Alzheimer CSF biomarkers in routine clinical setting

Acta Neurol Scand DOI: 1.1111/j.16-44.211.1592.x Ó 211 John Wiley & Sons A S ACTA NEUROLOGICA SCANDINAVICA Alzheimer CSF biomarkers in routine clinical setting Tabaraud F, Leman JP, Milor AM, Roussie JM, Barrie` re G, Tartary M, Boutros-Toni F, Rigaud M. Alzheimer CSF biomarkers in routine clinical setting. Acta Neurol Scand: DOI: 1.1111/j.16-44.211.1592.x. Ó 211 John Wiley & Sons A S. Objectives Our work was aimed to evaluate AlzheimerÕs disease diagnosis improvement using cerebrospinal fluid biomarkers (CSF) in neurological daily practice. Materials and Methods For this purpose, 15 patients clinically and neurochemically classified as having or cognitive impairment with or without other dementia type were included in the study. The following CSF peptides were studied, blindly to the clinical diagnosis: beta-amyloid 1 42 peptide (Ab 1 42 ), Tau (T-tau), threonine-181 hyperphosphorylated tau protein (P-tau 181 ), and beta-amyloid 1 4 peptide (Ab 1 4 ). From these measurements, InnotestÒ Amyloid Tau Index (IATI) was calculated for each patient. Results This assessment allowed to separate 83 biochemical profiles of and 67 non-alzheimerõs disease (non-), both and non- categories match with clinical data amounting to 73% and 9%, respectively. Among mild cognitive impairment (MCI) patients, CSF biomarkers led to discriminate those who are likely to be. We devoted a special section to Ab 1 4 which is not a routine parameter but can help to confirm a pathological amyloid process as Ab 1 42 Ab 1 4 ratio underlining the real decline of the Ab 1 42. Conclusions The interest of biomarkers and their ability to solve awkward cases were carefully noticed all the more when a discrepancy between clinical and CSF biological data was involved. The final proposed algorithm allowed to identify pathogenic forms of according to the prevailing role of hyperphosphorylated tau or amyloid beta peptide. F. Tabaraud 1, J. P. Leman 1, A. M. Milor 1, J. M. Roussie 2, G. Barri re 2, M. Tartary 2, F. Boutros-Toni 2, M. Rigaud 2 1 Clinical Center, Clinique du Colombier, Limoges, France; 2 ASTRALAB Clinical Laboratory, Limoges, France Key words: AlzheimerÕs disease;ab 1 42 peptide; Ab 1 4 peptide; T-tau; P-tau 181 ; InnotestÒ Amyloid Tau Index M. Rigaud, ASTRALAB, 7-11 Avenue de Lattre de Tassigny, 87 Limoges, France Tel.: 33 5 55 3 29 3 Fax: 33 5 55 48 85 1 e-mail: rigaud.michel@ yahoo.fr. Accepted for publication August 23, 211 Introduction AlzheimerÕs disease () is the most common cause of dementia increasing twofold every 5 years after 65 years, finally rising to 4% after 9 years (1). AlzheimerÕs disease () is a progressive, neurodegenerative disease anatomically defined by abnormal clumps, tangled fiber bundles involving deposition of amyloid beta peptide and intra neuronal lesions with neurofibrillary degeneration owing to the accumulation of hyperphosphorylated tau (1). Increasing age and genetic background are known risk factors for. The 4 allele of Apolipoprotein E (APOE) is the major genetic factor associated with early onset (2). A large majority of patients are not eligible for systematic APOE genotyping, this test therefore being of limited use in daily practice. Till now, diagnosis criteria (DMS IV and NINCDS-RDA) (3) are primarily exclusion criteria, poorly specific of the pathology, and lacking sensitivity in early stages. In addition to memory impairment, new diagnosis criteria have been added: Magnetic Resonance Imaging (MRI) showing hippocampal or temporal pole atrophy, abnormal levels of CSF biomarkers, particular aspect of functional neuroimaging, identified gene mutation in familial cases (4 6). Since 28, such recommendations are advocated by H.A.S (Haute Autorite de Santé, French Health Authority). It is admitted that, before dementia stage, there is a progressive cognitive decline reported as heterogeneous concept of MCI (mild cognitive impair- 1

Tabaraud et al. ment) (7). The issue of earlier diagnosis is all the more an important matter as new pharmacological approaches to slow the disease process (labeled as modifying disease treatments) greatly expand. In, numerous data have been reported on the CSF biomarkers as witnesses of the pathological process. Patients with present with a characteristic pattern: decreased beta-amyloid 1 42 peptide (Ab 1 42 ), increased total tau (T-tau), and threonine-181 hyperphosphorylated tau protein (Ptau 181 ) (8 1). Performances obtained by different laboratories dealing with assessment of CSF biomarkers are reported in Table 1 (11 21). The pitfall concerning biomarkers value differences needs to be underlined, and besides a uniform standardization of pre- and analytical procedures, multicentre comparison is required to assess sensitivity and specificity of methods. Cerebrospinal fluid biomarkers (CSF) biomarkers quantification has shown the benefits of Ab 1 42, T-tau and P-tau 181 measurement for diagnosis of Alzheimer-type dementia in clinical routine (1). Changes of these specific proteins, amyloid, and tau in CSF are connected with the amount of brain damage (19, 22, 23). P-tau 181 and T-tau increase is associated with neocortical neurofibrillary pathology (23); Ab 1 42 reduction is correlated to the density of neuritic plaques (19). There is a link between functional imaging, cortical amyloid labeling by PIB (C Pittsburgh Compound B, radio tracer), and Ab 1 42 decrease (24). Evaluation of cerebral metabolism by PET or SPECT showed that the increase of total tau and P-tau 181 in Alzheimer patientsõ CSF was linked to hypometabolism in temporal regions (25). Table 1 Concentrations of Aß 1 42, T-tau, and P-tau 181 in CSF obtained from literature studies References No of patients Aß 1 42 T-tau P-tau 181 Hulstaert et al. (11) 15 556 239 ND Andreasen et al. (12) 28 523 18 759 417 ND Hampel et al. (13) 93 545 23 725 266 ND Ibach et al. (14) 76 463 218 615 362 87 29 Ibach et al. (14) 76 53 348 65 Vanderstichele 94 378 (285 494) 61 (416 868) 84 (54 16) et al. (15) Parnetti et al. (16) 1 411 148 628 314 136 59 Lanari and 18 42 195 42 318 91 31 Parnetti (17) Henneman 31 474 113 811 486 92 38 et al. (18) Tapiola et al. (19) 123 515 35 52.5 Wallin et al. (2) 151 398 97 62 349 77 33 Kester et al. (21) 47 474 (335 62) 482 (393 853) 77 (5 13) These concentrations (mean SD or cut-off values) were determined using Innogenetics assays. Results are expressed in ng l. Wiltfang et al. (26) proposed Ab 1 4 peptide measurement as a tool to overcome inter-individual variability, then checking the value of Ab 1 42 Ab 1 4 ratio. Ab 1 4, the predominant form of soluble Ab peptides in brain and CSF, is not a routine parameter. We evaluated it in a forwardlooking purpose. This analysis enabled to assess more precisely amyloid disease profile for patients whose initial CSF biomarker profiles are not discriminating. Our work was aimed to evaluate diagnosis improvement using CSF in neurological daily practice. As far as this matter is considered, as post-mortem confirmation is scarcely available, follow-up examination was carried to confirm the clinical diagnosis. Materials and methods Patients and clinical assessments Between October 28 and March 21, 15 patients were included: 61 men, 89 women with an average age of 72.2 years (44 86). Patients consulted a neurologist for cognitive disorders assessment. Patient clinical evaluation consisted of history, evolution, effects on daily life activities, clinical and neurological examination, neuropsychological rating (Mini mental state examination (MMSE), clock test, five word test of Dubois, verbal fluency), blood tests as recommended by the H.A.S, and radiological assessment with either CT scan or MRI. A complete neuropsychological check was performed: memory tests including Grober Buschke test, executive functions tests (TMT, BREF: short frontal evaluation test, STROOP test) and, when necessary, other evaluations. Petersen criteria were used for the diagnosis of MCI (7), NINCDS-RDA criteria for the diagnosis of (3), Neary criteria for the diagnosis of fronto-temporal dementia (27), and McKeithÕs criteria for Lewy bodies dementia (28). The diagnosis of mixed dementia was established for patients with vascular risk factors, imaging showing stroke sequelae or major vascular lesions, i.e., leukopathy with cortical and or subcortical atrophy without selective hippocampal topography. The neuropsychological assessment of these patients indicated an overall impairment of cognitive functions with executive functions, praxis, memory and phasic disorders being involved without dominant hippocampal damage. The initial clinical diagnosis was set out without knowledge of CSF biomarker results. Patients were grouped into three clinical categories:, non-, and MCI. 2

AlzheimerÕs disease CSF biomarkers Biological samples After lumbar puncture (the patients or their caregivers gave written information consent in accordance with the Helsinki Declaration of 1975), CSF was directly collected in a polypropylene tube and sent to laboratory within 4 h, centrifuged at 25 rpm for 15 min. Aliquots in polypropylene tubes were stored at )8 C. CSF frozen samples were analyzed after thawing. Biochemical evaluation All markers (Ab 1 42, T-tau, P-tau 181 ) were assayed by ELISA following supplier recommendations (InnotestÒ; Innogenetics NV, Gent, Belgium). Ab 1 4 was quantified by ELISA (Human Amyloid b (1 4) (N) Assay kit-ibl-japan handed out by Innogenetics NV, Belgium). Ab 1 42 (29) and T-tau (19, 3) mean values, respectively, 5 and 35 ng l have been proposed as pathological thresholds. The IATI is an index derived from the Hulstaert discrimination line: IATI = (measured Ab 1 42 ) (24 + 1.18measured Tau) (11, 12). A control subject with normal Ab 1 42 and T-tau values has an IATI > 1. A patient with possible, with a lowered Ab 1 42 value and increased tau value, has an IATI < 1. A 95% sensitivity to diagnose patients vs normal patients other neurological disorders has been associated with a IATI value <.8. IATI value >1.2 allows to rule out with a 95% specificity and classify among Ônormal other neurological disordersõ profiles (31, 32). P-tau 181 cut-off value used in this study was 5 ng l (19). The CSF profile is characterized by a biochemical algorithm. If ÔIATI <.8 and P- tau 181 >5ng lõ, CSF profile is considered as biochemical profile. When results are not included in this algorithm, the pattern is qualified as Ôother profileõ. If ÔIATI > 1.2 and P- tau 181 <5ng lõ, CSF profile is considered as normal. If ÔIATI <.8 or P-tau 181 >5ng lõ, CSF profile is not considered as typical or distinctive. Both normal and non-discriminating profiles are biochemically classified as non-. The biochemical classification is thereafter compared to the clinical one. Statistical analysis The distribution of parameter values between both groups (IATI <.8 + P-tau 181 >5ng l, other CSF profiles) was analyzed using a Kolmogorov Smirnov test. Owing to size and asymmetrical distribution of groups, a non-parametric test was conducted (Kruskal Wallis) for group comparison. Results All patients were clinically classified: of the 15 patients, 97 were, 29 non-, the others 24 patients belonging to the MCI group. All CSF samples were frozen and analyzed after thawing. The biochemical classification was then applied to the entire cohort. Results are presented in Table 2. Eighty-three patients had an biochemical profile (IATI <.8 + P-tau 181 > 5 ng l): 71 upon 97, 3 upon 29 non-, and 9 upon 24 MCI clinical diagnoses. In the cohort, 67 patients had other biochemical profiles: 26 upon 97, 26 upon 29 non-, and 15 upon 24 MCI patients. Relative to or non- clinical diagnoses (not including MCI), there was a mismatch for 29 cases. Concordance between first clinical feeling and CSF profile was 77% overall. In and non- groups, the match was 73% and 9%, respectively. Using Kolmogorov Smirnov test, a significant distribution difference between the two CSF profile groups was found for T-tau, Aß 1 42, IATI, P-tau 181, Aß 42 P-tau 181 (P <.1). The comparison of age and MMSE between these two groups showed no significant difference for MMSE (data not shown). Table 3 (upper part) shows T-tau, Aß 1 42, IATI, P-tau 181,Aß 42 P-tau 181 average values in patients with biochemical profile (IATI <.8 + P- tau 181 >5ng l) for the clinically patients (71 97) and for those with other CSF profile and who are non-clinically (26 29). The Kruskal Wallis statistical analysis (Fig. 1) confirmed the significant difference between and non- groups for all parameters (P <.1). Table 3 (middle part) delineates the CSF biomarker values Table 2 CSF profiles and clinical categories Clinical profile Non- MCI Total Age Mean 73.1 7.7 69.3 72.4 SD 7.2 6.2 1.5 7.74 MMSE Mean 2.7 22 27.6 22.14 SD 4.4 4.4 1.5 4.78 IATI <.8 + P-tau 181 >5ng l 71 3 9 83 Other CSF profiles 26 26 15 67 Total 97 29 24 15 Mismatches between CSF biochemical profiles and clinical diagnoses. 3

Tabaraud et al. Table 3 Correlation between CSF biomarkers and clinical diagnosis Biochemical profile Clinical profile T-tau ng l Aß 1 42 ng l IATI P-tau 181 ng l Aß 1 42 P-tau 181 Aß 1 42 Aß 1 4 Ab 1 4 ng l (n = 71) 574 351 344 128.41.18 9 35 4.1 1.7.34.21 11424 3495 Non- (n = 26) 237 129 763 365 1.47.57 51 19 15.6 5.8.7.21 11357 4823 Non- (n = 3) 1226 496.29 159 3.1.28 18 633 429.43 99 4.33.22 1924 39 338.48 62 5.45.35 966 Non- Groupe A (n = 8) 272 86 27 122.49.21 36 9 7.34 2.41.3.2 8671 2532 Groupe B (n = 8) 379 12 67 258 1..1 73 18 9.65 2.2.5.2 12933 2537 Groupe C (n = 1) 276 86 73 119 1.3.24 52 15 14.75 4.1.6.15 12472 462 for three patients showing an biochemical profile despite the fact they were clinically non-. Table 3 (lower part) reports results concerning 26 clinically patients belonging to biochemical group Ôother combinationsõ. They are classified into three groups (A, B, and C). Statistical analysis using Kruskal Wallis test was applied for group comparison. Group A corresponded to eight patients having low P-tau but a disrupted IATI (IATI <.8) owing to Aß 1 42 low value. This group was significantly different from group concerning T-tau and P-tau 181 suggesting that these parameters were not the pathological process predominant factor. Group B gathered eight patients with positive P-tau 181 (>5 ng l) and IATI included between.8 and 1.2 and normal Aß 1 42 (>5 ng l). Group C was constituted by ten patients with IATI superior to 1.2 or by those with P-tau 181 level lower than 5 ng l. Group C classified as clinical had biological markers significantly different (P <.1) from group. Their biochemical data were statistically similar to those of the non- population. Owing to the observed discrepancies, Ab 1 4 quantification was performed to gain additional information either through its own value or Aß 1 42 Aß 1 4 ratio when CSF profile was non-discriminant (IATI <.8 or P-tau 181 >5ng l). According to Wiltfang (26), Aß 1 4 value and Aß 1 42 Aß 1 4 ratio better correlate with the total Ab peptides load than the patient pathological status. Using Kolmogorov Smirnov test, Ab 1 4 values within the and non- groups were not different (P =.584) but a significant distribution difference between the two CSF profile groups was found for Aß 1 42 Aß 1 4 (P <.1). Hence, statistical analysis for Aß 1 4 measurement (Kruskal Wallis) did not confirm the significant difference between and non- groups. The cut-off Aß 1 42 Aß 1 4 ratio value issued from our statistical analysis was.5. For each of the three groups (A, B, and C), the Aß 1 42 Aß 1 4 ratio helped us to classify patients. Thus in group A, Aß 1 42 Aß 1 4 ratio underlined a prevailing pathological amyloid process. In group B, a significant difference with regard to group for Aß 1 42 Aß 1 4 ratio indicated absence of pathological amyloid process suggesting a major pathogenic role of T-tau and P-tau 181 (P <.17 and.54, respectively, for T-tau and P-tau 181 vs the non- group). Moreover for three patients of this group, Aß 1 42 Aß 1 4 ratio indicated that the initial value of Aß 1 42 could be considered low if referred to global Aß peptides concentration. In group C, Aß 1 42 Aß 1 4 ratio was above the.5 cut-off value in accordance with the non- biochemical profile. As far as MCI is concerned, because first clinical examination is unable to predict disease development, we classified them in a different section (Table 4). Of 24 patients clinically classified as MCI, nine presented with a characteristic profile. The 15 remaining did not exhibit biochemical profile. For every one of them, as regards P-tau 181, Aß 1 42, IATI values, obvious differences were shown between progressive and stable disease (respectively P =.1, P =.3, P =.1). Furthermore, there are no significant differences, on the one hand between and progressive MCI and, on the other, between non- and stable MCI (data not shown). Discussion The purpose of this study was to appraise contribution of CSF biomarkers in daily clinical practice. For either public or liberal memory consultation centers, conclusions seemed similar. When biomarkers fit in with clinical assessment, they allow to assert more firmly the clinical impression, thus contributing to a faster and more reliable diagnosis. Numerous studies have shown the CSF biomarkers combination to be more effective than cognitive profile alone (2). Our data demonstrated the ability of the elected combination, i.e., IATI (<.8) and P-tau 181 (>5 ng l) to conclude to AlzheimerÕs pathology 4

AlzheimerÕs disease CSF biomarkers 3.6 3.2 2.8 IATI 25 2 P-tau181 2.4 2 15 1.6 1.2.8.4 1 5 A 1 42 T-tau 25 245 2 21 175 15 14 1 5 15 7 35 35 A 1 42 /P-tau181.125 A 1 42 /A 1 4 3 25 2 15 1 5.1.75.5.25 3 A 1 4 25 2 15 1 5 Figure 1. Kruskal Wallis statistical analysis. Determination of clinical-biochemical correlation or discrepancy. non- group differs significantly from group (P <.1). Table 4 CSF biological values in evolutive and stable MCI T-tau ng l Aß 1 42 ng l IATI P-tau 181 ng l Aß 1 42 P-tau 181 Aß 1 42 Aß 1 4 Ab 1 4 ng l Evolutive MCI (n = 9) 695 261 396 133.42.12 114 41 3.99 2.4.29.1 15982 5173 Stable MCI (n = 15) 234 64 763 233 1.47.4 49 13 15.84 4.94.72.18 1943 292 (73%), and turn it out with a 9% reliability. A more firmly set diagnosis thus allowed better coping with patient and family. Analysis of and non- population (defined by the clinico-biochemical concordance) showed, for each of the parameters or ratio, similar 5

Tabaraud et al. values to those described in the literature. The analysis of Receiver Operating Characteristic (ROC) curves analysis (data not shown) for each parameter between the two reference groups sets up following thresholds: 474 ng l (Sens. 95%; Spe.92%) for Aß 1 42 ; 336 ng l (Sens.75%; Spe.88%) for T-tau; and 57 ng l (Sens.87%; Spe.69%) for P-tau 181.The respective 35 and 5 ng l thresholds for Tau and Aß 1 42 thus perfectly apply to the two groups. P-tau 181 thresholds usually vary between 5 and 6 ng l (3,32).Toincreasesensitivity,wechoseto use the 5 ng l limit. Likewise, the 9 threshold Aß 1 42 P-tau 181 ratio value, first advocated by Welge et al. (33), correctly separates different groups (Sens. 98.6%; Spe.85%). Concerning Aß 1 4 peptide dosage, its only value was not contributive to diagnosis but enabled us to establish Aß 1 42 Aß 1 4 ratio. This later used with a.5 threshold can be of additional help to confirm the amyloid involvement in the pathogenic process (Sens. 9.5%; Spe. 8%). Owing to a distinct methodology, our threshold differs from WiltfangÕs one (26). Biochemical results, if limited to Aß 1 42, T-tau, P-tau 181, are unable to provide every time definite endings. This lack of information can be overcome with help of the Aß 1 42 Aß 1 4 ratio, enabling to separate two groups of patients (A and B) depending upon etiopathogenic pathways, either Ab amyloid or T- tau and P-tau 181. Although non-systematic, this dosage seems to be able to spare complementary imaging, thus reducing costs. Additional Aß 1 4 measurements were performed on previously frozen aliquots. We also focused on atypical cases. Three patients clinically considered as non- showed a typical CSF profile without any MRI atrophy. Follow-up eventually brought up evidence for development. Numerous records have clearly shown that CSF biomarkers are susceptible to disclose an incipient disease (3, 34). Pure usual dementia forms are not the rule in elderly people (35). Senile amyloid patches are evidenced by post-mortem examination of patients with ParkinsonÕs disease (36). Thus, routine CSF biomarkers use is advisable when clinical assessment is puzzling, and facing ParkinsonÕs disease cognitive impairment, we were able to evidence typical profile in some patients. Of 24 MCI patients 9 had an profile, meeting up well with hippocampus atrophy on MRI examination. Biomarkers are a convincing argument to predict disease progression from MCI to (2, 3). Follow-up (18 months) of these nine MCI patients confirmed the prodromal diagnosis established by CSF biomarkers, thus underlining their relevance in the earliest phase of the disease. Of 15 stable MCI, one of them showed hippocampus atrophy along with normal CSF biomarkers. As a clinical diagnosis mistake was turned down, further close monitoring is required. Conclusion This study confirms the paramount significance of CSF biomarkers measurements in diagnosis in routine practice. Pathognomonic results allow to Systematic clinical investigation neuropsychological evaluation Systematic brain MRI CSF biomarkers looking for histological signature Approach to diagnosis by exclusion: elimination of an associated pathology T-tau -amyloide 1 42 P-tau 181 by ELISA Innotests Looking for possible positive arguments: qualitative evaluation and quantitative volumetric evaluation of internal temporal structure IATI > 1.2 and P-tau 181 <5 ng/l IATI <.8 and P-tau 181 >5 ng/l IATI <.8 or P-tau 181 >5 ng/l Normal CSF profile profile Plausible profile If 3 biomarkers profile non discriminant Ratio Aβ 1 42 /Aβ 1 4 <.5 Pathological amyloid process Figure 2. Biomarkers integration in daily clinical practice. 6

AlzheimerÕs disease CSF biomarkers assert the diagnosis in the possible or likely forms and, moreover, in the occurrence of associated pathologies (vascular disease, psychiatric, basal ganglia disease, and others) synonymous with a wandering clinical diagnosis. Non-specific CSF biomarkers results require further investigation as other diagnoses have to be looked for. Including CSF biomarkers into the diagnostic processes seems to be relevant as summarized in the Fig. 2 algorithm. Acknowledgments We acknowledge Wereminski N. (EISAI SAS), Dejans D., Pelpel M., (INNOGENETICS France) for their logistical and technical supports and Drs Golfier-Pichon F., Lubeau M., Chazot F., Croguennec J. for their participation to the clinical protocol. This work was granted by Caisse Primaire dõassurance Maladie de la Haute-Vienne Caisse Primaire dõassurance Maladie de la Corre` ze and Caisse dõepargne Auvergne Limousin. Conflict of interest The authors declare no conflict of interest. References 1. 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