Neurofilament light chain

Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n 5 103/42) and Oxford (ALS/control, serum: n 5 64/36; paired CSF: n 5 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r 5 0.78, p , 0.0001). Blood NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98– 7.94, p , 0.001). Conclusion: Blood-derived NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. Neurology® 2015;84:2247–2257 GLOSSARY ALS 5 amyotrophic lateral sclerosis; CI 5 confidence interval; Nf 5 neurofilament; NfH 5 neurofilament heavy chain; NfL 5 neurofilament light chain; PRB 5 progression rate at baseline; PRL 5 progression rate at last visit. Various factors militate against the development of reliable biomarkers in amyotrophic lateral sclerosis (ALS), including clinical heterogeneity, variable rate of progression, and the lack of a recognizable preclinical state of this fatal neurodegenerative disorder. An easily accessible and reproducible prognostic biomarker would help patient stratification, improving assessment of individual prognosis and care-planning. It might also have potential as a pharmacodynamic measure of therapeutic response. *These authors contributed equally to this work. From the Centre for Neuroscience & Trauma (C.-H.L., G.G., J.K., A.M.), Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London; Sobell Department of Motor Neuroscience and Movement Disorders (C.-H.L., L.G.), Departments of Neuroinflammation (A.P.), Neurodegenerative Disease (P.F.), Molecular Neuroscience (K.S.), and Clinical Neuroscience (R.O.), and MRC Centre for Neuromuscular Diseases (R.O., L.G.), UCL Institute of Neurology, London; MRC Integrative Epidemiology Unit (C.M.-W.), University of Bristol; Nuffield Department of Clinical Neurosciences (E.G., K.T., M.R.T.), University of Oxford; Department of Medical Statistics (N.P.), London School of Hygiene and Tropical Medicine, London, UK; UmanDiagnostics (N.N.), Umeå, Sweden; Medicine Clinical Trial Unit (M.F.), Musgrove Park Hospital, Taunton, UK; National Hospital for Neurology and Neurosurgery (R.O., R.H., A.M.), London, UK; Neurology (J.K.), University Hospital Basel, Switzerland; North-East London and Essex MND Care and Research Centre (A.M.), London; and Basildon and Thurrock University Hospitals NHS Foundation Trust (A.M.), Basildon, UK. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. The Article Processing Charge was funded by RCUK. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2015 American Academy of Neurology 2247 a 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. The longitudinal assessment of a putative biomarker would allow a more reliable interpretation of the biomarker’s behavior when monitoring treatment response. Blood-based biomarkers are preferable because they require minimally invasive collection compared to CSF sampling. Neurofilaments (Nfs), the main byproducts of neuroaxonal breakdown, are potential “universal” biomarkers of neurodegeneration. Nf levels in CSF from patients with ALS increase significantly compared to other neurodegenerative disorders or to ALS-mimics, and show a robust interlaboratory reproducibility compared to other biomarkers. Nf bioavailability and measurement depend on matrix-related biological phenomena such as protein aggregation, as recently reported. In this study, we examined the prognostic value in ALS of neurofilament light chain (NfL), one of the main constituents of neurons and axons, building on previous small cross-sectional studies to evaluate the temporal profile of NfL expression in plasma, serum, and CSF from patients with ALS. METHODS Standard protocol approvals, registrations, and patient consents. Approvals were obtained from the East London and the City Research Ethics Committee 1 (09/ H0703/27) and South Central Oxford Ethics Committee B (08/H0605/85). All participants provided written consent (or gave verbal permission for a carer to sign on their behalf). Participants and sampling. This study included 103 patients with ALS and 42 healthy controls from a London cohort and 64 patients with ALS and 36 healthy controls from an Oxford cohort. Patients with ALS were diagnosed according to standard criteria, having been examined by experienced ALS neurologists (London cohort: A.M., K.S., R.O., R.H., M.F.; Oxford cohort: M.R.T., K.T.). Those with a family history of ALS or frontotemporal dementia, or known to carry a genetic mutation linked to ALS or frontotemporal dementia, were excluded to minimize any potential biases. Healthy controls were typically spouses and friends of patients. Exclusion criteria included neurologic comorbidities likely to affect Nf bioavailability. Baseline NfL levels were measured in plasma, serum, and CSF samples. Serial plasma samples and clinical information were obtained, on average, every 2 to 4 months from 67 of the 103 patients with ALS recruited in London. Serum and CSF samples (where possible) were collected every 6 months from 43 and 24 of the 64 patients with ALS in Oxford. No selection criteria were applied to individuals with ALS sampled longitudinally, other than their willingness to donate further samples. Symptom onset was defined as first patient-reported weakness. Progression rate was calculated at baseline (PRB) or last visit (PRL) as 48 minus the ALS Functional Rating Scale–Revised score, divided by the disease duration from onset of symptoms. Progression rate less than 0.5, between 0.5 and 1.0, and more than 1.0 (point/month) was defined as slow (ALS-slow), intermediate (ALS-intermediate), and fast progressing ALS (ALS-fast), respectively. Use of riluzole (or not) at the time of sampling was recorded. Sample analysis. Plasma, serum, and CSF samples were processed and aliquoted within 1 hour from collection and frozen at 280°C, following standard consensus procedures. An electrochemiluminescence immunoassay was used to quantify NfL as previously described; the investigators were blinded to clinical data. ALS and control samples were evenly distributed on each plate and measured in duplicate at the same dilution. Each plate contained calibrators (0–10,000 pg/mL) and quality controls. The interassay coefficients of variance were mostly below 10% and the mean intraassay coefficients of variance were below 10%. Linearity of the NfL assay was established (0–50,000 pg/mL) as previously reported. Statistical analysis. Continuous variables were summarized in median (interquartile range); hence, nonparametric analysis for group comparisons and correlation analysis. Receiver operating characteristic curve analysis was used to assess assay sensitivity/ specificity. We used log rank analysis to compare survival (fixed date was used to censor data for survival analysis) and multilevel random intercept models with a linear slope to examine NfL longitudinal trajectories (MLwiN version 2.30, from Stata version 13.1; runmlwin command) for the first 15 months of the follow-up period in 3 ALS progression subgroups: slow, intermediate, and fast progressors. A natural log transformation was used to normalize the measurements. Each ALS progression group was included as a categorical fixed effect; we also included an interaction between the ALS progression categories and time to assess whether the rate of change in NfL differed by ALS progression rate. NfL change was jointly modeled with the time to death within the 15-month follow-up period to account for any informative dropout. Cox regression analysis of survival by NfL at baseline and other covariates was tested in the London and Oxford cohorts separately and then combined (adjusting for study center). The matched serum and plasma NfL levels from healthy controls in a previous study showed high correlation (n5 25, Spearman r5 0.93, p, 0.0001) and strong agreement using Bland-Altman method comparison (bias: 3.92; serum-plasma; 95% confidence interval [CI]: 22.41, 10.25; 95% limits of agreement: 226.15, 33.99; Kuhle et al., unpublished data). We conducted analyses of the 2 cohorts combined, using the corresponding NfL data (serum or plasma) from each cohort. However, in recognition that NfL data from the 2 cohorts were different (albeit highly correlated) measures, we used cohortspecific tertile cutoff levels, and we adjusted Cox regression and Kaplan–Meier survival analyses by center. A p value of less than 0.05 was considered statistically significant. RESULTS Demographic and clinical characteristics of the London and Oxford cohorts are summarized in table e-1 on the Neurology® Web site at Neurology.org. Table 1 reports the baseline blood and CSF NfL levels along with the demographic and clinic characteristics of the cohorts. Cross-sectional analyses. NfL levels in CSF (Oxford cohort), serum (Oxford cohort), and plasma (London cohort).NfL levels were higher in patients with ALS than in controls in all biofluids measured (p , 0.0001; figure 1, A–C, left). Receiver operating characteristic analysis showed 2248 Neurology 84 June 2, 2015 a 2015 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Table 1 Summary of blood NfL levels (London, Oxford, and combined cohorts) and of CSF NfL levels (Oxford) used for cross-sectional analysis ALS, NfL levels Controls, NfL levels London (plasma) (n 5 103) Oxford (serum) (n 5 64) Combined (blood) (n 5 67) Oxford (CSF) (n 5 38) London (plasma) (n 5 42) Oxford (serum) (n 5 36) Combined (blood) (n 5 78) Oxford (CSF) (n 5 20)