CSF Tau and Aβ42: Logical Biomarkers for Alzheimer’s Disease?

Neurobiology of Aging, Vol. 19, No. 2, pp. 117–119, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00

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CSF Tau and Ab42: Logical Biomarkers for Alzheimer’s Disease? D. GALASKO Department of Neurosciences (Neurology), University of California, San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161 THE concept of Alzheimer’s Disease (AD) as a clinical diagnosis made only after excluding every other potential cause of dementia and confirmed only by autopsy is obsolete. Clinico-pathologic studies have shown accuracy of clinical diagnosis of 85% or higher (4,10,13,19). Diagnosing AD at an early or very early stage is more difficult, although selective psychometric tests have high sensitivity and specificity in distinguishing AD from elderly normals, and can show abnormalities presymptomatically. These findings originate from expert research centers and use resources exceeding those of routine clinical practice. Viewed against this background, biological markers can find a niche in clinical assessment, for example by “confirming” (in reality strengthening) a clinical diagnosis, or aiding in differential diagnosis. Because symptomatic AD is associated with substantial synaptic and neuronal loss in critical brain areas, early or preventative treatment is a priority. To complement this, very early or presymptomatic diagnosis must be a major goal of biological marker studies. In addition, biological markers provide insight into mechanisms of AD, and potentially can be used to monitor the effects of treatment. Issues of cost and convenience are important if biomarkers are to find widespread clinical use, but are secondary to the marker first proving its potential in rigorously conducted studies. To illustrate some of the progress and problems in developing and validating biomarkers (15), recent studies of cerebrospinal fluid (CSF) Ab42 and tau (1–3,7–9,12,14,16 –18,20,21) will be discussed. Similar principles apply to other types of markers, such as neuroimaging tests. First, markers should have a rationale or plausibility. The defining lesions of AD are senile plaques containing deposits of Ab, especially longer forms of this protein ending at amino acid 42 (Ab42), and neurofibrillary tangles (NFT) composed mainly of tau protein. Because CSF is in contact with the brain, measuring Ab42 and tau may provide indices related to amyloid deposition and axonal changes or tangle formation. A precise and reliable assay is required. In the case of Ab42 and tau, sandwich enzyme-linked immunosorbent assays (ELISAs) using specific monoclonal antibodies are needed to detect levels in the pg to low ng/mL range (14,20,21). Monoclonals that detect Ab42 but not Ab40 have been a key development. In addition to establishing the high sensitivity and very low variability of ELISAs, studies of repeated CSF measures of Ab42 and tau over 4 – 6 weeks have shown excellent test-retest reliability. It is important to know what the marker is measuring. For CSF studies, purifying the protein or marker from antemortem CSF is very helpful. CSF tau has been measured using several different ELISA whose antibodies detect all isoforms regardless of phosphorylation

state. Recent immunopurification of tau from CSF revealed only fragments rather than full length tau, which were not highly phosphorylated (9). Efforts to measure highly phosphorylated forms of tau in CSF are likely to fail, unless fragments from other regions of tau also exist in CSF. Do markers need to be adjusted or normalized for other variables? For CSF markers, caudal-cranial concentration gradients, diffusion from serum to CSF, the need to correct for total protein concentration, and effects of storage (including freezing and thawing) must be evaluated. In this regard, CSF tau is remarkably stable and can be measured accurately in samples stored frozen for years. Ab42 is more prone to loss during storage. Neither marker is detectable in serum at higher concentrations than in CSF, shows a CSF gradient, or correlates with CSF protein concentration. Some CSF markers were initially studied in autopsy CSF. This is of limited use, because postmortem proteolysis has profound effects on the constituents of CSF, and the terminal stages of AD may not be relevant to early AD, when the biomarker is needed. Initial clinical studies should show that markers distinguish probable AD from age-comparable normal controls. Sample size is important, as shown by a power analysis. To detect a difference between AD and controls of $1 standard deviation of the overall group (a minimal definition of a difference that may be clinically useful), with 80% power and 2-sided significance of 0.05, needs 20 subjects per group. Assuming diagnostic inaccuracy of 10 –15%, the number should be enlarged to at least 25 per group. If covariates such as age, sex, or apolipoprotein E (ApoE) genotype are to be analyzed, a much larger series is needed. Gold standard diagnoses are required to calibrate the biomarker. Patients with AD should meet research criteria (e.g., NINCDS-ADRDA probable AD) and should be free of additional neurological diagnoses such as stroke. Patients should not be taking medications known to affect the marker(s). Controls should be age-matched, free of significant neurological disease, and should receive cognitive testing. Ideally, controls should receive follow-up. Only if insufficient normal controls are available may disease controls be used; dementia must be excluded. A group of nondemented patients with various neurological disorders (e.g., Parkinson’s disease, multiple sclerosis, or peripheral neuropathy) also should be studied to establish whether general neurological factors influence levels of the marker. Having found a significant difference between AD and controls once, the most important next step is to replicate the finding in another series of patients. For example, over 15 studies have all shown that CSF tau is increased in AD, and at least 4 studies have shown that Ab42 is decreased. 117

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This unanimity contrasts with studies of markers such as CSF a-1-antichymotrypsin, or pupil dilatation in response to tropicamide, where conflicting positive and negative studies have been reported. The question of discriminating between AD and other dementias is important but complex. Unfortunately, clinical criteria for many of these disorders are less well-validated than those for AD, making the gold standard less trustworthy. A more important problem, highlighted by recent autopsy studies, is that AD often coincides with many dementing disorders, including vascular dementia, dementia with Lewy bodies, progressive supranuclear palsy, and others (4 – 6,10,11). Unless criteria for non-AD dementias can categorically exclude coexisting AD, interpretation of biomarkers in these conditions may be difficult. Autopsy confirmaton is the ultimate gold standard, but requires extensive resources and very long follow-up if the biomarker is measured at an early stage of the disease. For CSF tau, studies have shown increased levels, above the normal limit, in about 10 –20% of neurological controls (1,2,8,12,14,17,21). These may be falsely positive, i.e., tau is liberated into CSF in some patients with non-AD disorders, or truly positive, i.e., some patients have AD. The very high levels of CSF tau seen after large acute strokes suggest that a key determinant of tau release into CSF is the extent or rate of axonal or neuronal damage. Defining a normal range of values for the marker is critical. Needless to say, the larger the number of normals from whom this range is derived, the better. Several options are available, depending on whether assumptions of the normal distribution are met or not. If levels of the marker appear normally distributed, then the mean 6 2 or 3 standard deviations can be used. This may be overly conservative, especially if the sample of normals is small. ROC analysis can be generated to yield cutoffs that optimizes sensitivity, specificity, or both, as needed. For an AD biomarker, specificity is more important than sensitivity to avoid falsely labeling normals. This approach has generally been used to select cufoffs in studies of CSF tau, where sensitivity is about 60 – 85% for specificity of 95% (9,10,13). A combination of markers may provide greater diagnostic accuracy than each individually, especially in a complex and slowly-evolving disorder like AD. If more than one marker is considered simultaneously, several techniques are available, including logistic regression, discriminant function analysis, and classification tree analysis. Covariates that may influence levels of the marker should be carefully examined. Of fundamental importance are age, gender, and severity of dementia estimated by cognitive test scores. For example, CSF tau increases and Ab42 decreases slightly as dementia severity increases, as indexed by scores on the MiniMental State Examination (MMSE). In normals, age is associated with a minimal increase in CSF tau and no change in Ab42. Therefore, these markers do not need adjustment for age. Another factor is heterogeneity among patients with AD, for example the presence or absence of Parkinsonian signs, or genetic factors

associated with AD, such as ApoE genotype. Finally, comorbid illnesses may potentially affect marker levels, as may medications. For example, studies of markers based on inflammation should analyze whether acute or chronic inflammatory disorders or anti-inflammatory medications influence their levels. The utility of the marker in very mild AD should be carefully determined. Because definitions of mild AD differ, comparing studies (and markers) is difficult. For the MMSE, a widely used cognitive screening test, a cutoff of 23/30 or lower for AD has been recommended. Very mild AD can be arbitrarily defined by MMSE scores .23. To ensure that patients with higher MMSE scores are in fact demented, more detailed cognitive testing, informant interview, and ideally follow-up to document progression over time are essential. Two recent studies used this approach and found 70 – 80% of AD patients with MMSE scores $24/30 had increased levels of CSF tau (3,16). Initial promise needs to be reaffirmed in larger-scale studies, to obtain more precise estimates of cutoff points, sensitivity, and specificity. Predictive or preclinical identification of people destined to develop AD will be important as therapies are developed aimed at delaying onset. Although the analogy with serum cholesterol and coronary artery risk is easy to make, the logistics of proving that a marker predicts AD 5 to 10 years before its onset are formidable. A biomarker study is conceptually difficult and very expensive to carry out in this situation, but so also is a therapeutic study aimed at preventing AD from developing in normals. For example, in a elderly cohort enriched with risk factors for AD (e.g., positive family history and the ApoE e4 allele) initially studied while cognitively normal, even if the risk of dementia is increased tenfold, the incidence of AD will still be low (,15% per year), requiring hundreds of subjects and several years of follow-up to obtain an answer. Such a biomarker study would be invaluable, and economy of scale is obtained by coupling it to existing or planned epidemiologic studies of incident AD, or to preventive therapeutic studies. The cost and convenience of the biomarker test looms prominently here: asymptomatic subjects will be less willing to agree to a CSF test than to a blood test. Can a biomarker be used as a surrogate measure of outcome in a clinical trial for AD? There are technical obstacles: CSF markers are limited by the inconvenience of lumbar puncture, (especially when this needs to be repeated); neuroimaging studies are hampered by expense, and difficult with standardization and reliability when repeated in the same patient or attempted at many centers. Longitudinal data from markers piloted on a small group of patients in short- to medium-term treatment studies are needed to show feasibility before this approach is attempted in a large multi-center clinical trial. Many biological markers have been proposed for AD, among which CSF tau and Ab42 show considerable promise. This commentary emphasizes the need for rigorous evaluation of markers to define their potential applications in clinical practice.

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