METHODS AND KITS FOR DIAGNOSING MILD COGNITIVE IMPAIRMENT
20230025293 · 2023-01-26
Inventors
Cpc classification
G01N2333/70596
PHYSICS
G01N2333/70575
PHYSICS
G01N2800/52
PHYSICS
International classification
Abstract
Provided is a method for diagnosing a subject having or at risk of having mild cognitive impairment (MCI), including stimulating T cells in a biological sample obtained from the subject with an amyloid β peptide or a fragment thereof and evaluating a magnitude of a T cell response toward the amyloid β peptide or the fragment thereof. Also provided is a kit for diagnosing MCI by using the method.
Claims
1. A method for diagnosing a subject having or at risk of having mild cognitive impairment (MCI), comprising: stimulating T cells in a biological sample obtained from the subject with an amyloid β peptide or a fragment thereof; and evaluating a magnitude of a T cell response toward the amyloid β peptide or the fragment thereof.
2. The method according to claim 1, wherein the evaluating comprises measuring a level of a biomarker from the stimulated T cells, and wherein the biomarker is at least one selected from the group consisting of CD107a, IFNγ, IL-2, TNFα, and any combinations thereof.
3. The method according to claim 2, wherein the evaluating comprises measuring levels of at least two of CD107a, IFNγ, IL-2, and TNFα from the stimulated T cells.
4. The method according to claim 2, wherein the evaluating further comprises comparing the measured level of the biomarker to a reference level, and wherein a greater level of the biomarker as compared to the reference level is indicative of a higher likelihood that the subject is afflicted with or at risk of MCI.
5. The method according to claim 2, wherein the biomarker is expressed from CD4+ T cells.
6. The method according to claim 1, wherein the biological sample is a tissue sample or a fluid biological sample.
7. The method according to claim 1, wherein the biological sample is obtained from blood, plasma, serum, urine, sputum, saliva, cerebrospinal fluid, sweat, stool extract, tears, peritoneal fluid or brain of the subject.
8. The method according to claim 1, wherein the T cells are present in peripheral blood mononuclear cells (PBMCs) from the biological sample.
9. The method according to claim 1, wherein the amyloid β peptide comprises an amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 12.
10. The method according to claim 1, wherein the fragment of the amyloid β peptide comprises at least nine consecutive amino acids of an amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 12.
11. The method according to claim 1, wherein the fragment of the amyloid β peptide comprises an amino acid sequence represented by any one of SEQ ID NOs. 2 to 11.
12. The method according to claim 1, wherein the stimulating comprises incubating the T cells with the amyloid β peptide or the fragment thereof for at least 3 hours.
13. The method according to claim 1, wherein the subject is a mammal.
14. The method according to claim 13, wherein the mammal is selected from the group consisting of a human, a dog, a cat, a cow, a sheep, a pig, a horse, a monkey, a rodent, a murine, a rabbit, and a guinea pig.
15. The method according to claim 1, which is used to monitor a response, a side effect, or a combination thereof of the amyloid β peptide, the fragment thereof or an aggregate-related treatment.
16. The method according to claim 1, which is used to predict an efficacy of the amyloid β peptide, the fragment thereof or an aggregate-related treatment.
17. , The method according to claim 1, which is used to guide a therapeutic decision of the amyloid β peptide, the fragment thereof or an aggregate-related treatment.
18. The method according to claim 17, wherein the aggregate-related treatment is monoclonal antibody therapy.
19. The method according to claim 15, wherein the side effect is selected from the group consisting of weakness, headache, fever, chills, nausea, vomiting, diarrhea, rashes, low blood pressure, and any combination thereof.
20. The method according to claim 1, which is used to monitor at least one amyloid-related disease selected from the group consisting of Alzheimer disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, cerebral amyloid angiopathy, inflammatory cerebral amyloid angiopathy and cerebral amyloidoma.
21. The method of claim 1, which is used to evaluate the T cell response induced by the amyloid β peptide or the fragment thereof.
22. A kit for diagnosing a subject having or at risk of having mild cognitive impairment (MCI), comprising: an amyloid β peptide or a fragment thereof; and at least one reagent specific to at least one biomarker selected from the group consisting of CD107a, IFNγ, IL-2, and TNFα.
23. The kit according to claim 22, wherein the at least one reagent is an antibody specifically binding to a corresponding biomarker thereof based on antigen-antibody interaction.
24. The kit according to claim 22, wherein the amyloid β peptide has an amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 12.
25. The kit according to claim 22, wherein the fragment of the amyloid β peptide comprises at least 10 consecutive amino acids of an amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 12.
26. The kit according to claim 22, wherein the fragment of the amyloid β peptide has an amino acid sequence represented by any one of SEQ ID NOs. 2 to 11.
27. The kit according to claim 22, further comprising an instruction for use.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The present disclosure can be more fully understood by reading the following descriptions of the embodiments, with reference made to the accompanying drawings.
[0017]
[0018]
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0019] The following examples are used to exemplify the present disclosure. A person of ordinary skill in the art can understand the other advantages of the present disclosure, based on the specification of the present disclosure. The present disclosure can also be implemented or applied as described in different examples. It is possible to modify and/or alter the following examples for carrying out this disclosure without contravening its scope for different aspects and applications.
[0020] It is noted that, as used in this disclosure, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise. For example, when separating items in a list, “or” or “and/or”shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
[0021] As used herein, the term “essentially” refers to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or greater of the said feature. For example, the term “consisting essentially of” used herein may indicate a peptide consisting of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or greater of the amino acids of the referential sequence.
[0022] As used herein, the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, which are included in the present disclosure, yet open to the inclusion of unspecified elements. For example, a composition, mixture, process or method that comprises a list of elements or actions is not necessarily limited to only those elements or actions, but may include other elements or actions not expressly listed, or inherent to such composition, mixture, process, or method.
[0023] As used herein, the phrases “at least one” and “one or more” may have the same meaning and include one, two, three, or more. For example, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).
[0024] As used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of time periods, temperatures, operating conditions, ratios of amounts, and the likes disclosed herein should be understood as modified in all instances by the term “about.”
[0025] As used herein, the terms “subject,” “individual” and “patient” may be interchangeable and refer to an animal, e.g., a mammal. For example, the term “subject” may refer to both the male and female gender unless one gender is specifically indicated. Further, the term “patient” may refer to a “subject” who is suspected to be, or afflicted with a disease or condition. In some embodiments, the subject to be tested with the method of the present disclosure may be a human, a domestic animal (e.g., a dog, a cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rodent, a murine, a rabbit, a guinea pig, or the like).
[0026] As used herein, the term “biological sample” refers to any sample including: tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); or cell fractions, fragments or organelles (such as those obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include brain tissue, whole blood, serum, plasma, urine, sputum, saliva, cerebrospinal fluid, sweat, stool extract, synovial fluid, tears, peritoneal fluid, or any combination thereof.
[0027] As used herein, the term “diagnosing,” “diagnostic,” and “diagnosis” refer to methods by which a skilled artisan may estimate and/or determine the probability (“a likelihood”) of whether or not a subject is suffering from a given disease or condition. That such a diagnosis is “determined” is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. A skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold may indicate a greater likelihood of the occurrence of a disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.
[0028] As used herein, the term “development” or “progression” of a disease refers to initial manifestations and/or ensuing progression of the disease. Development of the disease may be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. The term “development” includes occurrence, recurrence, and onset of a disease, condition or disorder. As used herein, the term “onset” or “occurrence” of a target disease, condition or disorder includes initial onset and/or recurrence thereof.
[0029] In at least one embodiment, the present disclosure is directed to a method for diagnosing a subject having or at risk of having mild cognitive impairment (MCI), comprising stimulating T cells in a biological sample obtained from the subject with an amyloid β peptide or a fragment thereof, and evaluating a magnitude of a T cell response toward the amyloid β peptide or the fragment thereof.
[0030] In at least one embodiment of the present disclosure, the evaluating comprises measuring a level of a T cell response biomarker from the stimulated T cells, such as CD4+ T cells. In some embodiments, the evaluating further comprises comparing the measured level of the biomarker to a reference level, wherein a greater level of the biomarker as compared to the reference level is indicative of a higher likelihood that the subject is afflicted with or at risk of MCI.
[0031] As used herein, a “reference level” may be an absolute value, a relative value, a range of values, an average value, a median value, a mean value, a statistic value, a cut-off or discriminating value, or a value as compared to a particular control or baseline value. The reference level may be based on an individual sample value, such as a value obtained from a sample from a subject other than the individual tested or from a “normal” individual that is an individual identified as having a healthy status or an individual not diagnosed with MCI.
[0032] In at least one embodiment of the present disclosure, the T cells to be stimulated with the amyloid β peptide or the fragment thereof may be present in peripheral blood mononuclear cells (PBMCs) of the subject. In some embodiments, the method described herein may further comprise providing a biological sample (such as a fluid biological sample or a tissue sample) of the subject and isolating the PBMCs from the biological sample.
[0033] In at least one embodiment of the present disclosure, the method described herein may further comprise performing an additional assay for diagnosis of MCI, thereby increasing the diagnostic accuracy. The additional assay for diagnosis of MCI may be well known in the art. For example, the clinical criteria used for diagnosis of MCI include (1) memory complaints corroborated by an informant; (2) objective memory impairment beyond that expected for age and education; (3) normal general cognitive function; (4) intact activities of daily living; and (5) the subject failing to meet criteria for dementia.
[0034] In at least one embodiment, the present disclosure is also directed to a kit for diagnosing a subject having or at risk of having MCI. As used herein, the term “kit” includes, but is not limited to, at least one reagent for detecting, identifying, and/or analyzing a biological sample from a subject, and a legend providing instructions to the user on how to use the kit. The kit provided herein may be in suitable packaging, including, but not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a medical device which is applicable and well known in this field with no limitation.
[0035] In at least one embodiment of the present disclosure, the kit comprises an amyloid β peptide or a fragment thereof, and at least one reagent that is specific to at least one T cell response biomarker, such as cluster of differentiation 107a (CD107a), interferon-γ (IFNγ), interleukin 2 (IL-2), and tumor necrosis factor-α (TNFα). In some embodiments, the reagent can be an antibody that specifically binds to the corresponding biomarker thereof based on antigen-antibody interaction, such as an anti-CD107a antibody, an anti-IL-2 antibody, an anti-TNFα antibody, and an anti-IFNγ antibody.
[0036] In at least one embodiment, the kit of the present application may comprise instructions for use in accordance with any of the methods described herein. The included instructions may comprise a description of incubating T cells with the amyloid β peptide or the fragment thereof. The kit may further comprise a description of evaluating the magnitude of an amyloid-specific T cell response by measuring the level of the T cell response biomarker. In some embodiments, the reagent contained in the kit is detectable for measuring the level of the T cell response biomarker. The biomarkers labeled with the detectable reagent may be quantitated by using assays including, but not limited to, flow cytometry, magnetic-activated cell sorting (MACS) and enzyme-linked immunospot (ELISPOT) assay.
[0037] As used herein, the phrase “specifically binds” to a target refers to a binding reaction that is determinative of the presence of a molecule in the presence of a heterogeneous population of other biologics. Therefore, under designated immunoassay conditions, a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample.
[0038] Many examples have been used to illustrate the present disclosure. The examples below should not be taken as a limit to the scope of the present disclosure.
EXAMPLES
Example 1
Sample Processing and PBMCs Isolation
[0039] For blood sampling, human subjects were recruited from Far Eastern Memorial Hospital (FEMH) in this study. The study was approved by the FEMH's institutional ethical committee (Case No. FEMH 105147-F) and written informed consent were acquired from all participants. Detailed information on study participants was listed in Table 1 below.
TABLE-US-00001 TABLE 1 Demographic data of the human subjects MCI Normal Characteristics (N = 69) (N = 69) P-value Age, years, mean (SD) 72.1 (4.9) 71.8 (5.5) 0.79 Female, no. (%) 38 (55.0) 40 (58.0) 0.73 Education, years, 5.4 (3.1) 6.7 (3.6) 0.029 mean (SD) Married, no. (%) 57 (83.0) 51 (74.0) 0.22 Never smoking, no. (%) 51 (74.0) 51 (74.0) 0.73 Hypertension, no. (%) 36 (52.0) 35 (51.0) 0.86 Cardiovascular diseases, 16 (24.0) 10 (15.0) 0.19 no. (%) BMI, kg/m.sup.2, mean (SD) 24.9 (3.0) 25.4 (3.3) 0.28 GDS > 5, no. (%) 13 (19.0) 8 (12.0) 0.24 MMSE, mean (SD) 24.9 (3.0) 25.4 (3.3) <0.001 APOE4, no. (%) 14 (20.0) 10 (14.0) 0.37 Amyloid pool total 0.9 (0.7) 0.5 (0.6) <0.001 response, mean (SD) Amyloid full-length total 0.8 (0.7) 0.3 (0.3) <0.001 response, mean (SD) SD: standard deviation; BMI: body mass index; GDS: geriatric depression scale; MMSE: mini-mental state examination; APOE4: apolipoprotein E4.
[0040] On the day of blood sampling, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS gradient centrifugation following the manufacturer's protocol (GE Healthcare) and used in the subsequent experiment.
Example 2
T Cell Stimulation and Quantification of a T Cell Response Against Amyloid
[0041] The use of protein-spanning mixtures of overlapping peptides was efficient for immunostimulation of T-lymphocytes and diagnostic applications. In this disclosure, the overlapping 15mer peptides derived from the full-length amyloid β protein (Aβ1-42) were created and used for T cell stimulation. The cytomegalovirus (CMV) pp65 protein peptide pool (JPT Peptide Technologies, Germany) and chemical phorbol myristate acetate (PMA)/ionomycin were used as positive controls for PBMC stimulation.
[0042] The amino acid sequence of the full-length amyloid β protein (Aβ1-42) from JPT Peptide Technologies were DAEFRHDSGYEVHHQKLVFFGEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO. 1). The peptide pool composed of 10 sequences created based on 15mer amino acid length with sequential 3 amino acid shifts of the full-length amyloid β protein (Aβ1-42) included: DAEFRHDSGYEVHHQ (Aβ1-15, SEQ ID NO. 2), FRHDSGYEVHHQKLV (Aβ4-18, SEQ ID NO. 3), DSGYEVHHQKLVFFG (Aβ7-21, SEQ ID NO. 4),
[0043] YEVHHQKLVFFGEDV (Aβ10-24, SEQ ID NO. 5), HHQKLVFFGEDVGSN (Aβ13-27, SEQ ID NO. 6), KLVFFGEDVGSNKGA (Aβ16-30, SEQ ID NO. 7), FFGEDVGSNKGAIIG (Aβ19-33, SEQ ID NO. 8), EDVGSNKGAIIGLMV (Aβ22-36, SEQ ID NO. 9), GSNKGAIIGLMVGGV (Aβ25-39, SEQ ID NO. 10) and KGAIIGLMVGGVVIA (Aβ28-42, SEQ ID NO. 11).
[0044] The full-length amyloid β protein as well as the amyloid peptide pool and control CMV peptide pool were used for PBMC cell stimulation (1 mg/mL per peptide) along with co-stimulation of anti-CD28/CD49d, anti-CD107a, Golgi Stop (containing monensin, BD Biosciences) and GolgiPlug (containing brefeldin A, BD Biosciences) for six hours at 37° C. Subsequently, cells were stained with anti-CD3, anti-CD8, anti-CD4 and Live/Dead Cell Viability Assay Kit (Invitrogen) for 20 minutes before fixation with Cytofix/Cytoperm buffer (BD Biosciences). Cells were fixed, washed, and stained with anti-CD40L, anti-IL-2, anti-TNFα, and anti-IFNγ. Results were acquired using a multicolor flow cytometer (Beckman Coulter Cytoflex) at the Far Eastern Memorial Hospital Core Lab. Flow cytometry results were analyzed using FlowJo (Tree Star).
[0045] After gated on live CD3+ cells, CD4+ and CD8+ cells were analyzed separately for cytokine expression in response toward each stimulation. A combinatorial gating strategy based on the gates of each effector function was further analyzed for co-expression pattern using the FlowJo Boolean gate platform to derive the statistics of combinatorial function expression pattern. Each single function and total functional response of T cells against each stimulation was derived and compared. The representative images of T cell cytokine production in response toward amyloid protein were shown in
[0046] Magnitudes of PBMC CD4+ T cell response toward amyloid β peptide pool and amyloid β full-length peptide were tested for their sensitivity and specificity for distinguishing MCI from normal. Correctly classified participants, sensitivity, and specificity were calculated using the cut-off that produced the highest index from Liu's method for MCI versus normal. Total response was enumerated as the percentage of T cells among total CD4+ T cells responding toward the amyloid β peptide pool and amyloid β full-length peptide stimulation with at least one measured biomarker (i.e., CD40L, CD107a, IFNγ, IL-2 or TNFα).
[0047] The area under the receiver operating characteristic curve (ROC) was used to examine the discriminative performance of the biomarkers. Confidence intervals (CI) for areas under curve (AUCs) were calculated based on 2,000 bootstrap samples using the non-parametric method. The cut-offs for amyloid pool and amyloid full-length peptide responses were established at the highest product of sensitivity and specificity to discriminate between MCI and age-matched normal in each ROC analysis (Liu's method). The results of ROC analyses were shown in Tables 2 and 3 below and
TABLE-US-00002 TABLE 2 ROC analyses of T cell response biomarkers for amyloid β peptide pool response Biomarker Correctly (% CD4 + Cut- classified Sensi- Speci- T cell) AUC (95% CI) off participants tivity ficity Total 0.72 (0.63-0.8) 0.53 0.69 0.65 0.72 response CD40L 0.48 (0.38-0.57) 0.1 0.51 0.39 0.62 CD107a 0.83 (0.75-0.89) 0.036 0.82 0.87 0.77 IFNγ 0.84 (0.76-0.9) 0.062 0.80 0.74 0.86 IL-2 0.77 (0.69-0.85) 0.0485 0.72 0.78 0.67 TNFα 0.61 (0.51-0.7) 0.081 0.62 0.70 0.55
TABLE-US-00003 TABLE 3 ROC analyses of T cell response biomarkers for amyloid β full-length peptide response Biomarker Correctly (% CD4 + classified Sensi- Speci- T cell) AUC (95% CI) Cut-off participants tivity ficity Total 0.83 (0.75-0.89) 0.314 0.8 0.81 0.78 response CD40L 0.46 (0.37-0.56) 0.056 0.5 0.46 0.54 CD107a 0.86 (0.79-0.91) 0.06 0.81 0.81 0.81 IFNγ 0.77 (0.68-0.84) 0.064 0.73 0.64 0.83 IL-2 0.79 (0.7-0.86) A single cut-off cannot be determined because there are multiple cut-offs with the same sensitivity and specificity TNFα 0.7 (0.61-0.79) 0.048 0.68 0.68 0.68
[0048] From the above, it is found that CD4+ T cell responses toward amyloid β full-length peptide as well as amyloid β peptide pool can be used to distinguish elderly MCI and normal elderly. The method provided herein is based on stimulation of PBMCs and measurements of a cytokine effector response in stimulated T cells by multicolor flow cytometry, and thus is easier to be standardized when compared to proliferation assays. Because blood T cell responses toward amyloid peptide are rare events (with less than 0.1% of T cells among CD4+ cells), the multi-marker method provided herein increases the absolute value of measurements to more than 0.1% and shows persistent ability to distinguish MCI from normal. Accordingly, the method of the present disclosure can be utilized to categorize elderly individuals with risk for developing MCI and used to investigate its predictive power for the development of any amyloid-related neurodegenerative diseases.
[0049] While some of the embodiments of the present disclosure have been described in detail above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the embodiments shown without substantially departing from the teaching and advantages of the present disclosure. Such modifications and changes are encompassed in the scope of the present disclosure as set forth in the appended claims.
[0050] All references and publications cited and discussed herein are hereby incorporated by reference in their entirety and to the same extent as if each reference or publication was individually incorporated by reference.
REFERENCE
[0051] [1] Ferri C P, Prince M, Brayne C, et al. Global prevalence of dementia: a Delphi consensus study. Lancet 2005; 366(9503):2112-7. [0052] [2] Morris J C. Early-stage and preclinical Alzheimer disease. Alzheimer Disease and Associated Disorders 2005; 19(3): 163-5. [0053] [3] Gauthier S, Reisberg B, Zaudig M, et al. Mild cognitive impairment. Lancet 2006; 367(9518):1262-70. [0054] [4] Petersen R C. Mild cognitive impairment: current research and clinical implications. Seminars in Neurology 2007; 27(1):22-31. [0055] [5] Petersen R C, Smith G E, Waring S C, et al. Mild cognitive impairment: clinical characterization and outcome. Archives of Neurology 1999; 56(3):303-8. [0056] [6] Masters C L, Beyreuther K. The neuropathology of Alzheimer's disease in the year 2005. In: Beal M F, Lang A E, Ludolph A C, eds. Neurodegenerative Diseases: Neurobiology, Pathogenesis and Therapeutics. Cambridge: Cambridge University Press 2005. [0057] [7] Rabinovici G D, Jagust W J. Amyloid imaging in aging and dementia: testing the amyloid hypothesis in vivo. Behavioural Neurology 2009; 21(1): 117-28. [0058] [8] Small G W, Kepe V, Ercoli L M, et al. PET of brain amyloid and tau in mild cognitive impairment. The New England Journal of Medicine 2006; 355(25):2652-6. [0059] [9] Villemagne V L, Chetelat G. Neuroimaging biomarkers in Alzheimer's disease and other dementias. Ageing Research Reviews 2016; 30:4-16. [0060] [10] Pike K E, Savage G, Villemagne V L, et al. Beta-amyloid imaging and memory in non-demented individuals: evidence for preclinical Alzheimer's disease. Brain 2007; 130(Pt 11):2837-44. [0061] [11] Ong K, Villemagne V L, Bahar-Fuchs A, et al. .sup.18F-florbetaben Aβ imaging in mild cognitive impairment. Alzheimer's Research and Therapy 2013; 5(1):4. [0062] [12] Ma Y, Zhang S, Li J, et al. Predictive accuracy of amyloid imaging for progression from mild cognitive impairment to Alzheimer disease with different lengths of follow-up: a meta-analysis. Medicine (Baltimore) 2014; 93(27):e150.