Combined CSF tau, p-tau181 and amyloid-b 38/40/42 for diagnosing Alzheimer s disease

J Neural Transm (2009) 116:203 212 DOI 10.1007/s00702-008-0177-6 ALZHEIMER S DISEASE AND RELATED DISORDERS - ORIGINAL ARTICLE Combined CSF tau, p-tau181 and amyloid-b 38/40/42 for diagnosing Alzheimer s disease Volker Welge Æ Oliver Fiege Æ Piotr Lewczuk Æ Brit Mollenhauer Æ Hermann Esselmann Æ Hans-Wolfgang Klafki Æ Stefanie Wolf Æ Claudia Trenkwalder Æ Markus Otto Æ Johannes Kornhuber Æ Jens Wiltfang Æ Mirko Bibl Received: 17 July 2008 / Accepted: 17 December 2008 / Published online: 14 January 2009 Ó Springer-Verlag 2009 Abstract Cerebrospinal fluid (CSF) concentrations of amyloid-b (Ab) 1-38, 1-40, 1-42, total-tau and phospho-tau in samples from 156 patients with Alzheimer s disease (AD) (n = 44), depressive cognitive complainers (DCC, n = 25) and various other forms of non-alzheimer dementias (NAD, n = 87) were analyzed by electrochemiluminescence and enzyme linked immunosorbent assay, respectively. A significant decrease of CSF Ab1-42 was the most powerful single marker for differentiation of AD from DCC, yielding accuracies of beyond 85%. Increased p-tau and the ratio Ab1-42/Ab1-38 yielded accuracies of beyond 80 and 85%, respectively, to discriminate AD versus NAD. Combining p-tau with Ab1-42/Ab1-38 resulted in a sensitivity of 94% for detection of AD and 85% specificity for excluding NAD. Decreased CSF Ab1-42 represents a core V. Welge O. Fiege H.-W. Klafki J. Wiltfang M. Bibl (&) Klinik für Psychiatrie und Psychotherapie, Universität Duisburg-Essen, Rheinische Kliniken Essen, Virchowstr. 174, 45147 Essen, Germany e-mail: Mirko.Bibl@lvr.de; Melanie.Kownatka@lvr.de P. Lewczuk H. Esselmann J. Kornhuber Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany B. Mollenhauer S. Wolf Department of Psychiatry, University of Goettingen, von-siebold-str. 5, 37075 Göttingen, Germany C. Trenkwalder University of Goettingen, Paracelsus-Elena Klinik, Kassel, Germany M. Otto Department of Neurology, University of Ulm, Steinhövelstr. 1, Ulm, Germany biomarker for AD. The lack of specificity for exclusion of NAD can be most effectively compensated by the ratio Ab1-42/Ab1-38. The ratio Ab1-42/Ab1-38/p-tau powerfully discriminates AD versus NAD and fulfils the accuracy requirements for an applicable screening and differential diagnostic AD biomarker. Keywords Alzheimer s disease Cerebrospinal fluid Amyloid-b peptides Biomarkers ELISA Electrochemiluminescence Abbreviations Ab peptides Ab-SDS-PAGE/ immunoblot AD CSF DCC ELISA MMSE NINCDS-ADRDA NINDS-SPSP Introduction Amyloid-beta peptides Amyloid-beta-sodium-dodecylsulphate-polyacrylamide-gel electrophoresis with western immunoblot Alzheimer s disease Cerebrospinal fluid Depressive cognitive complainers Enzyme linked immunosorbent assay Mini-mental-status examination National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer s Disease and Related Disorders Association National Institute of Neurological Disorders and Stroke and the Society for PSP The differential diagnosis of Alzheimer s disease (AD) on clinical grounds is often challenging, and misclassifications

204 V. Welge et al. are frequent, particularly outside of specialized centers. AD biomarkers in cerebrospinal fluid (CSF) are currently under intensive investigation. Ab peptides as well as tau proteins have shown diagnostic potential to support the clinical diagnosis of AD. Both are related to the neuropathological hallmarks of AD: extracellular deposition of amyloid-beta (Ab) peptides into neuritic plaques (Glenner and Wong 1984), preferentially peptides ending at alanine-42 (Ab42), and formation of intracellular neurofibrillary tangles (NFT s). The major constituent of NFT s is tau, a microtubule-associated protein that undergoes excessive phosphorylation (phosphorylated tau, p-tau) at multiple sites (e.g. p-tau199, p-tau181 and p-tau231) and aggregates into paired helical filaments in AD (Lee et al. 1991). Ab peptides, mainly Ab1-40, Ab1-38 and Ab1-42 (Wiltfang et al. 2002) as well as tau proteins physiologically appear in CSF. Ab42 was found to be selectively reduced in AD, whereas the decrease of Ab42 was accompanied by diminished overall Ab peptide levels (e.g. Ab1-40 or Ab1-38) in other dementias (Bibl et al. 2006a, b, 2007a, b). Additionally, CSF levels of total-tau (tau) and p-tau are elevated in AD. Irrespective of which p-tau epitope was measured, CSF p-tau seems to be more specific for AD than total tau levels, which most probably simply reflect the rate of neurodegeneration. The diagnostic power of these AD biomarkers or combinations thereof depends on the pre-analytical handling of CSF samples, the assay method and the investigated diagnostic groups. Correspondingly, the reported accuracies vary between different studies (Blennow 2004). Forthcoming high throughput capable multiplexing platforms call for promising biomarker candidates that may be combined in these assay formats. Here we addressed the question of which combination of biomarkers will be most suitable to differentiate AD from differential diagnostic relevant disorders like depression with cognitive complaints and dementias other than AD. We simultaneously investigated the diagnostic power of Ab1-38, Ab1-40, Ab1-42, p-tau181 and tau for AD differential diagnosis by use of commercially available high throughput assays. Patients and methods A total of 156 CSF samples were referred to our laboratory between 1999 and 2004 and investigated. CSF concentrations of Ab1-38 (n = 111), Ab1-40 (n = 105), Ab1-42 (n = 156), p-tau181 (n = 143) and tau (n = 147) were measured. Aliquots of most of these samples had been studied previously under another objective (Bibl et al. 2007a). Cerebrospinal fluid of AD, other dementias and depressive patients came from the memory clinic of the University of Goettingen or from wards. Parkinson s disease dementia (PDD) and dementia with Lewy bodies (DLB) patients came from the Paracelsus-Elena Klinik, Kassel. A psychiatrist and a neurologist rendered diagnoses based on thorough anamnesis, clinical examination, neuropsychological assessment, clinical records and best clinical judgment. The investigators were blinded to the neurochemical outcome measures. Investigations were carried out with the informed consent of patients or their authorized caregiver. The study was conducted under the guidelines of the Declaration of Helsinki (World Medical Organisation 1996) and approved by the ethics committee of the University of Goettingen and Hessen. Test methods Preanalytical treatment of CSF Cerebrospinal fluid was drawn by lumbar puncture into polypropylene vials and centrifuged (1,000g, 10 min, 4 C). Aliquots of 200 ll were kept at room temperature for a maximum of 24 h before storage at -80 C for subsequent phospho-tau, Ab1-38 and Ab1-40 ELISA. CSF for Ab 1-42- and total-tau ELISA analysis was stored at?4 C and analyzed within 2 days. ELISA for phospho-tau, total-tau protein and Ab 1-42 Enzyme linked immunosorbent assay for phospho-tau at Thr181 was conducted as described previously (Vanmechelen et al. 2000). Briefly, the HT7 monoclonal antibody (MAb) directed against both normal tau and phospho-tau was used for capturing and biotinylated MAb AT270 for detection. According to published standard methods (Hulstaert et al. 1999), the Innotest htau Antigen ELISA and Innotest b-amyloid (1-42), ELISA Innogenetics (Ghent, Belgium) served for quantification of CSF tau and Ab 1-42, respectively. Electrochemiluminescence detection (MSD) of Ab1-38 Electrochemiluminescence detection of Ab1-38 in CSF was performed according to the manufacturer s recommendations (Meso Scale Discovery). In brief, Multi-Spot 4, 96 well plates, precoated with MAb 6E10 were blocked with solution A. The plate was then incubated with a reference peptide dilution series or 100 ll CSF for 1 h, followed by incubation with c-terminal SULFO-TAG Ab 1-38 antibody and finally Read Buffer, for 1 h each. Washing steps with 19 Tris buffer were performed in between. The emitted light was measured at *620 nm.

Biomarkers in AD 205 Participants Depressive cognitive complainers (DCC) Twenty-five depressive cognitive complainers (10 men and 15 women) underwent lumbar puncture for differential diagnosis of cognitive complaints during the course of disease. The diagnosis of depression was made according to the criteria of DSM IV, and cognitive impairment was assessed by MMSE at minimum. Patients with persistent cognitive decline for more than 6 months, MMSE score below 26 or clear focal atrophy in brain imaging (CT or MRI) were excluded. Patients with Alzheimer s disease Forty-four patients (15 men and 19 women) fulfilled DSM IV criteria and NINCDS-ADRDA criteria for clinical diagnosis of AD (McKhann et al. 1984). Structural (CT or MRI) or functional (SPECT or PET) brain imaging, respectively, displayed global cortical atrophy or temporal, parietotemporal, frontotemporal focal atrophy or marked hypometabolism of these regions. Patients with non-alzheimer dementias (NAD) Eighty-seven patients (54 men and 33 women) fulfilled the DSM IV criteria for dementia. Frontotemporal dementia (FTD, n = 23) and primary progressive aphasia (PPA, n = 6) were diagnosed according to the consensus criteria (Neary et al. 1998). Structural (CT or MRI) or functional (SPECT or PET) brain imaging revealed frontal or frontotemporal focal atrophy or marked hypometabolism in case of FTD and left anterior temporal focal atrophy or marked hypometabolism in case of PPA. Vascular dementia (VAD) was diagnosed in 30 patients according to NINDS-AIREN criteria (Roman et al. 1993) and signs of relevant vascular disease in structural brain imaging (CT or MRI). DLB and PDD was diagnosed in three and five patients, respectively, according to the respective consensus criteria (Gibb and Lees 1998; McKeith et al. 1996). Normal pressure hydrocephalus according to the proposed criteria of (Ishikawa 2004) was diagnosed in eight patients. Two patients were diagnosed with progressive supranuclear palsy and one with corticobasal degeneration, according to the established criteria (Litvan 1997). Three patients with sporadic Creutzfeldt Jakob s disease were evaluated according to established criteria (WHO: World Health Organisation 1998). Korsakow s syndrome (n = 6) was diagnosed according to the criteria of Oslin et al. (1998). The mean age and MMSE score of patient groups (DCC, AD and NAD) are given in Table 1. Table 1 Absolute abundances of total and phospho-tau as well as abundances of Ab peptides in the CSF of the diagnostic groups Diagnosis DCC (n = 30) AD (n = 44) NAD (n = 87) Mean ± SD Mean ± SD Mean ± SD Age 63.6 ± 10.9 69.8 ± 8.49 66.7 ± 9.28 MMSE 28.7 ± 1.40 18.7 ± 5.60 19.8 ± 7.54 Total tau 0.21 ± 0.12 0.57 ± 0.32 0.41 ± 0.45 Phospho-tau181 0.048 ± 0.02 0.10 ± 0.04 0.051 ± 0.02 Ab1-38 (MSD) a 0.75 ± 0.24 0.89 ± 0.32 0.66 ± 0.20 Ab1-40 (ELISA) b 5.78 ± 2.34 5.46 ± 2.10 4.85 ± 1.85 Ab1-42 (ELISA) b 0.83 ± 0.24 0.36 ± 0.15 0.59 ± 0.27 Ab1-42/1-40 c 0.16 ± 0.07 0.07 ± 0.03 0.15 ± 0.07 Ab1-42/1-38 c 1.21 ± 0.44 0.41 ± 0.16 1.05 ± 0.47 a Ab peptide concentrations as measured by electrochemiluminescence detection (MSD) (ng/ml) b Ab peptide concentrations as measured by ELISA (ng/ml) c Ab peptide ratio: Ab1-42 relative to the concentration of Ab1-40 and Ab1-38, respectively Statistical analysis Amyloid-beta peptides, tau and phospho-tau were expressed as absolute (ng/ml). Patient groups were characterized by mean and standard deviation (SD). The Mann Whitney U test and Kruskal Wallis test were employed for comparisons of diagnostic groups. Correlations were estimated by Spearman s Rho. The two-sided level of significance was taken as P \ 0.05. The global diagnostic accuracies were assessed by the area under the curve (AUC) of receiver operating characteristic curve (ROC). Cut-off points were determined at the maximum Youden index, providing a sensitivity of C85%. The statistical software package SPSS, version 10.0, served for computations. Results Test results The mean age of DCC was significantly younger than that of the AD group (P = 1.3 9 10-2 ). The mean age of NAD did not differ from DCC and AD, respectively. The mean MMSE score did not significantly differ between the dementia groups. Neurochemical phenotype of AD versus DCC Ab1-42 was selectively decreased in AD (P = 6.4 9 10-10 ), whereas Ab1-40 levels were virtually unchanged between both groups. Ab1-38 tended to be increased, but failed the level of significance. Consequently, the ratios

206 V. Welge et al. Fig. 1 Receiver operator curves for detection of AD among DCC based on a single-marker test using Ab1-42, tau and p-tau, respectively Fig. 2 Receiver operator curves for detection of AD among NAD based on a two-marker test using Ab1-42/Ab1-38 and Ab1-42/p-tau, respectively, as referenced to Ab1-42 Ab1-42/Ab1-40 (P = 2.2 9 10-6 ) and Ab1-42/Ab1-38 (P = 1.8 9 10-8 ) showed a decrease in AD (Table 1). CSF total-tau (P = 7.4 9 10-8 ) and phospho-tau (P = 2.7 9 10-7 ) were elevated in AD. Neurochemical phenotype of AD versus NAD Ab1-42 was selectively decreased in AD (P = 2.2 9 10-7 ). Ab1-40 levels tended to be increased in AD, but failed to reach the level of significance. In contrast, Ab1-38 was significantly increased in AD (P = 5.4 9 10-4 ). Consequently, the ratios Ab1-42/Ab1-40 (P = 5.1 9 10-9 ) and Ab1-42/Ab1-38 (P = 4.0 9 10-12 ) showed a pronounced decrease in AD (Table 1). Cerebrospinal fluid total-tau (P = 1.5 9 10-4 ) and phospho-tau (P = 4.2 9 10-11 ) were elevated in AD. Neurochemical phenotype of NAD versus DCC In NAD, all investigated Ab peptides showed decreased levels as compared to DCC, but only Ab1-42 reached the level of significance (P = 1.2 9 10-4 ). As a consequence, the ratios Ab1-42/Ab1-40 and Ab1-42/Ab1-38 were not significantly altered in dementias other than AD. CSF total-tau (P = 1.4 9 10-2 ) was elevated in NAD, but not phospho-tau. Correlations Throughout all investigated patient groups, the different Ab peptides were strongly correlated to each other, except for DCC, where a correlation between Ab1-42 to Ab1-40 and Ab1-38, respectively, was lacking. P-tau positively correlated with all other biomarkers, except for Ab1-42. Consequently, the ratios Ab1-42/Ab1-40 and Ab1-42/Ab1-38 were negatively correlated with p-tau. Tau was positively correlated to p-tau in all diagnostic groups and negatively correlated to the ratios Ab1-42/Ab1-40 and Ab1-42/Ab1-38 only in AD. Tau was positively correlated with age only in DCC, whereas Ab1-42 was negatively correlated with age only in NAD. In AD, none of the investigated markers, alone or in combination, was correlated to the severity of dementia in AD. In contrast, high tau and low Ab1-42 levels correlated with the MMSE score in NAD. There was no correlation of gender with any of the investigated biomarkers. All reported correlations reached the level of high significance (P \ 0.01). Estimates Diagnosis of AD Decreased CSF Ab1-42 enabled diagnostic accuracies of beyond 85%, being the most promising single marker for differentiation of AD from DCC (AUC = 0.95). Its accuracy tended to improve by combining with tau (AUC = 0.98), p-tau (AUC = 0.98) or Ab1-38 (AUC = 0.97). Increased p-tau was the most suitable single marker to discriminate AD from NAD, yielding accuracies of beyond

Biomarkers in AD 207 80% (AUC = 0.87). The combination of p-tau and Ab1-42 (p-tau/ab1-42) improved the accuracy of each biomarker alone (AUC = 0.89). By combining decreased Ab1-42 with Ab1-38 (Ab1-42/ Ab1-38), the accuracy of Ab1-42 alone could be improved to beyond 85%. The resulting enlargement of the AUC to 0.93 very narrowly failed the level of significance. Combining p-tau with Ab1-42/Ab1-38 resulted in a sensitivity of 94% for AD detection and a specificity of 85% for excluding other dementias. The AUC was significantly enlarged to 0.94. Remarkably, the latter two combinations were the only ones that reached the benchmark of 85% for both sensitivity and specificity in discriminating AD from NAD (Figs. 1, 2, 3). Discussion Here, we evaluated the most common CSF core biomarkers for AD in comparison to DCC and NAD by use of commercially available high throughput methods, namely electrochemiluminescence and ELISA. We confirmed our previous findings of disease specific CSF Ab peptide patterns in AD showing selective reduction of CSF Ab1-42, accompanied by slight increase of Ab1-38 and virtually unchanged Ab1-40 levels (Wiltfang et al. 2002; Bibl et al. 2007a). In agreement with the current literature, levels of tau and p-tau were elevated in AD (Blennow 2004). Ab peptides 1-38, 1-40 and 1-42 for the diagnosis of AD In agreement with previous studies, the three different Ab peptides were strongly correlated to each other (Wiltfang Fig. 3 Scatterplot of AD, NAD and DCC divided by their absolute p- tau levels and the ratio of Ab1-42/Ab1-38 et al. 2002; Bibl et al. 2007a; Schoonenboom et al. 2005; Mehta and Pirttila 2005), except for Ab1-38 and Ab1-42 in DCC. These findings suggest a tight regulation of Ab generation and turnover. The reduction of CSF Ab1-42 is a central and reproducible feature of AD. Various studies have confirmed its diagnostic value for AD as compared to patients without dementia (e.g. DCC) or mentally healthy controls (controls) (Blennow 2004). Correspondingly, CSF Ab1-42 gave excellent contrasts for differentiation of AD from DCC, one of the most frequent differential diagnostic challenges in clinical practice. Owing to overlapping values with FTD, VAD and DLB (Blennow 2004), the specificity of this biomarker declined markedly for the discrimination of AD from other dementias (Hulstaert et al. 1999). The concentrations of total CSF Ab and its major constituent, Ab1-40, remained unchanged between AD and controls (Motter et al. 1995). Using methods other than ELISA, even increased levels of CSF Ab1-40 have been reported in AD (Wiltfang et al. 2002; Lewczuk et al. 2003). Thus, a selective reduction of Ab1-42 in AD can be assumed (Motter et al. 1995).and correspondingly its ratio to Ab1-40 (Ab1-42/Ab1-40) was markedly decreased in AD (Blennow 2004). Meanwhile, comparative studies on the accuracy of Ab1-42 and its ratio to Ab1-40 have been conducted, which yielded conflicting results. Using different methods of measurement, we and others found improved accuracies for the ratio Ab1-42/Ab1-40 as compared to Ab1-42 levels alone (Bibl et al. 2006b; Lewczuk et al. 2004; Shoji et al. 1998; Kanai et al. 1998; Shoji et al. 2000; Shoji and Kanai 2001). This was most evident in the case of differential diagnosis of AD as opposed to other dementias, where CSF Ab1-42 is reduced along with other Ab peptides (Bibl et al. 2007a), or detecting MCI patients that are at high risk of developing AD (Wiltfang et al. 2007). Two previous studies have challenged the proposed diagnostic advantage of CSF Ab ratios as compared to absolute Ab1-42 levels (Schoonenboom et al. 2005; Mehta and Pirttila 2005). The authors quantified CSF Ab1-38 levels by a novel ELISA and reported comparable accuracies of Ab1-42, Ab1-42/Ab1-40 and Ab1-42/Ab1-38 for the differentiation of AD and controls. Here, the application of different carboxyterminally and/or aminoterminally specific anti-ab antibodies with distinct affinities and specificities may have contributed to discrepant results. Moreover, they did not examine patients with non- Alzheimer dementias for comparison. Unlike in AD, the reduction of Ab1-42 is often accompanied by an overall drop of CSF Ab in non-alzheimer dementias (Bibl et al. 2006a, b, 2007a, b, 2008a, b). Consequently, we found a larger AUC for Ab1-42/Ab1-40 in the differential diagnosis of AD from NAD as compared to Ab1-42 alone.

208 V. Welge et al. Thus, it appears that the benefit of calculating Ab1-42/ Ab1-40 is most evident for this application. The ratio of Ab1-42 to Ab1-38 has been examined less thoroughly before. Using Ab-SDS-PAGE/immunoblot, we were the first to report the regular presence of Ab1-38 in CSF, and we found a disease-specific percentage increase of (Ab1-38%) in AD as compared to NAD (Wiltfang et al. 2002; Bibl et al. 2007a). We confirmed this finding, which supports Ab1-38 as an additional biomarker in several studies, including the present one (Bibl et al. 2006a, b, 2007a, b). In contrast to Ab1-42 levels alone, the evaluation of Ab1-42/Ab1-38 resulted in accuracies of beyond 85% for the differentiation of both diagnostic groups and the corresponding enlargement of AUCs only narrowly failed the level of significance. The benefit from Ab1-42/ Ab1-38 mainly concerned the differential diagnosis of AD among NAD. The sensitivity of AD detection among DCC could also be improved, but at the cost of some specificity. In summary, our results suggest that the assessment of Ab1-42/Ab1-40 or Ab1-42/Ab1-38 improves the specificity of Ab1-42 levels alone, in particular for the differential diagnosis between AD and other dementias. We have previously shown that freezing of samples may hamper ELISA test accuracies and increase variance of the test results (Bibl et al. 2004). In the present study, Ab1-42 was measured in fresh CSF samples, whereas samples had been frozen prior to analysis of Ab1-38 and Ab1-40. It remains to be shown whether the diagnostic accuracy of these markers can be further improved by avoiding freezing of the CSF samples prior to quantification of Ab1-38 and Ab1-40. The following considerations make CSF Ab ratios, rather than sole Ab1-42 levels, promising biomarker candidates for early and differential dementia diagnostics as well as for therapeutic monitoring of forthcoming disease modifying inventions: (1) Ab1-40/Ab1-42 was not influenced by age, whereas each peptide separately showed a U-shaped natural course in normal aging (Shoji et al. 2001); (2) In contrast to absolute Ab peptide values, the relative abundances remained largely stable after different preanalytical procedures (Bibl et al. 2004, Lewczuk et al. 2006); (3) The absolute CSF Ab peptide concentrations show a remarkable inter-individual variance, while the relative distributions of different Ab species are regulated within very narrow limits (Wiltfang et al. 2002; Bibl et al. 2007a; Wiltfang et al. 2007) (4) Distinct g-secretase activities may be reflected most adequately by the ratio Ab1-42/Ab1-40 Wiltfang et al. 2001) or Ab1-38/Ab1-40 (Weggen et al. 2001). Tau and Ab1-42 Numerous studies have reported a moderately improved accuracy of the tau/ab1-42 ratio for the diagnosis of AD as compared to each biomarker alone (Blennow 2004). The mean sensitivity and specificity from 12 independent studies, including a total of 767 AD patients and 428 control subjects, was 89 and 90%, respectively. In comparison, 20 and 16 independent studies on AD diagnosis among control subjects revealed lower accuracies for tau (Sensitivity 81% and Specificity 92%) and Ab1-42 (Sensitivity 86% and Specificity 89%) as single biomarkers, respectively (Blennow 2004). We largely confirmed these results in our patient sample (see Table 2) and similar to other studies (Hulstaert et al. 1999), we found lower specificities when these parameters were applied to discriminate AD from NAD (see Table 2). Instead of using tau/ab1-42, Shoji et al. (1998) introduced the AD index (tau 9 Ab40/Ab42) and found reasonable accuracies (Sensitivity 69% and Specificity 88%) for differentiating AD from a reference group that included non-alzheimer dementias as well as neurological disorders (Shoji et al. 1998). This index was used for evaluation of both the GTT1 (Sensitivity 71% and Specificity 83%) and GTT2 study (Sensitivity 81% and Specificity 87%) (Shoji et al. 2000). In summary, these findings were comparable to what we found by combining Ab1-42, Ab1-40 and tau for the detection of AD among all other patients (Sensitivity 97% and Specificity 75%). By exchanging Ab1-40 to Ab1-38, we were able to increase the specificity of the index to 80% at the same sensitivity. P-tau 181 and Ab 1-42 The elevated concentration of CSF p-tau is believed to be closely related to AD pathology and to be relatively specific for AD. The most common ELISAs for p-tau, p-tau181, p-tau199 and p-tau231 revealed similar accuracies in a large comparative CSF study (Hampel et al. 2004). The mean sensitivity and specificity from 16 studies, investigating different sites of phosphorylation in a total of 1084 AD patients and 504 control subjects, were 81 and 91%, respectively. The largest study on p-tau181 for discriminating AD from other dementias gave a sensitivity and specificity of 85 and 81%, respectively (Hampel et al. 2004), supporting p-tau as a relatively specific marker. In our study, we observed very similar accuracies for the detection of AD among DCC and NAD. On the other hand, these data indicate that p-tau181 crucially lacks sensitivity as a screening test, or, in other words: When a minimum specificity of 85% is required for AD detection, the sensitivity declines to 80% or below, meaning that AD may be missed in at least every fifth patient. Two previous studies investigated the combination of p-tau and Ab 1-42 for enhancing the accuracy of each marker alone in differentiating AD from non-alzheimer dementias (Maddalena et al. 2003, De Jong et al. 2006).

Biomarkers in AD 209 Table 2 Cut off points, sensitivities and specificities for each differential diagnostic testing Differential diagnosis Parameter Cut off Sensitivity (%) Specificity Youden index AUC 95%-CI AD versus DCC Ab1-42 0.473 89 92 0.81 0.950 0.895 1.005 tau 0.253 90 76 0.66 0.903 0.827 0.979 p-tau181 0.059 90 80 0.70 0.885 0.801 0.966 Ab1-42/tau 2.017 98 96 0.94 0.981 0.952 1.010 Ab1-42/p-tau181 8.718 95 92 0.87 0.979 0.954 1.005 Ab1-42/Ab1-40 0.113 91 82 0.73 0.910 0.823 0.997 Ab1-42/Ab1-38 0.748 100 89 0.89 0.970 0.940 1.012 Ab1-42/Ab1-40/tau 0.375 97 88 0.85 0.955 0.897 1.014 Ab1-42/Ab1-38/tau 2.098 97 94 0.91 0.979 0.944 1.014 Ab1-42/Ab1-40/p-tau181 1.613 85 88 0.73 0.913 0.832 0.993 Ab1-42/Ab1-38/p-tau181 11.00 94 88 0.82 0.955 0.901 1.008 AD versus NAD Ab1-42 0.458 86 64 0.50 0.774 0.693 0.894 tau 0.266 87 48 0.35 0.711 0.619 0.803 p-tau181 0.065 87 83 0.70 0.874 0.801 0.947 Ab1-42/tau 1.451 92 60 0.52 0.773 0.692 0.853 Ab1-42/p-tau181 8.157 95 75 0.70 0.894 0.837 0.951 Ab1-42/Ab1-40 0.099 85 76 0.61 0.871 0.791 0.945 Ab1-42/Ab1-38 0.650 89 86 0.75 0.931 0.882 0.980 Ab1-42/Ab1-40/tau 0.357 97 71 0.68 0.847 0.764 0.929 Ab1-42/Ab1-38/tau 2.001 97 75 0.72 0.880 0.810 0.950 Ab1-42/Ab1-40/p-tau181 1.605 85 81 0.66 0.908 0.846 0.970 Ab1-42/Ab1-38/p-tau181 11.34 94 85 0.79 0.942 0.895 0.989 AD versus all other Ab1-42 0.457 86 70 0.56 0.813 0.744 0.882 tau 0.266 87 54 0.41 0.755 0.674 0.837 p-tau181 0.065 87 83 0.70 0.876 0.808 0.945 Ab1-42/tau 1.351 87 69 0.61 0.820 0.754 0.886 Ab1-42/p-tau181 6.43 85 85 0.70 0.965 0.868 0.961 Ab1-42/Ab1-40 0.099 85 77 0.62 0.881 0.817 0.945 Ab1-42/Ab1-38 0.650 89 87 0.76 0.942 0.901 0.982 Ab1-42/Ab1-40/tau 0.260 86 76 0.62 0.874 0.806 0.942 Ab1-42/Ab1-38/tau 1.53 86 81 0.67 0.905 0.849 0.961 Ab1-42/Ab1-40/p-tau181 1.605 85 83 0.68 0.909 0.850 0.968 Ab1-42/Ab1-38/p-tau181 9.65 88 88 0.76 0.945 0.902 0.989 The first study included 30 non-alzheimer dementias and found contrasts of below 80% for each marker alone, but an improvement to a sensitivity and specificity of 80 and 73%, respectively, by combining p-tau and Ab 1-42 (Maddalena et al. 2003). The second focused on the discrimination of vascular dementias (n = 25) from AD (n = 61) and reported a similar effect, although on a remarkably higher level of accuracy for p-tau and Ab 1-42 alone and consequently for the combination thereof (Sensitivity 100% and Specificity 85%) (De Jong et al. 2006). In our study, the accuracies of Ab1-42/p-tau were in between the results of these two studies, most likely owing to the inclusion of various forms of NAD, but also a large number of VAD patients. Ab1-42/p-tau yielded insignificantly higher AUCs than each marker alone for the differentiation of AD and DCC. Otherwise, the ratio did not improve the accuracy of p-tau alone for the differentiation of AD and NAD. Therefore, Ab ratios, rather than absolute Ab1-42, should be considered for a combination assay with p-tau, as the selective reduction of Ab1-42 seems to be most closely related to AD-specific pathomechanisms. Promising biomarker combinations for multiplex assays In summary, our results support the hypothesis that combinations of Ab1-42 with other AD-specific biomarkers may enhance the accuracy of absolute Ab1-42 CSF concentrations alone. A sensible combination of biomarkers should satisfy the accuracy requirements for an applicable AD biomarker (i.e. sensitivity and specificity C85%) and

210 V. Welge et al. additionally improve the AUC of absolute biomarker concentration. Moreover, the differential diagnostic question will particularly determine which combination to choose. For the differentiation of AD from DCC, none of the tested biomarker combinations was able to significantly enhance the AUC of Ab1-42 alone, which yielded contrasts of beyond 85%. Thus, our results indicate that absolute Ab1-42 levels readily fulfill the requirements for an AD biomarker for this differential diagnostic question, which is in line with the current literature. In contrast, none of the tested biomarkers alone was able to fulfill the requirements for the discrimination of AD versus NAD. The same held true when AD was tested among a combined group of DCC and NAD. Solely biomarker combinations that considered Ab1-38 and/or p-tau in addition to Ab1-42 were able to discriminate AD among NAD or a combined group of DCC and NAD with accuracies of 85% at minimum. Significant enlargement of the AUC of Ab1-42 alone could be achieved by combining Ab1-42/Ab1-38 to p-tau (Ab1-42/ Ab1-38/p-tau) for both differential diagnostic questions. In case of screening for AD among all other patients, the ratio of Ab1-42/Ab1-38 enlarged the AUC of Ab1-42, even when p-tau levels were left aside. From these data, we conclude that Ab1-42/Ab1-38/p-tau is most suitable to screen for AD, when another kind of dementia syndrome is differentially diagnostically relevant. Conclusions In line with previous studies, we consider multiparametric testing of CSF biomarkers to be a useful tool for neurochemical dementia diagnostics. Yielding contrasts of 85% or greater, decreased CSF Ab1-42 levels represent a core biomarker for AD and the best suited single-marker screen for AD among non demented subjects. Ab1-42 lacks specificity for the discrimination of AD from NAD, which may be compensated by using the ratios Ab 1-42/Ab1-38 or Ab 1-42/Ab1-38/p-tau, respectively. The latter combination fulfils the accuracy requirements for an applicable AD biomarker for differentiation from NAD or a combined group of DCC and NAD. Limitations of the study The reliance on clinical diagnosis limits our results because of potential misclassification. Another point of concern is the lack of a single standardized sample pre-treatment protocol. Moreover, the methods used for measurement varied between Ab1-38 and Ab1-40 or Ab1-42, respectively. Acknowledgments This study was supported by the following grants from the German Federal Ministry of Education and Research (BMBF): Kompetenznetz Demenzen (01 GI 0420), HBPP-NGFN2 (01 GR 0447), and the Forschungsnetz der Früh- und Differenzialdiagnose der Creutzfeldt-Jakob-Krankheit und der neuen Variante der CJK (01 GI 0301), and by the EU grants cneupro (contract no. LSHM-CT-2007-037950), and neurotas (contract no. LSHB-CT- 2006-037953). MB was supported by the Research program, Faculty of medicine, Georg-August-University Göttingen. MO was supported by grants from the German Federal Ministry of Education (German CJD therapy study FK 01KO0506), EU (anteprion 019090) and Landesstiftung Baden Württemberg).The authors would like to thank Sabine Paul, Birgit Otte and Heike Zech for excellent technical assistance. Conflict of interest statement There are no actual or potential conflicts of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could have inappropriately influenced (bias) our work. The study was conducted under the guidelines of the Declaration of Helsinki (World medical Organisation 1996) and approved by the ethics committee of the University of Goettingen and Hessen. References Bibl M, Mollenhauer B, Esselmann H, Lewczuk P, Klafki HW, Sparbier K, Smirnov A, Cepek L, Trenkwalder C, Rüther E, Kornhuber J, Otto M, Wiltfang J (2006a) CSF amyloidb-peptides in Alzheimer s disease, dementia with Lewy bodies and Parkinson s disease dementia. Brain 129:1177 1187 Bibl M, Mollenhauer B, Esselmann H, Lewczuk P, Trenkwalder C, Brechlin P, Rüther E, Kornhuber J, Otto M, Wiltfang J (2006b) CSF diagnosis of Alzheimer s disease and dementias with Lewy bodies. J Neural Transm 113:1771 1778 Bibl M, Mollenhauer B, Lewczuk P, Esselmann H, Wolf S, Trenkwalder C, Otto M, Stiens G, Ruther E, Kornhuber J, Wiltfang J (2007a) Validation of amyloid-b peptides in CSF diagnosis of neurodegenerative dementias. Mol Psy 12:671 680 Bibl M, Mollenhauer B, Wolf S, Esselmann H, Lewczuk P, Kornhuber J, Wiltfang J (2007b) Reduced CSF carboxyterminally truncated Ab petides in frontotemporal lobe degenerations. J Neural Transm 114:621 628 Bibl M, Esselmann H, Otto M, Lewczuk P, Cepek L, Rüther E, Kornhuber J, Wiltfang J (2004) Cerebrospinal fluid (CSF) amyloid beta (Ab) peptide patterns in Alzheimer s disease (AD) patients and non-demented controls depend on sample pretreatment: Indication of carrier- mediated epitope masking of Ab peptides. Electrophoresis 25:2912 2918 Bibl M, Mollenhauer B, Esselmann H, Schneider M, Lewczuk P, Welge V, Gross M, Falkai P, Kornhuber J, Wiltfang J (2008a) Cerebrospinal fluid neurochemical phenotypes in vascular dementias: original data and mini-review. Dement Geriatr Cogn Disord 25:256 265 Bibl M, Lewczuk P, Esselmann H, Mollenhauer B, Klafki HW, Welge V, Wolf S, Trenkwalder C, Otto M, Kornhuber J, Wiltfang J (2008b) CSF amyloid-b 1-38 and 1-42 in FTD and AD: biomarker performance critically depends on the detergent accessible fraction. Proteomics Clin Appl 2:1548 1556 Blennow K (2004) Cerebrospinal fluid protein biomarkers for Alzheimer s disease. NeuroRx. 1:213 225 De Jong D, Jansen RWMM, Kremer BPH, Verbeek MM (2006) Cerebrospinal fluid amyloid b42/phosphorylated tau ratio discriminates between Alzheimer s disease and vascular dementia. J Gerontol A Biol Sci Med Sci 61:755 758

Biomarkers in AD 211 Gibb WR, Lees AJ (1988) The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson s disease. J Neurol Neurosurg Psychiatry 51:745 752 Glenner GG, Wong CW (1984) Alzheimer s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885 890 Hampel H, Buerger K, Zinkowski R, Teipel SJ, Goernitz A, Andreasen N, Sjoegren M, DeBernardis J, Kerkman D, Ishiguro K, Ohno H, Vanmechelen E, Vanderstichele H, McCulloch C, Moller HJ, Davies P, Blennow K (2004) Measurement of phosphorylated tau epitopes in the differential diagnosis of Alzheimer s disease a comparative study. Arch Gen Psychiatry 61:95 102 Hulstaert F, Blennow K, Ivanoiu A, Schoonderwaldt HC, Riemenschneider M, De Deyn PP, Bancher C, Cras P, Wiltfang J, Mehta PD, Iqbal K, Pottel H, Vanmechelen E, Vanderstichele H (1999) Improved discrimination of AD-patients using ß-amyloid (1-42) and tau levels in CSF. Neurology 52:1555 1562 Ishikawa M (2004) Guideline committee for idiopathic normal pressure hydrocephalus, Japanese society of normal pressure hydrocephalus. Neurol Med Chir 44:222 223 Kanai M, Matsubara E, Isoe K, Urakami K, Nakashima K, Arai H, Sasaki H, Abe K, Iwatsubo T, Kosaka T, Watanabe M, Tomidokoro Y, Shizuka M, Mitsushima K, Nakamura T, Igeta Y, Ikeda Y, Amari M, Kawarabayashi T, Ishiguro K, Harigaya Y, Wakabayashi K, Okamoto K, Hirai S, Shoji M (1998) Longitudinal study of cerebrospinal fluid levels of tau, Ab1-40, and Ab1-42(43) in Alzheimer s disease: a study in Japan. Ann Neurol 44:17 26 Lee VMY, Balin BJ, Otvos L, Trojanowski JQ (1991) A68. A major subunit of paired helical filaments and derivatized forms of normal tau. Science 251:675 678 Lewczuk P, Beck G, Esselmann H, Bruckmoser R, Zimmermann R, Fiszer M, Bibl M, Maler JM, Kornhuber J, Wiltfang J (2006) Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid b peptides. Clin Chem 52:332 334 Lewczuk P, Esselmann H, Otto M, Maler JM, Henkel AW, Henkel MK, Eikenberg O, Antz C, Krause WR, Reulbach U, Kornhuber J, Wiltfang J (2004) Neurochemical diagnosis of Alzheimer s dementia by CSF Abeta 42, Abeta42/Abeta40 ratio and total tau. Neurobiol Aging 25:273 281 Lewczuk P, Esselmann H, Meyer M, Wollscheid V, Neumann M, Otto M, Maler JM, Rüther E, Kornhuber J, Wiltfang J (2003) The amyloid-b (Ab) peptide pattern in cerebrospinal fluid in Alzheimer s disease: evidence of a novel carboxyterminally elongated Ab peptide. Rapid Commun Mass Spectrom 17:1291 1296 Litvan I (1997) Progressive supranuclear palsy and corticobasal degeneration. Baillieres Clin Neurol 6:167 185 Maddalena A, Papassotiropoulos A, Mueller-Tillmanns B, Jung HH, Hegi T, Nitsch RM, Hock C (2003) Cerebrospinal fluid amyloid b42/phosphorylated tau ratio discriminates between Alzheimer s disease and vascular dementia. Arch Neurol 60:1202 1206 McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM (1984) Clinical diagnosis of Alzheimer s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer s Disease. Neurology 34:939 944 McKeith IG, Galasko D, Kosaka K, Perry EK, Dickson DW, Hansen LA, Salmon DP, Lowe J, Mirra SS, Byrne EJ, Lennox G, Quinn NP, Edwardson JA, Ince PG, Bergeron C, Burns A, Miller BL, Lovestone S, Collerton D, Jansen EN, Ballard C, de Vos RA, Wilcock GK, Jellinger KA, Perry RH (1996) Consensus guidlines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop. Neurology 47:1113 1124 Mehta PD, Pirttila T (2005) Increased cerebrospinal fluid A beta38/a beta42 ratio in Alzheimer s disease. Neurodegener Dis 2:242 245 Motter R, Vigo-Pelfrey C, Kholodenko D, Barbour R, Johnson-Wood K, Galasko D, Chang L, Miller B, Clark C, Green R, Olson D, Southwick P, Wolfert R, Munroe B, Lieberburg I, Seubert P, Schenk D (1995) Reduction of beta-amyloid peptide42 in the cerebrospinal fluid of patients with Alzheimer s disease. Ann Neurol 38:643 648 Neary D, Snowden JS, Gustafson L, Passant U, Stuss D, Black S, Freedmann M, Kertesz A, Robert PH, Albert M, Boone K, Miller BL, Cummings J, Benson DF (1998) Frontotemporal lobardegeneration. A consensus on clinical diagnostic criteria. Neurology 51:1546 1554 Oslin D, Aktinsson RM, Smith DM, Hendrie H (1998) Alcohol related dementia: proposed clinical criteria. Int J Ger Psychiatry 13:203 212 Roman GC, Tatemichi TK, Erkinjutti T, Cummings JL, Masedeu JC, Garcia JH, Amaducci L, Orgogozo JM, Brun A, Hofman A (1993) Vascular dementia: diagnostic criteria for research studies: report of the NINDS-AIREN International Workshop. Neurology 43:243 245 Schoonenboom NS, Mulder C, Van Kamp GJ, Mehta SP, Scheltens P, Blankenstein MA, Mehta PD (2005) Amyloid b 38, 40 and 42 species in cerebrospinal fluid: more of the same? Ann Neurol 58:139 142 Shoji M, Matsubara E, Kanai M, Watanabe M, Nakamuro T, Tomidokoro Y, Shizuka M, Wakabayashi K, Igeta Y, Ikeda Y, Mizushima K, Amari M, Ishiguro K, Kawarabayashi T, Harigaya Y, Okamoto K, Hirai S (1998) Combination assay of CSF tau, Ab1-40 and Ab1-42(43) as a biochemical marker of Alzheimer s disease. J Neurol Sci 158:134 140 Shoji M, Matsubara E, Kanai M, Ikeda M, Harigaya Y, Okamoto K, Hirai S (2000) Taps to Alzheimer s patients: a continuous Japanese study of cerebrospinal fluid biomarkers. Ann Neurol 48:402 Shoji M, Kanai M (2001) Cerebrospinal fluid Ab40 and Ab42: natural course and clinical usefulness. J Alzheimers Dis 25:273 281 Shoji M, Kanai M, Matsubara E, Tomidokoro Y, Shizuka M, Ikeda Y, Ikeda M, Harigaya Y, Okamoto K, Hirai S (2001) The levels of cerebrospinal fluid Ab40 and Ab42(43) are regulated agedependently. Neurobiol Aging 22:209 215 Vanmechelen E, Vanderstichele H, Davidsson P, Van Kerschaver E, Van Der Perre B, Sjögren M, Andreasen N, Blennow K (2000) Quantification of tau phosphorylated at threonine 181 in human cerebrospinal fluid: a sandwich ELISA with a synthetic phosphopeptide for standardization. Neurosci Lett 285:49 52 Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T, Kang DE, Marquez- Sterling N, Golde TE, Koo EH (2001) A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414:212 216 WHO: World Health Organisation (1998) Human transmissible spongiform encephalopathy. Wkly Epidemiol Rec 73:361 365 Wiltfang J, Esselmann H, Bibl M, Hüll M, Hampel H, Kessler H, Frölich L, Schröder J, Peters O, Jessen F, Luckhaus C, Perneczky R, Jahn H, Fiszer M, Maler JM, Zimmermann R, Bruckmoser R, Kornhuber J, Lewczuk P (2007) Amyloid b peptide ratio 42/40 but not Ab42 correlates with phospho-tau in patients with lowand high-csf Ab40 load. J Neurochem 101:1053 1059 Wiltfang J, Esselmann H, Cupers P, Neumann M, Kretzschmar H, Beyermann M, Schleuder D, Jahn H, Rüther E, Kornhuber J, Anneart W, De Strooper B, Saftig P (2001) Elevation of b-amyloid peptide 2-42 in sporadic and familial Alzheimer s disease and its generation in PS1 knockout cells. J Biol Chem 276:42645 42657

212 V. Welge et al. Wiltfang J, Esselmann H, Bibl M, Smirnov A, Otto M, Paul S, Schmidt B, Klafki HW, Maler M, Dyrks T, Bienert M, Beyermann M, Rüther E, Kornhuber J (2002) Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer s disease and patients with chronic neuroinflammation. J Neurochem. 81:481 496 World Medical Organisation (1996) Declaration of Helsinki. Br Med J 313:1448 1449

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