Plasma neurofilament light as a potential biomarker of neurodegeneration in Alzheimer’s disease

Plasma neurofilament light as a potential biomarker of neurodegeneration in Alzheimer’s disease Background: A growing body of evidence suggests that the plasma concentration of the neurofilament light chain (NfL) might be considered a plasma biomarker for the screening of neurodegeneration in Alzheimer’s disease (AD). Methods: With a single molecule array method (Simoa, Quanterix), plasma NfL concentrations were measured in 99 subjects with AD at the stage of mild cognitive impairment (MCI-AD; n = 25) or at the stage of early dementia (ADD; n = 33), and in nondemented controls (n = 41); in all patients, the clinical diagnoses were in accordance with the results of the four core cerebrospinal fluid (CSF) biomarkers (amyloid β (Aβ)1–42, Aβ42/40, Tau, and pTau181), interpreted according to the Erlangen Score algorithm. The influence of preanalytical storage procedures on the NfL in plasma was tested on samples exposed to six different conditions. Results: NfL concentrations significantly increased in the samples exposed to more than one freezing/thawing cycle, and in those stored for 5 days at room temperature or at 4 °C. Compared with the control group of nondemented subjects (22.0 ± 12.4 pg/mL), the unadjusted plasma NfL concentration was highly significantly higher in the MCI-AD group (38.1 ± 15.9 pg/mL, p < 0.005) and even further elevated in the ADD group (49.1 ± 28.4 pg/mL; p <0.001). A significant association between NfL and age (ρ =0.65, p < 0.001) was observed; after correcting for age, the difference in NfL concentrations between AD and controls remained significant (p = 0.044). At the cutoff value of 25.7 pg/mL, unconditional sensitivity, specificity, and accuracy were 0.84, 0.78, and 0.82, respectively. Unadjusted correlation between plasma NfL and Mini Mental State Examination (MMSE) across all patients was moderate but significant (r = −0.49, p < 0.001). We observed an overall significant correlation between plasma NfL and the CSF biomarkers, but this correlation was not observed within the diagnostic groups. Conclusions: This study confirms increased concentrations of plasma NfL in patients with Alzheimer’sdisease compared with nondemented controls. Keywords: Alzheimer’s disease, Neurofilament light, Biomarker, Plasma * Correspondence: Piotr.Lewczuk@uk-erlangen.de Department of Psychiatry and Psychotherapy, Lab for Clinical Neurochemistry and Neurochemical Dementia Diagnostics, Universitätsklinikum Erlangen, and Friedrich-Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany Department of Neurodegeneration Diagnostics, Department of Biochemical Diagnostics, Medical University of Bialystok, University Hospital of Bialystok, Bialystok, Poland Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 2 of 10 Background caregivers gave their written consent. Blood samples Cerebrospinal fluid (CSF) biomarkers have been extensively were obtained from 99 patients at the Department of studied as tools for an early diagnosis of Alzheimer’sdisease Psychiatry and Psychotherapy, Universitätsklinikum (AD) [1] and have proven to be cheaper, less demanding in Erlangen, and categorized into three groups: nondemen- terms of infrastructure and patient management, and most ted controls (Controls, n = 41), MCI with high probabil- probably capable of showing pathologic alterations slightly ity of AD pathology (MCI-AD, n = 25), and AD in early earlier than other diagnostic modalities such as, for example, dementia stage (ADD, n = 33). According to the revised positron emission tomography; the relative invasiveness of NINCDS-ADRDA criteria [11, 12], diagnoses were made the two modalities remains disputable [2]. In particular, the after clinical evaluation, neuropsychological testing with four core CSF biomarkers, amyloid β (Aβ)1–42, Aβ42/40 ra- the CERAD+ battery, magnetic resonance imaging tio, Tau, and pTau181, reliably support AD diagnostics (MRI), duplex sonography of the carotids and brain reflecting the hallmark AD pathologies, i.e., amyloidosis and arteries as well as electroencephalographic (EEG) re- neurodegeneration [3]. Lumbar puncture (LP) is a routine cordings. The distinction between dementia and MCI clinical procedure with a low incidence of complications [4]. was made on the basis of the clinical assessment; pa- Nevertheless, collection of CSF is accompanied by proced- tients with MCI had an objectively measureable cogni- ural efforts and inconvenience for subjects, ultimately tive decline but their independence regarding functional preventing its use as a screening tool in early, asymptomatic life abilities was preserved, which means that the revised cases and it can also be challenging to use for repetitive NINCDS-ADRDA diagnostic criteria of dementia were monitoring of the disease progression.Hence,there is a not fulfilled. In all cases, the clinical and neuropsycholo- strong need to develop blood-based biomarkers that, when gic diagnoses remained in accord with the results of the applied in a proper context of use, could serve as targeted four core biomarkers of neurochemical dementia and relative noninvasive screening tests [5]. diagnostics (NDD), all analyzed by enzyme-linked im- Neurofilaments (Nf) consist of three types of pro- munosorbent assays (ELISA): Aβ1–42 (IBL International, tein chains, differing in molecular mass: a light chain (NfL) Hamburg, Germany), Aβ42/40 (IBL International), Tau of 68 kD, an intermediate chain of 150 kD, and a heavy chain (Fujirebio Europe, Ghent, Belgium), and pTau181 (Fujirebio of 190 to 210 kD, and are major components of axonal cyto- Europe), interpreted according to the Erlangen Score skeleton [6]. Each subunit is composed of a double-stranded, algorithm (ES), [13, 14]. All control cases had normal CSF highly conserved α-helical core domain, bordered by a head results (ES ≤ 1) and all MCI-AD and ADD patients had N-terminus and a tail C-terminus. Nf are highly phosphory- profoundly pathological CSF results (ES = 4). Vascular lated proteins, and the degree of this phosphorylation deter- copathology was not an exclusion criterion in our study. mines the axon diameter [6]. Axonal damage leads to release Blood was collected by venipuncture into standard of Nf molecules into the extracellular space and, conse- polypropylene EDTA test tubes (Sarstedt, Nümbrecht, quently, into body fluids, such as the CSF or plasma. In line Germany) followed by centrifugation; the obtained plasma with this, increased blood NfL concentrations were reported was portioned into approximate 500 μL aliquots, fro- in neurodegenerative and neuroinflammatory disorders zen at −80 °C, and kept unthawed until the analyses. [7–9]. A very recent report by Mattsson et al. convincingly concludes that plasma NfL could be considered a neuro- Samples for testing the preanalytical storage conditions degeneration biomarker in AD [10]. For testing of the influence of the preanalytical storage In this study, we measured NfL concentrations in conditions, blood samples were collected from five do- plasma samples of AD patients at the stages of mild cogni- nors, and the plasma was generated as above. Immediately tive impairment (MCI) and early dementia (ADD), and thereafter, from each sample six aliquots were prepared, nondemented controls. In all our subjects the clinical and resulting in a matrix of 5 cases × 6 aliquots (conditions): a) neuropsychologic diagnoses were in accordance with the one aliquot was immediately frozen at −80 °C, and served results of their four core CSF biomarkers (Aβ1–42, Aβ42/ as a reference sample; b) one aliquot was frozen at −80 °C 40, Tau, and pTau181). For the validation of the assay, we and exposed to one thawing/refreezing (1 × F/T) cycle; c) further tested the influence of different preanalytical sam- one aliquot was frozen at −80 °C and exposed to two F/T - ple handling procedures (repetitive freezing/thawing, and cycles; d) one aliquot was frozen at −80 °C and ex- storage at room temperature or in a refrigerator) on the posed to three F/T cycles; e) one aliquot was stored concentrations of plasma NfL. for 5 days at 4 °C; and f) one aliquot was stored for 5 days in the dark at room temperature (~ 21 °C). Following the Methods isochronous testing strategy [15], after 5 days at either Patients and blood collection; diagnostic CSF tests room temperature or 4 °C, the aliquots (e) and (f) were The ethical committee of the University of Erlangen- transferred to the reference condition (−80 °C freezer) and Nuremberg approved the study, and all patients or their kept frozen until analyses. Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 3 of 10 Simoa assay If not stated otherwise, estimates in logistic models For analysis of NfL concentrations, the samples were were obtained with the maximum likelihood (ML) transported on dry ice to the Clinical Neurochemistry method. P < 0.05 was considered significant. All analyses Laboratory, Sahlgrenska University Hospital. The ana- were performed with Stata 14.2 (StataCorp, College Sta- lyses were performed using an in-house assay on the sin- tion, TX, USA). gle molecule array platform (Simoa; Quanterix, Lexington, MA, USA), as previously described in detail Results [16]. This method has an analytical sensitivity of Plasma NfL assay performance 0.62 pg/mL [7]. All measurements were performed by Two quality control (QC) samples spanning clinically board-certified laboratory technicians who were blinded relevant concentrations of the analyte were analyzed in to clinical data. duplicates two to three times in each run. For a QC sample with a concentration of 12.5 pg/mL, repeatability Statistical analysis was 8.9% and intermediate precision was 11.0%. For a The number of samples was estimated considering the be- QC sample with a concentration of 105.7 pg/mL, repeat- tween-group differences reported in the available litera- ability and intermediate precision were both 6.4%. ture, and with the significance and the power set to 0.05 and 0.9, respectively. If not stated otherwise, the results Concentrations of NfL in plasma samples under different are presented as averages ± standard deviations (SD) or storage conditions 95% confidence intervals (95% CI). Correlations between Concentrations of NfL in aliquots of the five plasma sam- continuous variables, unadjusted for other covariates, are ples with six different preanalytical treatment conditions presented as Pearson (r)or Spearman (ρ) correlation coef- are presented in Fig. 1. Compared to the reference aliquot, ficients, as indicated. Partial correlation was used to the concentrations of NfL under all conditions, except for estimate the correlation coefficients and their significances one cycle of thawing/refreezing, were slightly but signifi- between Mini Mental State Examination (MMSE) scores cantly higher (p < 0.05). After normalization, defining the and the concentrations of plasma NfL and the CSF bio- concentration in a reference aliquot as 1.00, the average markers, conditional on diagnostic categories. Differences relative concentrations and their standard deviations in the across multiple categories were tested with analysis of tested aliquots were: 1.07 ± 0.23 in the aliquots exposed to variance (ANOVA) followed by post-hoc tests with appro- one freezing/thawing cycle, 1.24 ± 0.23 in the aliquots priate corrections for multiple comparisons. thawed and refrozen twice, 1.41 ± 0.42 in the aliquots Association of the preanalytical storage conditions thawed and refrozen three times, 1.32 ± 0.26 in the aliquots and the NfL concentrations was tested with a vari- stored for 5 days at 4 °C, and 1.46 ± 0.21 in the aliquots ance component model with conditions crossed with stored for 5 days at room temperature. The ML estimates samples. A linear regression was used to model NfL of the random parameters of the variance-components plasma concentrations in the diagnostic groups, ad- model were ψ = 37.3 (variability of the random intercept, justed for patient age, including interaction term i.e., between-sample), and θ = 1.94 (variability of the level-1 age-by-disease status. The models were compared residual, i.e., within-sample), resulting in a within-sample with likelihood ratio (LR) test on indicated degrees of (intraclass) correlation ρ =0.95. freedom (df). The NfL concentration cutoff separating controls from Plasma NfL in nondemented controls, MCI-AD, and ADD AD patients was calculated at the maximized Youden patients index. Logistic regression was used to model the sensi- Demographic characterization of the three groups consid- tivity and the specificity as a function of age according ered in this study, the results of the CSF biomarkers, and to Coughlin et al. [17], slightly modified by introducing the plasma NfL concentrations are presented in Table 1. interaction term for age-by-disease status. The test's ac- Results of the NfL measurements in the three diagnostic curacy was defined as the probability of the agreement groups are presented in Fig. 2. Unadjusted for other between the result of the test and the disease status (re- covariates and compared with the control group of nonde- sponse = 1 if both positive or both negative, or 0 other- mented subjects (22.0 ± 12.4 pg/mL), average NfL concen- wise), and was modeled, conditional on age, with logistic trations were highly significantly higher in the MCI-AD regression. The area under the receiver operating char- group (38.1 ± 15.9 pg/mL, p < 0.005), and even further ele- acteristic (ROC) curve (AUC) was calculated with a non- vated in the ADD group (49.1 ± 28.4 pg/mL; p < 0.001). parametric method. Fisher’s linear discriminant analysis The concentrations in the ADD group was also higher (LDA) was used to calculate parameters of the line opti- compared with the MCI-AD group; however, this differ- mally separating the controls and the AD patients on ence became borderline insignificant after Scheffe’s the basis of their NfL concentrations and age. post-hoc correction for multiple comparisons (p =0.12). Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 4 of 10 Fig. 1 Neurofilament light chain (NfL) plasma levels of samples with different preanalytical treatment. *p < 0.05, versus the reference (Ref.) sample (frozen at −80 °C and unthawed). d days, F/T freeze/thaw cycle, n.s. not significant, RT room temperature Taking into consideration that a) according to the interaction term of age-by-disease status was insignifi- current concept of the AD continuum [18, 19], MCI-AD cant (β = 0.365, p = 0.361), and did not improve the is not a separate diagnostic entity but rather a stage of model fit (LR test χ (df = 1) = 0.87, p = 0.35), we excluded AD, and b) MCI-AD and ADD cases had, expectedly, it from further analyses. Using LDA, we finally estimated the same pattern of the pathologic CSF results, we the parameters of the line optimally separating the AD pooled the two groups into one diagnostic category (Alz- patients from the controls: NfL (pg/mL) = −5.25 × age heimer’s disease, AD) and contrasted it with the nonde- (years) + 367.7, which resulted in 81% of cases being mented controls. In Fig. 3, NfL concentrations in AD properly categorized (sensitivity and specificity of 0.86 and controls are plotted against age. Overall, we ob- and 0.76, respectively). served a highly significant association between NfL and age (ρ = 0.65, p < 0.001). After correcting for age, the dif- Sensitivity, specificity, and accuracy of plasma NfL as a ference in NfL concentrations between the two groups potential AD biomarker remained statistically significant (p = 0.044). Since the Figure 4 presents the performance of the plasma NfL concentration in the setting of AD patients with positive Table 1 Demographic characterization, CSF results, and NfL CSF biomarkers versus nondemented controls with concentrations in the diagnostic groups negative CSF biomarkers. Unadjusted for other variables, Controls MCI-AD ADD the AUC of the ROC curve in the setting controls vs dis- N (male + female) 41 (22 + 19) 25 (10 + 15) 33 (13 + 20) eased turned out reasonably large (0.853, 95% CI 0.772– 0.934; Fig. 4a). After introducing age to the model esti- Age (years) 52.5 ± 13.1 71.3 ± 8.4 70.8 ± 7.6 mating the ROC curve, the AUC increased insignifi- a b MMSE 29.3 ± 0.9 26.7 ± 2.1 21.2 ± 3.4 cantly (p = 0.055) to 0.920 (95% CI 0.869–0.970). At the CSF Aβ1–42 (pg/mL) 1025 ± 308 585 ± 116 536 ± 114 cutoff maximizing Youden index, 25.7 pg/mL, uncondi- CSF Aβ1–40 (pg/mL) 13,598 ± 4046 19,171 ± 5689 15,309 ± 4172 tional sensitivity, specificity, and accuracy were 0.84, CSF Aβ42/40 0.076 ± 0.01 0.032 ± 0.01 0.036 ± 0.006 0.78, and 0.82, respectively. Considering age differences CSF Tau (pg/mL) 198 ± 64.4 631 ± 214 558 ± 178 between the groups in this study, we consequently mod- eled the performance characteristics of the test as a CSF pTau181 (pg/mL) 37.3 ± 12.1 101.4 ± 29.8 89.9 ± 18.4 function of age (Fig. 4b). While the sensitivity increased Plasma NfL (pg/mL) 22.0 ± 12.4 38.1 ± 15.9 49.1 ± 28.4 with age, from 0.61 at 50 years to 0.91 at 80 years, the Values are shown as averages ± standard deviations or as numbers per group specificity decreased from 0.89 to 0.20, respectively, Aβ amyloid β, AD Alzheimer’s disease, ADD Alzheimer’s disease dementia, CSF cerebrospinal fluid, MCI mild cognitive impairment, MMSE Mini Mental State leaving the overall accuracy of the test practically un- Examination, NfL neurofilament light chain a altered (0.84 and 0.80 at the age of 50 and 80 years, Available in 22 cases Available in 23 cases respectively). Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 5 of 10 Fig. 2 Unadjusted neurofilament light chain (NfL) concentrations in the three diagnostic categories. A borderline insignificant (p = 0.11 after Scheffe correction) tendency was observed towards higher concentrations in Alzheimer’s disease dementia (ADD) compared with mild cognitive impairment Alzheimer’s disease (MCI-AD). The green horizontal line represents the cutoff at the maximized Youden index (25.7 pg/mL), leading to the unadjusted diagnostic accuracy of 82% in the setting of controls versus AD (MCI-AD and ADD). *p < 0.05 The unadjusted correlation between plasma NfL and Correlation of plasma NfL with the four core CSF MMSE score was moderate and highly significant (r = biomarkers −0.49, p < 0.001; Fig. 5). After controlling for the diag- Without taking diagnostic categories into consideration, nostic categories, the coefficient became weaker, albeit NfL in plasma correlated highly significantly (p < 0.001) still significant (r = −0.24, p = 0.036). None of the CSF with all CSF biomarkers except Aβ1–40 (as expected, biomarkers correlated significantly with the MMSE score positively with Tau and pTau181, and negatively with after controlling for the diagnostic categories (p > 0.2; Aβ1–42 and Aβ42/40; data not shown). This correlation data not shown). became insignificant for all CSF biomarkers (p > 0.35) Fig. 3 Neurofilament light chain (NfL) plasma levels plotted against age in nondemented controls (green) and the AD patients (a combined group of MCI-AD and ADD subjects, red). The black dotted line represents the optimal separation of the two groups according to Fisher’s LDA Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 6 of 10 Fig. 4 a Unadjusted ROC curve in the setting of controls vs. AD. b Sensitivity (blue), specificity (brown), and accuracy (green) as functions of age. AUC area under the curve, CI confidence interval, SE standard error when the diagnoses were taken into account. Figure 6 patients, whose diagnoses were in accordance with the presents, exemplarily, a significant correlation between pathological results of the CSF biomarkers, compared plasma NfL and CSF Tau across all patients taken with the nondemented control subjects, whose normal together (Fig. 6a) and a lack of within-groups cognitive status was in accordance with the unaltered re- correlation between the two analytes (overall p = 0.64) sults of their CSF biomarkers. Within the AD group, we when the three diagnostic groups are treated separ- observed a tendency towards higher NfL concentrations ately (Fig. 6b−d). in patients at the stage of early dementia compared with the stage of MCI. Plasma NfL concentrations correlated Discussion inversely with the global cognitive status measured by A growing body of literature postulates a potential appli- the MMSE results. Finally, we also provide evidence for cation of the plasma concentration of the light chain of the influence of the preanalytical sample handling on the the neurofilament protein as a screening tool for neuro- plasma NfL concentrations. degeneration. In our study, the plasma NfL concentra- We started our study by examining the influence of sam- tion was found to be significantly higher in the AD ple handling procedures on plasma NfL concentrations. Fig. 5 Correlation between plasma neurofilament light chain (NfL) concentrations and the Mini Mental State Examination (MMSE) results Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 7 of 10 Fig. 6 Plasma neurofilament light chain (NfL) plotted against cerebrospinal fluid (CSF) Tau. a. Overall moderate, highly significant correlation between plasma NfL and CSF Tau; lacking within-group correlation between plasma NfL and CSF Tau in the controls (b), MCI-AD (c), and ADD (d) We believe this is a critically important aspect; for example, seems reasonable to postulate sending the material to dis- asample’s storage and transportation to distant laboratories tant laboratories deeply frozen and avoiding more than one is a nontrivial issue and has practical implications. Com- intermediate thawing/refreezing cycle. pared with a reference sample (i.e., a deep-frozen aliquot In agreement with recently published studies on spor- stored unthawed until analysis), we observed a slight but adic AD [10, 26, 27] and familial AD (FAD) [28], we significant increase in the concentrations in the aliquots found increased plasma NfL concentrations in AD pa- thawed and refrozen twice or three times, kept for 5 days at tients in the dementia stage (ADD) as well as in MCI the room temperature, or stored in a refrigerator. In con- subjects with a high probability of underlying AD path- trast, one thawing/refreezing cycle did not systematically ology (MCI-AD), compared with nondemented controls. alter the NfL concentration, but resulted in nonsystematic The results of the current study confirm those reported changes, i.e., the concentrations increased in some samples by Mattsson et al. obtained with the same method and but decreased in others, resulting in increased variability in the same laboratory but on different patient cohorts but an unchanged average. Whereas in most cases a de- [10], not only in terms of the average concentrations crease in the concentration of a protein with storage time and their biological variability (coefficients of variation), or under thawing/refreezing is expected, it is known that but also in terms of the NfL performance as a potential some proteins, for example serum albumin, tend to in- plasma diagnostic test. In both studies, almost identical crease in concentration after repetitive thawing/refreezing areas under the ROC curves, contrasting AD patients [20]. Similarly, in our previous studies, an unsystematic versus nondemented controls, were obtained (0.853 and increase in CSF Aβ1–42, Aβ1–40, and Tau in some, but 0.87, respectively). A slightly higher average NfL concen- not all, aliquots exposed for more than two thawing/re- tration in the controls reported by Mattsson et al. com- freezing cycles was observed [21, 22]. As an explanation, a pared with the present study can be explained by the release of NfL monomers from aggregates, known to form fact that one-third of the controls in the previous in certain neurodegeneration disorders [23], might be paper showed Aβ positivity, whereas in the current study considered. Interestingly, the data on the stability of NfL in positive Aβ CSF results excluded a subject as a control. the CSF are ambiguous; whereas one study found rapid de- This is due to the fact that, in contrast to the papers cline of concentrations at room temperature or 4 °C [24], published previously, patients in the current study were no changes were observed in another study until 3 days, included only if their clinical and neuropsychological followed by a subsequent decrease [25]. In any case, it diagnoses stayed in agreement with the outcome of the Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 8 of 10 four core CSF biomarkers (Aβ1–42, Aβ42/40, Tau, and a slight decrease of the AUC from 0.87 to 0.79 when the pTau181) conservatively interpreted according to the Er- model was fitted with age, sex, and educational level, in- langen Score algorithm [13, 14]. We are aware that such stead of all variables considered in their study. We are not an approach has advantages and disadvantages; it en- aware of any report analyzing age-dependency of any ables more reliable stratification of the cases, but it ex- other metrics. We believe that the characteristics found in cludes the possibility of a direct comparison of the this study, with an age-dependent increase in the sensitiv- diagnostic utility of the plasma NfL with any of the CSF ity at the cost of the decreasing specificity clearly seen in biomarkers. the age range of 60–80 years (i.e., in the range when Our finding of a positive association between plasma neurodegeneration is most commonly considered in the NfL concentration and age is in agreement with diagnosis), further supports the postulated potential appli- previously reported studies on plasma [9, 10] and CSF cation of the plasma NfL as a screening tool for neurode- [29, 30]. A weak but significant association between generation, rather than as a test for confirming AD serum NfL and age at onset of a disease was also reported diagnosis. in primary progressive aphasia (PPA) [8]; however, another In line with the recently published results [10], we study did not find a correlation of NfL with age after found an inverse correlation of plasma NfL concentra- adjusting for the estimated age of onset of a disease in tions with MMSE results. In contrast, none of the CSF FAD [28]. Mechanisms of this age-dependent increase in biomarkers measured in this study correlated signifi- the NfL concentrations in body fluids, and also in persons cantly with the MMSE score after controlling for the without clinical signs of neurodegeneration, are unclear diagnostic categories. This finding supports our previous thus far. It was hypothesized that aging leads to a subclin- results of a lack of association between MMSE score and ical axonal degeneration and, in consequence, to the re- the CSF results [32, 33], and remains in agreement with lease of Nf molecules [29]. The same group also proposed the generally accepted assumption that the CSF bio- that subclinical cerebrovascular changes might be consid- markers do not correlate with disease progression at the ered as an explanation, since cerebrovascular pathology is stage of MCI and later [34]. Other studies provide evi- common and known to increase with age; finally, vascular dence that NfL plasma concentrations reflect the dynam- copathology is also commonly observed in AD [31]. Irre- ics of neurodegeneration processes measured with spective of the underlying mechanisms, the association of different metrics. Steinacker et al. found an association be- the NfL concentrations with age has implications for the tween increased NfL concentration and functional decline diagnosis-oriented interpretation of the results. First, an and progression of atrophy in the left frontal lobe of PPA age-dependent cutoff needs to be established, and calcula- patients [8], and Weston et al. reported an association of tion of such a cutoff is not a trivial task. Perhaps the best serum NfL concentration with the time from symptom approach is by applying such statistical tools such as LDA; onset in FAD [28]. Similarly, increased CSF NfL was found in such a case, however, the slope of the line discriminat- to correlate with decreased MMSE score and with faster ing the groups (i.e., the age-dependent cutoff) clearly brain atrophy over time, as measured by changes in depends on the distribution of the parameters in question whole-brain volume, ventricular volume, and hippocam- (here NfL concentrations and age) in these groups. If they pus volume in AD [35]. In multiple sclerosis, CSF NfL re- are not age-matched, as in our study and in some other flects acute axonal damage, and hence it might be reports [27, 28], a line best discriminating AD patients considered a prognostic biomarker (reviewed in [6]). from the controls has a negative slope, which might look Similar to the previously published findings [10], we contradictory to common sense (i.e., in spite of NfL con- observed a highly significant overall correlation of centration increasing with age, its cutoff decreases). This plasma NfL with CSF biomarkers for AD pathology would be different if the two groups were age-matched, as when the diagnostic categories were not considered. in the study by Mattsson et al. [10]. In such a case, the dis- Confirming the previous report, the significance of this criminatory line would have a positive slope (i.e., it would correlation disappeared when the diagnostic groups were increase with age). Secondly, metrics of the performance evaluated separately. Such a correlation pattern, with of the NfL concentrations as a potential diagnostic test overall significant correlation that is not observed within also depend on age. In this study, we observed an increase particular diagnostic groups, is not surprising when a in the sensitivity at the cost of a decrease in the specificity lack of association between the CSF biomarkers and the with increasing age, leaving overall accuracy practically disease dynamics as soon as the first cognitive symptoms unaltered. Furthermore, we observed a slight, borderline occur (i.e., from the MCI stage on) is taken into consid- insignificant increase of the area under the ROC curve eration [34]. with age. To the best of our knowledge, only Mattsson et Perhaps the most important limitation of our study is al. [10] evaluated the age-dependent AUC of the ROC the relatively small, age-unmatched groups, which we curve discriminating AD from healthy controls, observing tried to counterbalance by controlling for age in all Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 9 of 10 statistical analyses. It must be stressed, however, that Competing interests PL has received consultation and/or lecture honoraria from IBL International, such discrepancy between age of AD patients and non- Fujirebio Europe, AJ Roboscreen, and Roche. KB has served as a consultant or demented controls simply reflects the reality that AD on advisory boards for Alzheon, BioArctic, Biogen, Eli Lilly, Fujirebio Europe, IBL patients are older. International, Merck, Novartis, Pfizer, and Roche Diagnostics. KB and HZ are cofounders of Brain Biomarker Solutions in Gothenburg AB, a GU Ventures- based platform company at the University of Gothenburg. HZ has served on ad- Conclusion visory boards of Eli Lilly and Roche Diagnostics and has received travel support In conclusion, we confirmed increased concentrations of from Teva. CS and JP are full-time employees of Boehringer Ingelheim. All remaining authors declare that they have no competing interests. plasma NfL in Alzheimer’s disease; however, its future potential application as a biomarker will have to take its Publisher’sNote nonspecificity into account. We speculate that plasma Springer Nature remains neutral with regard to jurisdictional claims in NfL will not be able to replace the CSF biomarkers of published maps and institutional affiliations. neurodegeneration within a given diagnostic group, but Author details it could perhaps be considered as a potential screening Department of Psychiatry and Psychotherapy, Lab for Clinical tool between the groups. Finally, we extended previous Neurochemistry and Neurochemical Dementia Diagnostics, studies with a systematic test for the influence of the Universitätsklinikum Erlangen, and Friedrich-Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany. preanalytical sample handling procedures on the NfL Department of Neurodegeneration Diagnostics, Department of Biochemical concentrations in plasma. Diagnostics, Medical University of Bialystok, University Hospital of Bialystok, Bialystok, Poland. Clinical Neurochemistry Laboratory, Sahlgrenska University Abbreviations Hospital, Mölndal, Sweden. Department of Psychiatry and Neurochemistry, AD: Alzheimer’s disease; ADD: Alzheimer’s disease dementia; AUC: Area Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the under the curve; Aβ: Amyloid β; CI: Confidence interval; CSF: Cerebrospinal University of Gothenburg, Mölndal, Sweden. Boehringer Ingelheim Pharma fluid; df: Degrees of freedom; ES: Erlangen Score; FAD: Familial Alzheimer’s GmbH & Co. KG, Biberach an der Riss, Germany. Boehringer Ingelheim disease; F/T: Freeze/thaw; LDA: Linear discriminant analysis; LP: Lumbar International GmbH, Ingelheim am Rhein, Germany. Department of puncture; LR: Likelihood ratio; MCI: Mild cognitive impairment; ML: Maximum Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, likelihood; MMSE: Mini Mental State Examination; NfL: Neurofilament light UK. UK Dementia Research Institute at UCL, London, UK. chain; PPA: Primary progressive aphasia; QC: Quality control; ROC: Receiver operating characteristic; SD: Standard deviation Received: 25 April 2018 Accepted: 9 July 2018 Funding The research leading to these results has received support from the Innovative References Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372, 1. Olsson B, Lautner R, Andreasson U, Ohrfelt A, Portelius E, Bjerke M, Holtta M, resources of which are composed of a financial contribution from the European Rosen C, Olsson C, Strobel G, et al. CSF and blood biomarkers for the Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA diagnosis of Alzheimer's disease: a systematic review and meta-analysis. companies in kind contribution, the Swedish and European Research Councils, Lancet Neurol. 2016;15:673–84. the Swedish Alzheimer Foundation, the Swedish Brain Foundation, the Swedish 2. Lewczuk P, Kornhuber J. Do we still need positron emission tomography for State Support for Clinical Research (ALFGBG), the Torsten Söderberg early Alzheimer's disease diagnosis? Brain. 2016;139:e60. Foundation, the Knut and Alice Wallenberg Foundation, Frimurarestiftelsen, 3. Lewczuk P, Riederer P, O'Bryant SE, Verbeek MM, Dubois B, Visser PJ, Hjärnfonden, and Alzheimerfonden. Jellinger KA, Engelborghs S, Ramirez A, Parnetti L, et al. Cerebrospinal fluid and blood biomarkers for neurodegenerative dementias: an update of the consensus Availability of data and materials of the task force on biological markers in psychiatry of the World Federation of The datasets used for the analyses are available from the corresponding Societies of Biological Psychiatry. World J Biol Psychiatry. 2018;19:244–328. author on reasonable request. 4. Duits FH, Martinez-Lage P, Paquet C, Engelborghs S, Lleo A, Hausner L, Molinuevo JL, Stomrud E, Farotti L, Ramakers I, et al. Performance and Authors’ contributions complications of lumbar puncture in memory clinics: results of the PL: conceptualization and design of the study; collection of the samples and multicenter lumbar puncture feasibility study. Alzheimers Dement. 2016;12: the data; interpretation of the data including the statistical analyses; drafting/ 154–63. revising of the manuscript. NE: collection of the samples and the data; 5. O'Bryant SE, Mielke MM, Rissman RA, Lista S, Vanderstichele H, Zetterberg H, assisting in drafting/revising of the manuscript. UA: collection of the samples Lewczuk P, Posner H, Hall J, Johnson L, et al. Blood-based biomarkers in and the data; performing the analyses; assisting in drafting/revising of the Alzheimer disease: current state of the science and a novel collaborative manuscript. CS: conceptualization of the study; collection of the samples and paradigm for advancing from discovery to clinic. Alzheimers Dement. 2017; the data; interpretation of the data; assisting in drafting/revising of the 13:45–58. manuscript. JP: conceptualization of the study; collection of the samples and 6. Teunissen CE, Khalil M. Neurofilaments as biomarkers in multiple sclerosis. the data; interpretation of the data; assisting in drafting/revising of the Mult Scler. 2012;18:552–6. manuscript. PS and JMM: collection of the samples and the data; assisting in 7. Kuhle J, Barro C, Andreasson U, Derfuss T, Lindberg R, Sandelius A, Liman V, drafting/revising of the manuscript. JK: collection of the samples and the Norgren N, Blennow K, Zetterberg H. Comparison of three analytical data; interpretation of the data; assisting in drafting/revising of the platforms for quantification of the neurofilament light chain in blood manuscript. KB and HZ: collection of the data; performing the analyses; samples: ELISA, electrochemiluminescence immunoassay and Simoa. 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Increased neurofilament light chain blood levels in neurodegenerative neurological diseases. PLoS One. 2013;8:e75091. 28. Weston PSJ, Poole T, Ryan NS, Nair A, Liang Y, Macpherson K, Druyeh R, Malone IB, Ahsan RL, Pemberton H, et al. Serum neurofilament light in familial Alzheimer disease: a marker of early neurodegeneration. Neurology. 2017;89:2167–75. 29. Vagberg M, Norgren N, Dring A, Lindqvist T, Birgander R, Zetterberg H, Svenningsson A. Levels and age dependency of neurofilament light and http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Alzheimer's Research & Therapy Springer Journals

Plasma neurofilament light as a potential biomarker of neurodegeneration in Alzheimer’s disease

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Biomedicine; Neurosciences; Neurology; Geriatrics/Gerontology; Geriatric Psychiatry
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Abstract

Background: A growing body of evidence suggests that the plasma concentration of the neurofilament light chain (NfL) might be considered a plasma biomarker for the screening of neurodegeneration in Alzheimer’s disease (AD). Methods: With a single molecule array method (Simoa, Quanterix), plasma NfL concentrations were measured in 99 subjects with AD at the stage of mild cognitive impairment (MCI-AD; n = 25) or at the stage of early dementia (ADD; n = 33), and in nondemented controls (n = 41); in all patients, the clinical diagnoses were in accordance with the results of the four core cerebrospinal fluid (CSF) biomarkers (amyloid β (Aβ)1–42, Aβ42/40, Tau, and pTau181), interpreted according to the Erlangen Score algorithm. The influence of preanalytical storage procedures on the NfL in plasma was tested on samples exposed to six different conditions. Results: NfL concentrations significantly increased in the samples exposed to more than one freezing/thawing cycle, and in those stored for 5 days at room temperature or at 4 °C. Compared with the control group of nondemented subjects (22.0 ± 12.4 pg/mL), the unadjusted plasma NfL concentration was highly significantly higher in the MCI-AD group (38.1 ± 15.9 pg/mL, p < 0.005) and even further elevated in the ADD group (49.1 ± 28.4 pg/mL; p <0.001). A significant association between NfL and age (ρ =0.65, p < 0.001) was observed; after correcting for age, the difference in NfL concentrations between AD and controls remained significant (p = 0.044). At the cutoff value of 25.7 pg/mL, unconditional sensitivity, specificity, and accuracy were 0.84, 0.78, and 0.82, respectively. Unadjusted correlation between plasma NfL and Mini Mental State Examination (MMSE) across all patients was moderate but significant (r = −0.49, p < 0.001). We observed an overall significant correlation between plasma NfL and the CSF biomarkers, but this correlation was not observed within the diagnostic groups. Conclusions: This study confirms increased concentrations of plasma NfL in patients with Alzheimer’sdisease compared with nondemented controls. Keywords: Alzheimer’s disease, Neurofilament light, Biomarker, Plasma * Correspondence: Piotr.Lewczuk@uk-erlangen.de Department of Psychiatry and Psychotherapy, Lab for Clinical Neurochemistry and Neurochemical Dementia Diagnostics, Universitätsklinikum Erlangen, and Friedrich-Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany Department of Neurodegeneration Diagnostics, Department of Biochemical Diagnostics, Medical University of Bialystok, University Hospital of Bialystok, Bialystok, Poland Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 2 of 10 Background caregivers gave their written consent. Blood samples Cerebrospinal fluid (CSF) biomarkers have been extensively were obtained from 99 patients at the Department of studied as tools for an early diagnosis of Alzheimer’sdisease Psychiatry and Psychotherapy, Universitätsklinikum (AD) [1] and have proven to be cheaper, less demanding in Erlangen, and categorized into three groups: nondemen- terms of infrastructure and patient management, and most ted controls (Controls, n = 41), MCI with high probabil- probably capable of showing pathologic alterations slightly ity of AD pathology (MCI-AD, n = 25), and AD in early earlier than other diagnostic modalities such as, for example, dementia stage (ADD, n = 33). According to the revised positron emission tomography; the relative invasiveness of NINCDS-ADRDA criteria [11, 12], diagnoses were made the two modalities remains disputable [2]. In particular, the after clinical evaluation, neuropsychological testing with four core CSF biomarkers, amyloid β (Aβ)1–42, Aβ42/40 ra- the CERAD+ battery, magnetic resonance imaging tio, Tau, and pTau181, reliably support AD diagnostics (MRI), duplex sonography of the carotids and brain reflecting the hallmark AD pathologies, i.e., amyloidosis and arteries as well as electroencephalographic (EEG) re- neurodegeneration [3]. Lumbar puncture (LP) is a routine cordings. The distinction between dementia and MCI clinical procedure with a low incidence of complications [4]. was made on the basis of the clinical assessment; pa- Nevertheless, collection of CSF is accompanied by proced- tients with MCI had an objectively measureable cogni- ural efforts and inconvenience for subjects, ultimately tive decline but their independence regarding functional preventing its use as a screening tool in early, asymptomatic life abilities was preserved, which means that the revised cases and it can also be challenging to use for repetitive NINCDS-ADRDA diagnostic criteria of dementia were monitoring of the disease progression.Hence,there is a not fulfilled. In all cases, the clinical and neuropsycholo- strong need to develop blood-based biomarkers that, when gic diagnoses remained in accord with the results of the applied in a proper context of use, could serve as targeted four core biomarkers of neurochemical dementia and relative noninvasive screening tests [5]. diagnostics (NDD), all analyzed by enzyme-linked im- Neurofilaments (Nf) consist of three types of pro- munosorbent assays (ELISA): Aβ1–42 (IBL International, tein chains, differing in molecular mass: a light chain (NfL) Hamburg, Germany), Aβ42/40 (IBL International), Tau of 68 kD, an intermediate chain of 150 kD, and a heavy chain (Fujirebio Europe, Ghent, Belgium), and pTau181 (Fujirebio of 190 to 210 kD, and are major components of axonal cyto- Europe), interpreted according to the Erlangen Score skeleton [6]. Each subunit is composed of a double-stranded, algorithm (ES), [13, 14]. All control cases had normal CSF highly conserved α-helical core domain, bordered by a head results (ES ≤ 1) and all MCI-AD and ADD patients had N-terminus and a tail C-terminus. Nf are highly phosphory- profoundly pathological CSF results (ES = 4). Vascular lated proteins, and the degree of this phosphorylation deter- copathology was not an exclusion criterion in our study. mines the axon diameter [6]. Axonal damage leads to release Blood was collected by venipuncture into standard of Nf molecules into the extracellular space and, conse- polypropylene EDTA test tubes (Sarstedt, Nümbrecht, quently, into body fluids, such as the CSF or plasma. In line Germany) followed by centrifugation; the obtained plasma with this, increased blood NfL concentrations were reported was portioned into approximate 500 μL aliquots, fro- in neurodegenerative and neuroinflammatory disorders zen at −80 °C, and kept unthawed until the analyses. [7–9]. A very recent report by Mattsson et al. convincingly concludes that plasma NfL could be considered a neuro- Samples for testing the preanalytical storage conditions degeneration biomarker in AD [10]. For testing of the influence of the preanalytical storage In this study, we measured NfL concentrations in conditions, blood samples were collected from five do- plasma samples of AD patients at the stages of mild cogni- nors, and the plasma was generated as above. Immediately tive impairment (MCI) and early dementia (ADD), and thereafter, from each sample six aliquots were prepared, nondemented controls. In all our subjects the clinical and resulting in a matrix of 5 cases × 6 aliquots (conditions): a) neuropsychologic diagnoses were in accordance with the one aliquot was immediately frozen at −80 °C, and served results of their four core CSF biomarkers (Aβ1–42, Aβ42/ as a reference sample; b) one aliquot was frozen at −80 °C 40, Tau, and pTau181). For the validation of the assay, we and exposed to one thawing/refreezing (1 × F/T) cycle; c) further tested the influence of different preanalytical sam- one aliquot was frozen at −80 °C and exposed to two F/T - ple handling procedures (repetitive freezing/thawing, and cycles; d) one aliquot was frozen at −80 °C and ex- storage at room temperature or in a refrigerator) on the posed to three F/T cycles; e) one aliquot was stored concentrations of plasma NfL. for 5 days at 4 °C; and f) one aliquot was stored for 5 days in the dark at room temperature (~ 21 °C). Following the Methods isochronous testing strategy [15], after 5 days at either Patients and blood collection; diagnostic CSF tests room temperature or 4 °C, the aliquots (e) and (f) were The ethical committee of the University of Erlangen- transferred to the reference condition (−80 °C freezer) and Nuremberg approved the study, and all patients or their kept frozen until analyses. Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 3 of 10 Simoa assay If not stated otherwise, estimates in logistic models For analysis of NfL concentrations, the samples were were obtained with the maximum likelihood (ML) transported on dry ice to the Clinical Neurochemistry method. P < 0.05 was considered significant. All analyses Laboratory, Sahlgrenska University Hospital. The ana- were performed with Stata 14.2 (StataCorp, College Sta- lyses were performed using an in-house assay on the sin- tion, TX, USA). gle molecule array platform (Simoa; Quanterix, Lexington, MA, USA), as previously described in detail Results [16]. This method has an analytical sensitivity of Plasma NfL assay performance 0.62 pg/mL [7]. All measurements were performed by Two quality control (QC) samples spanning clinically board-certified laboratory technicians who were blinded relevant concentrations of the analyte were analyzed in to clinical data. duplicates two to three times in each run. For a QC sample with a concentration of 12.5 pg/mL, repeatability Statistical analysis was 8.9% and intermediate precision was 11.0%. For a The number of samples was estimated considering the be- QC sample with a concentration of 105.7 pg/mL, repeat- tween-group differences reported in the available litera- ability and intermediate precision were both 6.4%. ture, and with the significance and the power set to 0.05 and 0.9, respectively. If not stated otherwise, the results Concentrations of NfL in plasma samples under different are presented as averages ± standard deviations (SD) or storage conditions 95% confidence intervals (95% CI). Correlations between Concentrations of NfL in aliquots of the five plasma sam- continuous variables, unadjusted for other covariates, are ples with six different preanalytical treatment conditions presented as Pearson (r)or Spearman (ρ) correlation coef- are presented in Fig. 1. Compared to the reference aliquot, ficients, as indicated. Partial correlation was used to the concentrations of NfL under all conditions, except for estimate the correlation coefficients and their significances one cycle of thawing/refreezing, were slightly but signifi- between Mini Mental State Examination (MMSE) scores cantly higher (p < 0.05). After normalization, defining the and the concentrations of plasma NfL and the CSF bio- concentration in a reference aliquot as 1.00, the average markers, conditional on diagnostic categories. Differences relative concentrations and their standard deviations in the across multiple categories were tested with analysis of tested aliquots were: 1.07 ± 0.23 in the aliquots exposed to variance (ANOVA) followed by post-hoc tests with appro- one freezing/thawing cycle, 1.24 ± 0.23 in the aliquots priate corrections for multiple comparisons. thawed and refrozen twice, 1.41 ± 0.42 in the aliquots Association of the preanalytical storage conditions thawed and refrozen three times, 1.32 ± 0.26 in the aliquots and the NfL concentrations was tested with a vari- stored for 5 days at 4 °C, and 1.46 ± 0.21 in the aliquots ance component model with conditions crossed with stored for 5 days at room temperature. The ML estimates samples. A linear regression was used to model NfL of the random parameters of the variance-components plasma concentrations in the diagnostic groups, ad- model were ψ = 37.3 (variability of the random intercept, justed for patient age, including interaction term i.e., between-sample), and θ = 1.94 (variability of the level-1 age-by-disease status. The models were compared residual, i.e., within-sample), resulting in a within-sample with likelihood ratio (LR) test on indicated degrees of (intraclass) correlation ρ =0.95. freedom (df). The NfL concentration cutoff separating controls from Plasma NfL in nondemented controls, MCI-AD, and ADD AD patients was calculated at the maximized Youden patients index. Logistic regression was used to model the sensi- Demographic characterization of the three groups consid- tivity and the specificity as a function of age according ered in this study, the results of the CSF biomarkers, and to Coughlin et al. [17], slightly modified by introducing the plasma NfL concentrations are presented in Table 1. interaction term for age-by-disease status. The test's ac- Results of the NfL measurements in the three diagnostic curacy was defined as the probability of the agreement groups are presented in Fig. 2. Unadjusted for other between the result of the test and the disease status (re- covariates and compared with the control group of nonde- sponse = 1 if both positive or both negative, or 0 other- mented subjects (22.0 ± 12.4 pg/mL), average NfL concen- wise), and was modeled, conditional on age, with logistic trations were highly significantly higher in the MCI-AD regression. The area under the receiver operating char- group (38.1 ± 15.9 pg/mL, p < 0.005), and even further ele- acteristic (ROC) curve (AUC) was calculated with a non- vated in the ADD group (49.1 ± 28.4 pg/mL; p < 0.001). parametric method. Fisher’s linear discriminant analysis The concentrations in the ADD group was also higher (LDA) was used to calculate parameters of the line opti- compared with the MCI-AD group; however, this differ- mally separating the controls and the AD patients on ence became borderline insignificant after Scheffe’s the basis of their NfL concentrations and age. post-hoc correction for multiple comparisons (p =0.12). Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 4 of 10 Fig. 1 Neurofilament light chain (NfL) plasma levels of samples with different preanalytical treatment. *p < 0.05, versus the reference (Ref.) sample (frozen at −80 °C and unthawed). d days, F/T freeze/thaw cycle, n.s. not significant, RT room temperature Taking into consideration that a) according to the interaction term of age-by-disease status was insignifi- current concept of the AD continuum [18, 19], MCI-AD cant (β = 0.365, p = 0.361), and did not improve the is not a separate diagnostic entity but rather a stage of model fit (LR test χ (df = 1) = 0.87, p = 0.35), we excluded AD, and b) MCI-AD and ADD cases had, expectedly, it from further analyses. Using LDA, we finally estimated the same pattern of the pathologic CSF results, we the parameters of the line optimally separating the AD pooled the two groups into one diagnostic category (Alz- patients from the controls: NfL (pg/mL) = −5.25 × age heimer’s disease, AD) and contrasted it with the nonde- (years) + 367.7, which resulted in 81% of cases being mented controls. In Fig. 3, NfL concentrations in AD properly categorized (sensitivity and specificity of 0.86 and controls are plotted against age. Overall, we ob- and 0.76, respectively). served a highly significant association between NfL and age (ρ = 0.65, p < 0.001). After correcting for age, the dif- Sensitivity, specificity, and accuracy of plasma NfL as a ference in NfL concentrations between the two groups potential AD biomarker remained statistically significant (p = 0.044). Since the Figure 4 presents the performance of the plasma NfL concentration in the setting of AD patients with positive Table 1 Demographic characterization, CSF results, and NfL CSF biomarkers versus nondemented controls with concentrations in the diagnostic groups negative CSF biomarkers. Unadjusted for other variables, Controls MCI-AD ADD the AUC of the ROC curve in the setting controls vs dis- N (male + female) 41 (22 + 19) 25 (10 + 15) 33 (13 + 20) eased turned out reasonably large (0.853, 95% CI 0.772– 0.934; Fig. 4a). After introducing age to the model esti- Age (years) 52.5 ± 13.1 71.3 ± 8.4 70.8 ± 7.6 mating the ROC curve, the AUC increased insignifi- a b MMSE 29.3 ± 0.9 26.7 ± 2.1 21.2 ± 3.4 cantly (p = 0.055) to 0.920 (95% CI 0.869–0.970). At the CSF Aβ1–42 (pg/mL) 1025 ± 308 585 ± 116 536 ± 114 cutoff maximizing Youden index, 25.7 pg/mL, uncondi- CSF Aβ1–40 (pg/mL) 13,598 ± 4046 19,171 ± 5689 15,309 ± 4172 tional sensitivity, specificity, and accuracy were 0.84, CSF Aβ42/40 0.076 ± 0.01 0.032 ± 0.01 0.036 ± 0.006 0.78, and 0.82, respectively. Considering age differences CSF Tau (pg/mL) 198 ± 64.4 631 ± 214 558 ± 178 between the groups in this study, we consequently mod- eled the performance characteristics of the test as a CSF pTau181 (pg/mL) 37.3 ± 12.1 101.4 ± 29.8 89.9 ± 18.4 function of age (Fig. 4b). While the sensitivity increased Plasma NfL (pg/mL) 22.0 ± 12.4 38.1 ± 15.9 49.1 ± 28.4 with age, from 0.61 at 50 years to 0.91 at 80 years, the Values are shown as averages ± standard deviations or as numbers per group specificity decreased from 0.89 to 0.20, respectively, Aβ amyloid β, AD Alzheimer’s disease, ADD Alzheimer’s disease dementia, CSF cerebrospinal fluid, MCI mild cognitive impairment, MMSE Mini Mental State leaving the overall accuracy of the test practically un- Examination, NfL neurofilament light chain a altered (0.84 and 0.80 at the age of 50 and 80 years, Available in 22 cases Available in 23 cases respectively). Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 5 of 10 Fig. 2 Unadjusted neurofilament light chain (NfL) concentrations in the three diagnostic categories. A borderline insignificant (p = 0.11 after Scheffe correction) tendency was observed towards higher concentrations in Alzheimer’s disease dementia (ADD) compared with mild cognitive impairment Alzheimer’s disease (MCI-AD). The green horizontal line represents the cutoff at the maximized Youden index (25.7 pg/mL), leading to the unadjusted diagnostic accuracy of 82% in the setting of controls versus AD (MCI-AD and ADD). *p < 0.05 The unadjusted correlation between plasma NfL and Correlation of plasma NfL with the four core CSF MMSE score was moderate and highly significant (r = biomarkers −0.49, p < 0.001; Fig. 5). After controlling for the diag- Without taking diagnostic categories into consideration, nostic categories, the coefficient became weaker, albeit NfL in plasma correlated highly significantly (p < 0.001) still significant (r = −0.24, p = 0.036). None of the CSF with all CSF biomarkers except Aβ1–40 (as expected, biomarkers correlated significantly with the MMSE score positively with Tau and pTau181, and negatively with after controlling for the diagnostic categories (p > 0.2; Aβ1–42 and Aβ42/40; data not shown). This correlation data not shown). became insignificant for all CSF biomarkers (p > 0.35) Fig. 3 Neurofilament light chain (NfL) plasma levels plotted against age in nondemented controls (green) and the AD patients (a combined group of MCI-AD and ADD subjects, red). The black dotted line represents the optimal separation of the two groups according to Fisher’s LDA Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 6 of 10 Fig. 4 a Unadjusted ROC curve in the setting of controls vs. AD. b Sensitivity (blue), specificity (brown), and accuracy (green) as functions of age. AUC area under the curve, CI confidence interval, SE standard error when the diagnoses were taken into account. Figure 6 patients, whose diagnoses were in accordance with the presents, exemplarily, a significant correlation between pathological results of the CSF biomarkers, compared plasma NfL and CSF Tau across all patients taken with the nondemented control subjects, whose normal together (Fig. 6a) and a lack of within-groups cognitive status was in accordance with the unaltered re- correlation between the two analytes (overall p = 0.64) sults of their CSF biomarkers. Within the AD group, we when the three diagnostic groups are treated separ- observed a tendency towards higher NfL concentrations ately (Fig. 6b−d). in patients at the stage of early dementia compared with the stage of MCI. Plasma NfL concentrations correlated Discussion inversely with the global cognitive status measured by A growing body of literature postulates a potential appli- the MMSE results. Finally, we also provide evidence for cation of the plasma concentration of the light chain of the influence of the preanalytical sample handling on the the neurofilament protein as a screening tool for neuro- plasma NfL concentrations. degeneration. In our study, the plasma NfL concentra- We started our study by examining the influence of sam- tion was found to be significantly higher in the AD ple handling procedures on plasma NfL concentrations. Fig. 5 Correlation between plasma neurofilament light chain (NfL) concentrations and the Mini Mental State Examination (MMSE) results Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 7 of 10 Fig. 6 Plasma neurofilament light chain (NfL) plotted against cerebrospinal fluid (CSF) Tau. a. Overall moderate, highly significant correlation between plasma NfL and CSF Tau; lacking within-group correlation between plasma NfL and CSF Tau in the controls (b), MCI-AD (c), and ADD (d) We believe this is a critically important aspect; for example, seems reasonable to postulate sending the material to dis- asample’s storage and transportation to distant laboratories tant laboratories deeply frozen and avoiding more than one is a nontrivial issue and has practical implications. Com- intermediate thawing/refreezing cycle. pared with a reference sample (i.e., a deep-frozen aliquot In agreement with recently published studies on spor- stored unthawed until analysis), we observed a slight but adic AD [10, 26, 27] and familial AD (FAD) [28], we significant increase in the concentrations in the aliquots found increased plasma NfL concentrations in AD pa- thawed and refrozen twice or three times, kept for 5 days at tients in the dementia stage (ADD) as well as in MCI the room temperature, or stored in a refrigerator. In con- subjects with a high probability of underlying AD path- trast, one thawing/refreezing cycle did not systematically ology (MCI-AD), compared with nondemented controls. alter the NfL concentration, but resulted in nonsystematic The results of the current study confirm those reported changes, i.e., the concentrations increased in some samples by Mattsson et al. obtained with the same method and but decreased in others, resulting in increased variability in the same laboratory but on different patient cohorts but an unchanged average. Whereas in most cases a de- [10], not only in terms of the average concentrations crease in the concentration of a protein with storage time and their biological variability (coefficients of variation), or under thawing/refreezing is expected, it is known that but also in terms of the NfL performance as a potential some proteins, for example serum albumin, tend to in- plasma diagnostic test. In both studies, almost identical crease in concentration after repetitive thawing/refreezing areas under the ROC curves, contrasting AD patients [20]. Similarly, in our previous studies, an unsystematic versus nondemented controls, were obtained (0.853 and increase in CSF Aβ1–42, Aβ1–40, and Tau in some, but 0.87, respectively). A slightly higher average NfL concen- not all, aliquots exposed for more than two thawing/re- tration in the controls reported by Mattsson et al. com- freezing cycles was observed [21, 22]. As an explanation, a pared with the present study can be explained by the release of NfL monomers from aggregates, known to form fact that one-third of the controls in the previous in certain neurodegeneration disorders [23], might be paper showed Aβ positivity, whereas in the current study considered. Interestingly, the data on the stability of NfL in positive Aβ CSF results excluded a subject as a control. the CSF are ambiguous; whereas one study found rapid de- This is due to the fact that, in contrast to the papers cline of concentrations at room temperature or 4 °C [24], published previously, patients in the current study were no changes were observed in another study until 3 days, included only if their clinical and neuropsychological followed by a subsequent decrease [25]. In any case, it diagnoses stayed in agreement with the outcome of the Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 8 of 10 four core CSF biomarkers (Aβ1–42, Aβ42/40, Tau, and a slight decrease of the AUC from 0.87 to 0.79 when the pTau181) conservatively interpreted according to the Er- model was fitted with age, sex, and educational level, in- langen Score algorithm [13, 14]. We are aware that such stead of all variables considered in their study. We are not an approach has advantages and disadvantages; it en- aware of any report analyzing age-dependency of any ables more reliable stratification of the cases, but it ex- other metrics. We believe that the characteristics found in cludes the possibility of a direct comparison of the this study, with an age-dependent increase in the sensitiv- diagnostic utility of the plasma NfL with any of the CSF ity at the cost of the decreasing specificity clearly seen in biomarkers. the age range of 60–80 years (i.e., in the range when Our finding of a positive association between plasma neurodegeneration is most commonly considered in the NfL concentration and age is in agreement with diagnosis), further supports the postulated potential appli- previously reported studies on plasma [9, 10] and CSF cation of the plasma NfL as a screening tool for neurode- [29, 30]. A weak but significant association between generation, rather than as a test for confirming AD serum NfL and age at onset of a disease was also reported diagnosis. in primary progressive aphasia (PPA) [8]; however, another In line with the recently published results [10], we study did not find a correlation of NfL with age after found an inverse correlation of plasma NfL concentra- adjusting for the estimated age of onset of a disease in tions with MMSE results. In contrast, none of the CSF FAD [28]. Mechanisms of this age-dependent increase in biomarkers measured in this study correlated signifi- the NfL concentrations in body fluids, and also in persons cantly with the MMSE score after controlling for the without clinical signs of neurodegeneration, are unclear diagnostic categories. This finding supports our previous thus far. It was hypothesized that aging leads to a subclin- results of a lack of association between MMSE score and ical axonal degeneration and, in consequence, to the re- the CSF results [32, 33], and remains in agreement with lease of Nf molecules [29]. The same group also proposed the generally accepted assumption that the CSF bio- that subclinical cerebrovascular changes might be consid- markers do not correlate with disease progression at the ered as an explanation, since cerebrovascular pathology is stage of MCI and later [34]. Other studies provide evi- common and known to increase with age; finally, vascular dence that NfL plasma concentrations reflect the dynam- copathology is also commonly observed in AD [31]. Irre- ics of neurodegeneration processes measured with spective of the underlying mechanisms, the association of different metrics. Steinacker et al. found an association be- the NfL concentrations with age has implications for the tween increased NfL concentration and functional decline diagnosis-oriented interpretation of the results. First, an and progression of atrophy in the left frontal lobe of PPA age-dependent cutoff needs to be established, and calcula- patients [8], and Weston et al. reported an association of tion of such a cutoff is not a trivial task. Perhaps the best serum NfL concentration with the time from symptom approach is by applying such statistical tools such as LDA; onset in FAD [28]. Similarly, increased CSF NfL was found in such a case, however, the slope of the line discriminat- to correlate with decreased MMSE score and with faster ing the groups (i.e., the age-dependent cutoff) clearly brain atrophy over time, as measured by changes in depends on the distribution of the parameters in question whole-brain volume, ventricular volume, and hippocam- (here NfL concentrations and age) in these groups. If they pus volume in AD [35]. In multiple sclerosis, CSF NfL re- are not age-matched, as in our study and in some other flects acute axonal damage, and hence it might be reports [27, 28], a line best discriminating AD patients considered a prognostic biomarker (reviewed in [6]). from the controls has a negative slope, which might look Similar to the previously published findings [10], we contradictory to common sense (i.e., in spite of NfL con- observed a highly significant overall correlation of centration increasing with age, its cutoff decreases). This plasma NfL with CSF biomarkers for AD pathology would be different if the two groups were age-matched, as when the diagnostic categories were not considered. in the study by Mattsson et al. [10]. In such a case, the dis- Confirming the previous report, the significance of this criminatory line would have a positive slope (i.e., it would correlation disappeared when the diagnostic groups were increase with age). Secondly, metrics of the performance evaluated separately. Such a correlation pattern, with of the NfL concentrations as a potential diagnostic test overall significant correlation that is not observed within also depend on age. In this study, we observed an increase particular diagnostic groups, is not surprising when a in the sensitivity at the cost of a decrease in the specificity lack of association between the CSF biomarkers and the with increasing age, leaving overall accuracy practically disease dynamics as soon as the first cognitive symptoms unaltered. Furthermore, we observed a slight, borderline occur (i.e., from the MCI stage on) is taken into consid- insignificant increase of the area under the ROC curve eration [34]. with age. To the best of our knowledge, only Mattsson et Perhaps the most important limitation of our study is al. [10] evaluated the age-dependent AUC of the ROC the relatively small, age-unmatched groups, which we curve discriminating AD from healthy controls, observing tried to counterbalance by controlling for age in all Lewczuk et al. Alzheimer's Research & Therapy (2018) 10:71 Page 9 of 10 statistical analyses. It must be stressed, however, that Competing interests PL has received consultation and/or lecture honoraria from IBL International, such discrepancy between age of AD patients and non- Fujirebio Europe, AJ Roboscreen, and Roche. KB has served as a consultant or demented controls simply reflects the reality that AD on advisory boards for Alzheon, BioArctic, Biogen, Eli Lilly, Fujirebio Europe, IBL patients are older. International, Merck, Novartis, Pfizer, and Roche Diagnostics. KB and HZ are cofounders of Brain Biomarker Solutions in Gothenburg AB, a GU Ventures- based platform company at the University of Gothenburg. HZ has served on ad- Conclusion visory boards of Eli Lilly and Roche Diagnostics and has received travel support In conclusion, we confirmed increased concentrations of from Teva. CS and JP are full-time employees of Boehringer Ingelheim. All remaining authors declare that they have no competing interests. plasma NfL in Alzheimer’s disease; however, its future potential application as a biomarker will have to take its Publisher’sNote nonspecificity into account. We speculate that plasma Springer Nature remains neutral with regard to jurisdictional claims in NfL will not be able to replace the CSF biomarkers of published maps and institutional affiliations. neurodegeneration within a given diagnostic group, but Author details it could perhaps be considered as a potential screening Department of Psychiatry and Psychotherapy, Lab for Clinical tool between the groups. Finally, we extended previous Neurochemistry and Neurochemical Dementia Diagnostics, studies with a systematic test for the influence of the Universitätsklinikum Erlangen, and Friedrich-Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany. preanalytical sample handling procedures on the NfL Department of Neurodegeneration Diagnostics, Department of Biochemical concentrations in plasma. Diagnostics, Medical University of Bialystok, University Hospital of Bialystok, Bialystok, Poland. Clinical Neurochemistry Laboratory, Sahlgrenska University Abbreviations Hospital, Mölndal, Sweden. Department of Psychiatry and Neurochemistry, AD: Alzheimer’s disease; ADD: Alzheimer’s disease dementia; AUC: Area Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the under the curve; Aβ: Amyloid β; CI: Confidence interval; CSF: Cerebrospinal University of Gothenburg, Mölndal, Sweden. Boehringer Ingelheim Pharma fluid; df: Degrees of freedom; ES: Erlangen Score; FAD: Familial Alzheimer’s GmbH & Co. KG, Biberach an der Riss, Germany. Boehringer Ingelheim disease; F/T: Freeze/thaw; LDA: Linear discriminant analysis; LP: Lumbar International GmbH, Ingelheim am Rhein, Germany. Department of puncture; LR: Likelihood ratio; MCI: Mild cognitive impairment; ML: Maximum Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, likelihood; MMSE: Mini Mental State Examination; NfL: Neurofilament light UK. UK Dementia Research Institute at UCL, London, UK. chain; PPA: Primary progressive aphasia; QC: Quality control; ROC: Receiver operating characteristic; SD: Standard deviation Received: 25 April 2018 Accepted: 9 July 2018 Funding The research leading to these results has received support from the Innovative References Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372, 1. Olsson B, Lautner R, Andreasson U, Ohrfelt A, Portelius E, Bjerke M, Holtta M, resources of which are composed of a financial contribution from the European Rosen C, Olsson C, Strobel G, et al. CSF and blood biomarkers for the Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA diagnosis of Alzheimer's disease: a systematic review and meta-analysis. companies in kind contribution, the Swedish and European Research Councils, Lancet Neurol. 2016;15:673–84. the Swedish Alzheimer Foundation, the Swedish Brain Foundation, the Swedish 2. Lewczuk P, Kornhuber J. Do we still need positron emission tomography for State Support for Clinical Research (ALFGBG), the Torsten Söderberg early Alzheimer's disease diagnosis? Brain. 2016;139:e60. Foundation, the Knut and Alice Wallenberg Foundation, Frimurarestiftelsen, 3. Lewczuk P, Riederer P, O'Bryant SE, Verbeek MM, Dubois B, Visser PJ, Hjärnfonden, and Alzheimerfonden. Jellinger KA, Engelborghs S, Ramirez A, Parnetti L, et al. Cerebrospinal fluid and blood biomarkers for neurodegenerative dementias: an update of the consensus Availability of data and materials of the task force on biological markers in psychiatry of the World Federation of The datasets used for the analyses are available from the corresponding Societies of Biological Psychiatry. World J Biol Psychiatry. 2018;19:244–328. author on reasonable request. 4. Duits FH, Martinez-Lage P, Paquet C, Engelborghs S, Lleo A, Hausner L, Molinuevo JL, Stomrud E, Farotti L, Ramakers I, et al. Performance and Authors’ contributions complications of lumbar puncture in memory clinics: results of the PL: conceptualization and design of the study; collection of the samples and multicenter lumbar puncture feasibility study. Alzheimers Dement. 2016;12: the data; interpretation of the data including the statistical analyses; drafting/ 154–63. revising of the manuscript. NE: collection of the samples and the data; 5. O'Bryant SE, Mielke MM, Rissman RA, Lista S, Vanderstichele H, Zetterberg H, assisting in drafting/revising of the manuscript. UA: collection of the samples Lewczuk P, Posner H, Hall J, Johnson L, et al. Blood-based biomarkers in and the data; performing the analyses; assisting in drafting/revising of the Alzheimer disease: current state of the science and a novel collaborative manuscript. CS: conceptualization of the study; collection of the samples and paradigm for advancing from discovery to clinic. Alzheimers Dement. 2017; the data; interpretation of the data; assisting in drafting/revising of the 13:45–58. manuscript. JP: conceptualization of the study; collection of the samples and 6. Teunissen CE, Khalil M. Neurofilaments as biomarkers in multiple sclerosis. the data; interpretation of the data; assisting in drafting/revising of the Mult Scler. 2012;18:552–6. manuscript. PS and JMM: collection of the samples and the data; assisting in 7. Kuhle J, Barro C, Andreasson U, Derfuss T, Lindberg R, Sandelius A, Liman V, drafting/revising of the manuscript. JK: collection of the samples and the Norgren N, Blennow K, Zetterberg H. Comparison of three analytical data; interpretation of the data; assisting in drafting/revising of the platforms for quantification of the neurofilament light chain in blood manuscript. KB and HZ: collection of the data; performing the analyses; samples: ELISA, electrochemiluminescence immunoassay and Simoa. 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Alzheimer's Research & TherapySpringer Journals

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