J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2019-321321 on 13 October 2019. Downloaded from Multiple sclerosis ORIGINAL RESEARCH Dimethyl fumarate decreases neurofilament light chain in CSF and blood of treatment naïve relapsing MS patients Tobias Sejbaek ,1,2,3,4,5 Helle Hvilsted Nielsen,1,2,5 Natasha Penner,6 Tatiana Plavina,6 Jason P Mendoza,6 Nellie Anne Martin,2 Maria Louise Elkjaer,1,2 Mads Henrik Ravnborg,1 Zsolt Illes 1,2,5 ► Additional material is ABSTRact disease modifying therapies (DMTs), such as natal- published online only. To view Objectives In a prospective phase IV trial of the izumab, fingolimod, cladribine, daclizumab and please visit the journal online first-line oral treatment dimethyl fumarate (DMF), we alemtuzumab resulted in significant reduction in (http:// dx. doi. org/ 10. 1136/ 8–13 jnnp- 2019- 321321). examined dynamics of neurofilament light N( FL) chain NFL in the CSF and blood. Switch from inject- in serum, plasma and cerebrospinal fluid (CSF) samples ables to fingolimod reduced NFL concentration in 1Neurology, Odense collected over 12 months from relapsing-remitting the plasma, and switch to rituximab decreased NFL Universitetshospital, Odense, multiple sclerosis (RRMS) patients. NFL changes were levels in the CSF.14 15 In patients treated with natali- Denmark 2Department of Clinical related to disease activity. zumab, such reduction in NFL in the CSF correlated 16 Research, University of Southern Methods We examined NFL levels by single-molecule with brain atrophy changes. Therefore, NFL has Denmark, Odense, Denmark array in 88 CSF, 348 plasma and 131 sera from been suggested as a supplementary biomarker in the 3 Neurology, Hospital of South treatment-naïve RRMS patients (n=52), healthy controls management of MS.17 West Jutland, Esbjerg, Denmark 4 Prospective studies that investigate changes of The Department of Regional (n=23) and a placebo group matched by age, sex and Health Research, University of NFL (n=52). Plasma/sera were collected at baseline, and NFL in the blood and CSF during treatment with Southern Denmark, Odense, 1, 3, 6 and 12 months after DMF. CSF samples were first-line DMTs are lacking. In this prospective Denmark open label phase IV study, we investigated dynamic 5 collected at baseline and 12 months after DMF. MS Alliance of Southern Results NFL concentration in CSF, plasma and serum changes of NFL concentration in parallel samples Denmark, Esbjerg, Denmark 6Value Based Medicine, correlated highly (p<0.0001 for all), but plasma levels of serum, plasma and CSF taken from newly diag- Biogen Idec Inc, Cambridge, were only 76.9% of paired serum concentration. nosed treatment-naïve relapsing-remitting multiple Massachusetts, USA After 12 months of DMF treatment, NFL concentration sclerosis (RRMS) patients after treatment with decreased by 73%, 69% and 55% in the CSF, serum and dimethyl fumarate (DMF). We also compared NFL Correspondence to plasma (p<0.0001, respectively). Significant reduction levels in MS patients before and after DMF treat- Dr Tobias Sejbaek, Neurology, in blood was observed after 6 and 12 months treatment ment to healthy controls and placebo patients from Hospital of South West Jutland, Esbjerg 5000, Denmark; Tobias. compared with baseline (p<0.01 and p<0.0001, the randomised phase III trial: Pegylated interferon Sejbaek. Mathiesen@ rsyd. dk respectively) and to placebo (p<0.0001). Patients with β−1a for relapsing-remitting multiple sclerosis NFL above the 807.5 pg/mL cut-off in CSF had 5.0-times (ADVANCE).18 http://jnnp.bmj.com/ Received 30 May 2019 relative risk of disease activity (p<0.001). Revised 24 September 2019 Accepted 28 September 2019 Conclusions This study provides Class II evidence that METHODS first-line DMF reduces NFL in both blood and CSF after Study design 6 months and normalises CSF levels in 73% of patients. TREMEND (Tecfidera in Relapsing-Remitting High NFL concentration in CSF after a year reflected Multiple Sclerosis: Endothelial Dysfunction) was disease activity. NFL levels were higher in serum than in a prospective open label phase IV trial which plasma, which should be considered when NFL is used as enrolled newly diagnosed MS patients with RRMS on October 1, 2021 by guest. Protected copyright. a biomarker. from March 2014 till August 2016 (EudraCT 2014- ► http:// dx. doi. org/ 10. 1136/ 000254-11) (see online supplementary figure 1). jnnp- 2019- 321615 TREMEND was a study designed to identify poten- tial biomarkers in DMF-treated MS patients. The INTRODUCTION primary and secondary objectives were the change © Author(s) (or their Levels of the cytoskeleton protein neurofilament in the levels of biomarkers from baseline by month employer(s)) 2019. Re-use light (NFL) are increased in the cerebrospinal fluid 12 and 24 in subjects with RRMS receiving DMF. permitted under CC BY-NC. No (CSF) and blood in diseases associated with axonal Analysing NFL was one of the biomarker endpoints, commercial re-use. See rights damage.1 If measured by sensitive methods, blood and part of the original protocol. and permissions. Published 2–4 by BMJ. and CSF concentrations of NFL correlate. We included untreated (naïve) newly diag- Studies have shown that in the blood and CSF of nosed MS patients with RRMS according to the To cite: Sejbaek T, Nielsen patients with multiple sclerosis (MS), NFL concen- McDonald 2010 criteria (table 1).19 All patients HH, Penner N, et al. J Neurol tration is elevated at the time of diagnosis compared had oligoclonal bands (OCBs) in the CSF (n=52). Neurosurg Psychiatry Epub ahead of print: [please with healthy controls (HC) and is associated with The reason for including only patients with positive 5 include Day Month Year]. disease severity and prognosis. NFL levels are also OCBs was to ensure that participants had active doi:10.1136/jnnp-2019- increased during relapse and when new lesions are disease since absence of OCBs has been described 321321 detected by MRI.6 7 Treatment with highly effective a marker of low disease activity20 and support the Sejbaek T, et al. J Neurol Neurosurg Psychiatry 2019;0:1–7. doi:10.1136/jnnp-2019-321321 1 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2019-321321 on 13 October 2019. Downloaded from Multiple sclerosis Standard protocol approvals, registrations, patient consents Table 1 Demographics of study participants and baseline NFL and monitoring MS patients MS patients All patients and healthy controls gave written and oral consent. TREMEND ADVANCE The study was monitored according to the national laws by the DMF placebo Healthy controls (±SD) (±SD) (±SD) P value unit for good clinical practice at the hospital. Age (years) 34.1 (±8.7) 33.5 (±6.5) 38.2 (±11.2) ns Female/male 86.6% 90% 89.2% ns Data protection (%) This study was approved by the Danish Data Protection Agency Baseline CSF 2368 (±1947) N/A 416.8 (±191.2) P<0.0001 (journal no. 17/12684). NFL (pg/mL) Baseline 16.4 (±14.4) 17.5 (±14.0) 7.3 (±3.0) P<0.0001 Data availability plasma NFL Anonymised data will be shared on request from any qualified TREMEND (Tecfidera in Relapsing-Remitting Multiple Sclerosis: Endothelial investigator under approval from the Danish Data Protection Dysfunction) (n=52) and ADVANCE (Pegylated interferon β−1a for relapsing- remitting multiple sclerosis) (n=48)18; mean±SD is shown. P values represent Agency. difference between healthy controls (n=23) versus patients in the ADVANCE and TREMEND trial, respectively. There was no significant difference between the Statistical analyses ADVANCE and TREMEND trial. We described baseline characteristics with means and SDs for CSF, cerebrospinal fluid; DMF, dimethyl fumarate; MS, multiple sclerosis; N/A, not available; NFL, neurofilament light; ns, not significant. continuous variables and percentages for binary variables. Linear fit regression was performed using Spearman linear fit regression to calculate coefficients and linearity between NFL in CSF, plasma and serum. Data was checked for normality using inclusion of patients with MS even after a single clinical event.21 D'Agostino & Pearson normality test. Patients were recruited from two MS clinics of the MS Alli- Receiver operating characteristic (ROC) analysis was ance of Southern Denmark (Odense University Hospital and performed to identify cut-offs of NFL concentration in blood Hospital of South West Jutland), but the clinical examination and CSF that differentiate healthy controls and MS patients, and and sample preparation were performed by the same physician define their specificity and sensitivity. (TS) following identical protocols. We collected plasma and We used ordinary one-way analysis of variance and Holm-Si- sera at baseline, and 1, 3, 6 and 12 months after DMF treat- 22 dak’s multiple comparisons test in normal distributed data. In ment. Expanded Disability Status Scale (EDSS) scoring was the absence of Gaussian distribution, non-parametric analysis performed at the same time points and when clinical relapse was was performed with Kruskal-Wallis test and Dunn’s multiple suspected. CSF was collected at baseline (n=30) and optionally comparisons. 12 months after treatment (n=35). Altogether, we examined To analyse relative risk between high (above cut-off) and low 65 CSF samples, 108 serum samples and 230 plasma samples. (below cut-off) NFL levels in CSF and blood, we used Fish- Patients had brain MRI at baseline, 3 to 6 months after initia- er's exact test. Statistical analysis and data management were tion of DMF (re-baseline) and after 12 months table 1. Patients performed using GraphPad Prism 7. with relapses and/or new or enlarging T2-weighted lesions on MRI were defined as patients with disease activity (national guidelines).23 RESULTS We also collected CSF, plasma and serum from an age-matched NFL levels correlate in body fluids and are the lowest in http://jnnp.bmj.com/ and sex-matched healthy control cohort (n=23): patients plasma without neurological disease and neurological signs, and with To examine NFL in body fluids obtained from MS and HC, normal MRI and CSF results (table 1).