achieving greater than 70% accuracy with 25 biomarkers randomly selected from Table 1A, but only a 12% chance with 25 random non-Table 1A components. This increase in performance is also observable from two biomarkers; thus, the 70% accuracy threshold is achieved in 4% of cases with two biomarkers randomly selected from Table 1A, but in 0% of cases with non-Table 1A components. Thus, using at least two biomarkers from Table 1A significantly increases diagnostic performance to greater than 70% compared to other plasma components.

[0446] A multiple two-way ANOVA analysis confirmed that the biomarkers in Table 1A were superior to all plasma components (non-Table 1A components) for diagnosing Alzheimer's disease patients (Figure 15C).

[0447] It is interesting to note that the performance obtained with the five randomly selected biomarkers in Table 1A slightly exceeds that obtained with the 15 non-Table 1A components. Again, these results highlight the ability of the Table 1A biomarkers to distinguish Alzheimer's disease patients from non-Alzheimer's disease individuals.

[0448] Controls, including patients with other neurodegenerative diseases, support the specificity of the biomarkers in Table 1A for Alzheimer's disease from the asymptomatic stage.

[0449] conclusion In summary, we have identified plasma markers in rats with high useful value for distinguishing Alzheimer's disease rats from control rats (Example 1), which raises the important question of the transferability of these biomarkers identified in rats to humans.

[0450] Knowing that transgenic animal models are not very suitable for identifying blood biomarkers of AD, could we show that the AgenT rat model is superior for identifying biomarkers that carry information from the asymptomatic phase of the Alzheimer's state?

[0451] Analysis of all 119 "best in class" biomarkers previously identified in rats used in combination and compared with other combined plasma molecules (non-Table 1A components) shows high usefulness values ​​for all biomarkers in Table 1 A. Indeed, Example 2 shows that all combinations of biomarkers from Table 1 A provide clinical diagnostic value, even from combinations of as few as two biomarkers, that statistically exceeds the usefulness values ​​of non-Table 1A components.

[0452] Taken together, these data demonstrate that all biomarkers previously identified in rats (Table 1A) are useful and specific biomarkers of human AD, thus validating the effectiveness of both the AgenT rat model and the developed learning transfer approach used here for the first time.

Claims

1. 1. A computer system for determining whether a subject has an asymptomatic phase of Alzheimer's disease, the computer system comprising: (i) at least one processor; (ii) when executed by said processor, a. receiving input of levels, amounts, or concentrations of at least five biomarkers determined in a sample previously obtained from said subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers by organizing and / or modifying each level, amount, or concentration input via a machine learning algorithm to derive a probability score and / or classification label; the machine learning algorithm is trained on a training dataset; the training dataset comprises information relating to the levels, amounts, or concentrations of the same at least five biomarkers from samples previously obtained from subjects with a known Alzheimer's disease state; Steps and c. Producing an output that is a classification label or a probability score; at least one storage medium storing at least one code readable by said processor that causes said processor to perform Including, The biomarkers include mitochondrial 10 kDa heat shock protein, 14-3-3 protein, 1-methyladenosine, 1-methyl-5-imidazole acetate, 3,4-dihydroxybutyrate, 3-amino-2-piperidone, 3-methyl-2-oxobutyrate, 4-methyl-2-oxopentanoate, 5-methyl-2'-deoxycytidine, 5-methylthioadenosine (MTA), 5-oxoproline, mitochondrial adenylate kinase 4, allantoic acid, α-1B-glycoprotein, α-actinin-1, α-soluble NSF binding protein, anserine, anti-F4 / 80 kappa light chain variable region, apolipoprotein, arabonate / xylonate, and Arp2 / 3. Complex protein, biotinidase, BWK3, C4b-binding protein beta chain, calpain small subunit 1, calreticulin, carboxylesterase 1 family, carboxypeptidase B2, carnitine and conjugates, carnosine, cholate and conjugates, chromogranin A, clathrin heavy chain, coactosin-like protein, coagulation factor family, complement system family, creatine, creatinine, creatinine kinase family, cysteine-glutathione disulfide, dimethylarginine, dimethyl sulfone, EGF-containing fibulin-like extracellular matrix protein 1, EH domain-containing protein 3, elongation factor 1α, ergothioneine, erythronate, ethyl malonate, extracellular matrix protein 1, F-actin-capping protein subunit α-2, fibronectin 1, fibulin-1, fibulin-5, fructose, fructose-bisphosphate aldolase, FYN-binding protein 1, gelsolin, globin family, globulin family, glucuronate, glucose, glycosylphosphatidylinositol-specific phospholipase D1, HGF activator, histidine-rich glycoprotein, hyaluronidase, hydroxyproline, Ighg protein, integrin β, integrin subunit α V, interleukin-1 receptor accessory protein, keratin type II cytoskeleton 5, kininogen family, lipase, lumican, lysine and conjugates, lysyl oxidase-like1, macrophage migration inhibitory factor, major urinary protein, cytosolic malate dehydrogenase, mannan-binding lectin serine peptidase 2, mannose-binding protein A, microfibril-associated glycoprotein 4, multiple inositol polyphosphate phosphatase 1, myosin regulatory light chain polypeptide 9, myosin regulatory light chain RLC-A, N-acetylalanine, N-acetylasparagine, octadecanediate (C18), oleate / vaccenate (18:1), palmitate (16:0), palmitoleate (16:1n7), peptidyl-prolyl cis-trans isomerase A, peptidyl-prolyl cis-trans isomerase FKBP1A, peroxiredoxin 3, phenylalanine, polyubiquitin-C, prolyl hydroxyproline, proteasome complex family, rat MHC class I A computer system is provided that detects biomarkers selected from the group consisting of truncated cell surface antigen mRNA, retinoic acid receptor responder 2, retinol binding protein, ribonate (ribonolactone), ribulonate / xylulonate / lyxonate, sacsin molecular chaperone, serpin superfamily members, serum amyloid P component, sphingosine 1-phosphate, sulfate, mitochondrial superoxide dismutase [Mn], talin 2, tartronate (hydroxymalonate), thioredoxin, Tmprss13 protein, transferrin receptor protein 1, transthyretin, urinary protein 1, valerate and conjugates, voltage-dependent anion-selective channel protein 3, and Xaa-Pro aminopeptidase 2.

2. 1. A computer-implemented method for determining whether a subject has an asymptomatic phase of Alzheimer's disease, the method comprising: a. receiving an input of levels, amounts, or concentrations of at least five biomarkers selected from the group of biomarkers of claim 1 determined in a sample previously obtained from the subject; b. analyzing and transforming the level, amount, or concentration inputs of at least five biomarkers by organizing and / or modifying each level, amount, or concentration input via a machine learning algorithm to derive a probability score and / or classification label; the machine learning algorithm is trained on a training dataset; the training dataset comprises information relating to the levels, amounts, or concentrations of the same at least five biomarkers from samples previously obtained from subjects with a known Alzheimer's disease state; Steps and c. Producing an output that is a classification label or a probability score; A method comprising:

3. The computer system of claim 1 or the computer-implemented method of claim 2, wherein the training dataset comprises information related to the levels, amounts, or concentrations of the same at least five biomarkers of claim 1 from samples previously obtained from substantially healthy subjects and subjects known to be suffering from the asymptomatic stage of Alzheimer's disease.

4. 4. The computer system or computer-implemented method of claim 1, comprising providing a stratification of subjects suffering from asymptomatic stages of Alzheimer's disease into grades of the asymptomatic stages of Alzheimer's disease.

5. 5. The computer system or computer-implemented method of claim 4, wherein the asymptomatic stage grade of Alzheimer's disease is grade S1, S2, or S3.

6. 6. The computer system or computer-implemented method of claim 4 or 5, wherein step a. comprises receiving an input of levels, amounts, or concentrations of at least 14 biomarkers selected from the group of biomarkers of claim 1.

7. The training dataset comprises a variation profile of biomarkers for grades S1, S2, and S3 of asymptomatic Alzheimer's disease compared to substantially healthy subjects as set forth in the table below:

7. The computer system or computer-implemented method of any one of claims 1 to 6, wherein "+" means substantially higher (i.e., substantially higher by more than +5% compared to healthy subjects), "-" means substantially lower (i.e., substantially lower by more than -5% compared to healthy subjects), and "=" means substantially similar (i.e., less than ±5% compared to healthy subjects).

8. A computer program comprising processor readable software code adapted to perform the computer implemented method of any one of claims 2 to 7 when executed by said processor.

9. A non-transitory computer readable storage medium comprising code which, when executed by a computer, causes a processor to perform the computer-implemented method of any one of claims 2 to 7.

10. A molecular signature of the asymptomatic stage of Alzheimer's disease comprising at least five biomarkers selected from the group of biomarkers of claim 1.

11. The biomarkers include fructosyl lysine, integrin β, isobutyrylcarnitine (C4), myosin regulatory light chain RLC-A, talin 2, Igh-6 protein, octadecanediate (C18), ribonate (ribonolactone), mitochondrial 10 kDa heat shock protein, 5-hydroxylysine, mitochondrial adenylate kinase 4, calreticulin, creatine kinase type B, ergothioneine, peptidyl-prolyl cis-trans isomerase FKBP1A, globin c2, integrin subunit αV, myoglobin, and retinoids.

11. The molecular signature of Alzheimer's disease in an asymptomatic stage of claim 10, wherein the biomarker is selected from the group of biomarkers consisting of phosphate receptor responder 2, Tmprss13 protein, transferrin receptor protein 1, apolipoprotein E protein, Arp2 / 3 complex 34 kDa subunit, carnitine, isovalerylcarnitine (C5), coagulation factor VII, serine (or cysteine) proteinase inhibitor clade A member 4, serum amyloid P component, allantoic acid, calpain small subunit 1, carboxypeptidase B2, carnosine, clathrin heavy chain, complement C6, extracellular matrix protein 1, fructose-bisphosphate aldolase, keratin type II cytoskeleton 5, mannose-binding protein A, N-acetylasparagine, ribulonate, and Xaa-Pro aminopeptidase 2.

12. 1. A method for determining whether a subject has an asymptomatic stage of Alzheimer's disease, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers of claim 1 in a sample previously obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; A method comprising:

13. 1. A method for predicting the progression of subclinical phase of Alzheimer's disease in a subject, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers of claim 1 in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; A method comprising:

14. 1. A method for determining an individualized course of treatment for a subject suffering from the asymptomatic stage of Alzheimer's disease, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers of claim 1 in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; A method comprising:

15. 1. A method for stratifying subclinical stages of Alzheimer's disease in a subject into different subclinical stage grades, comprising: a) determining a molecular signature by measuring the level, amount, or concentration of at least five biomarkers selected from the group of biomarkers of claim 1 in a sample obtained from the subject; b) comparing the molecular signature obtained in step a) with a reference signature; c) stratifying the subject into subclinical grades of Alzheimer's disease based on the correlation between the reference signature and the molecular signature; A method comprising:

16. 16. The method of claim 15, wherein the different asymptomatic stage grades are S1, S2, or S3 grades.

17. 17. The method of claim 15 or 16, wherein the molecular signature comprises at least 14 biomarkers selected from the group of biomarkers of claim 1.

18. 18. The method of any one of claims 12 to 17, wherein the reference signature comprises the same levels, amounts, or concentrations of at least five biomarkers measured in a sample previously obtained from a substantially healthy subject.

19. 18. The method of any one of claims 12 to 17, wherein the reference signature comprises the same levels, amounts, or concentrations of at least five biomarkers measured in samples previously obtained from a population of substantially healthy subjects.

20. 20. The method of any one of claims 12 to 19, wherein the comparison in step b) is made by comparing the variations in level, amount or concentration of at least five biomarkers in said molecular signature and said reference signature with a variation profile of biomarkers in grades S1, S2 and S3 of asymptomatic stages of Alzheimer's disease compared to substantially healthy subjects as set forth in the table of claim 7.

21. The method of any one of claims 12 to 20, wherein the molecular signature comprises the biomarkers of claim 11.

22. The method of any one of claims 12 to 21, wherein the comparison in step b) is performed using at least one machine learning algorithm.

23. 23. The method of claim 22, wherein the at least one machine learning algorithm is selected from the group comprising an artificial neural network (ANN), a perceptron algorithm, a deep neural network, a clustering algorithm, a k-nearest neighbors (k-NN), a decision tree algorithm, a random forest algorithm, a linear regression algorithm, a linear discriminant analysis (LDA) algorithm, a quadratic discriminant analysis (QDA) algorithm, a support vector machine (SVM), a Bayesian algorithm, a simple rule algorithm, a clustering algorithm, a meta-classifier algorithm, a Gaussian mixture model (GMM) algorithm, a nearest centroid algorithm, an extreme gradient boosting (XG Boost) algorithm, a linear mixed-effects model algorithm, and combinations thereof.

24. 24. The method of claim 22 or 23, wherein the at least one machine learning algorithm is trained with a training dataset comprising information related to the levels, amounts, or concentrations of the same at least five biomarkers of claim 1 from samples previously obtained from substantially healthy subjects and subjects known to be suffering from the asymptomatic stage of Alzheimer's disease.

25. 25. The method of any one of claims 22 to 24, wherein the at least one machine learning algorithm is trained with a training dataset comprising biomarker variation profiles for asymptomatic stages of Alzheimer's disease grades S1, S2, and S3 compared to substantially healthy subjects as set forth in the table of claim 7.

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