ARTICLE OPEN ACCESS CLASS OF EVIDENCE Serum Neurofilament Levels and PML Risk in Patients With Multiple Sclerosis Treated With Natalizumab Nicolás Fissolo, PhD, Beatrice Pignolet, PhD, Jordi Rio, MD, PhD, Patrick Vermersch, MD, PhD, Correspondence: Dr. Fissolo nicolas.fissolo@vhir.org Aurélie Ruet, MD, PhD, Jerome deSèze, MD, PhD, Pierre Labauge, MD, PhD, Sandra Vukusic, MD, PhD, Caroline Papeix, MD, Laurent Martinez-Almoyna, MD, Ayman Tourbah, MD, PhD, Pierre Clavelou, MD, PhD, Thibault Moreau, MD, PhD, Jean Pelletier, MD, PhD, Christine Lebrun-Frenay, MD, PhD, Bertrand Bourre, MD, Gilles Defer, MD, PhD, Xavier Montalban, MD, David Brassat, MD, PhD, and Manuel Comabella, MD, PhD Neurol Neuroimmunol Neuroinflamm 2021;8:e1003. doi:10.1212/NXI.0000000000001003 Abstract MORE ONLINE Objectives Class of Evidence The study aimed to assess the potential for serum neurofilament light chain (NFL) levels to Criteria for rating predict the risk of progressive multifocal leukoencephalopathy (PML) in natalizumab (NTZ)- therapeutic and diagnostic studies treated patients with multiple sclerosis (MS) and to discriminate PML from MS relapses. NPub.org/coe Methods NFL levels were measured with single molecule array (Simoa) in 4 cohorts: (1) a prospective cohort of patients with MS who developed PML under NTZ therapy (pre-PML) and non-PML NTZ-treated patients (NTZ-ctr); (2) a cohort of patients whose blood was collected during PML; (3) an independent cohort of non-PML NTZ-treated patients with serum NFL deter- minations at 2 years (replication cohort); and (4) a cohort of patients whose blood was collected during exacerbations. Results Serum NFL levels were significantly increased after 2 years of NTZ treatment in pre-PML patients compared with NTZ-ctr. The prognostic performance of serum NFL levels to predict PML development at 2 years was similar in the NTZ-ctr group and replication cohort. Serum NFL levels also distinguished PML from MS relapses and were 8-fold higher during PML compared with relapses. Conclusions These results support the use of serum NFL levels in clinical practice to identify patients with relapsing-remitting MS at higher PML risk and to differentiate PML from clinical relapses in NTZ-treated patients. Classification of Evidence This study provides Class I evidence that serum NFL levels can identify NTZ-treated patients with MS who will develop PML with a sensitivity of 67% and specificity of 80%. From the Servei de Neurologia-Neuroimmunologia (N.F., M.C.), Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d’Hebron (VHIR), Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Spain; CRC-SEP Neurosciences Centre Hospitalier Universitaire Toulouse (B.P., D.B.), CPTP INSERM UMR 1043 CNRS UMR 5282 et Université de Toulouse III, UPS, France; Servei de Neurologia-Neuroimmunologia (J.R., X.M.), Centre d’Esclerosi Múltiple de Catalunya (Cemcat), Hospital Universitari Vall d’Hebron, Barcelona, Spain; Univ. Lille (P.V.), Inserm U1172, CHU Lille, FHU Imminent, France; Université (A.R.), Bordeaux; CHU de Bordeaux (A.R.), INSERM-CHU CIC-P 0005, & Services de Neurologie; Neurocentre Magendie (A.R.), INSERM U1215; Department of Neurology ( J.dS), Hôpital Civil, Strasbourg; Department of Neurology (P.L.), CHU Montpellier; Department of Neurology CHU Lyon (S.V.); Department of Neurology (C.P.), Hôpital de la Salpétrière, Paris; Chi Aix en Provence (L.M.-A.); Department of Neurology and Faculté de Médecine de Reims (A.T.), CHU de Reims, URCA; LPN EA2027 Université Paris VIII (A.T.), Saint-Denis; Department of Neurology (P.C.), CHRU Clermont Ferrand; Department of Neurology (T.M.), CHU Dijon; Aix-Marseille Univ (J.P.), APHM, Hôpital de la Timone, Pôle de Neurosciences Cliniques, Service de Neurologie, CNRS, CRMBM UMR 7339, Marseille; Service de Neurology (C.L.-F.), CHU de Nice Pasteur2, Université Nice Cote d’Azur UR2CA URRIS, Nice; Neurologie (B.B.), CHU Rouen; and Neurologie (G.D.), CHU Caen, France. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary AUC = area under the ROC curve; MS = multiple sclerosis; NFL = neurofilament light chain; NTZ = natalizumab; PML = progressive multifocal leukoencephalopathy; RRMS = relapsing-remitting multiple sclerosis. Despite its proved efficacy for patients with highly active The table summarizes the main demographic and baseline relapsing-remitting multiple sclerosis (RRMS),1 the use of clinical characteristics of patients included in the study. natalizumab (NTZ) is limited due to the increased risk of progressive multifocal leukoencephalopathy (PML).2 In ad- Standard Protocol Approvals, Registrations, dition to existing PML risk stratification algorithms based on and Patient Consents NTZ treatment duration, previous immunosuppressive ther- Written informed consent was obtained from each participant. apies, and JC virus index,2,3 other biomarkers may certainly The study was approved by the local hospital ethics committees, contribute to estimate the risk of PML at an individual level. BIONAT cohort; ClinicalTrials.gov identifier: NCT00942214, and Vall d’Hebron Hospital (EPA(AG)57/2013(3834)). Numerous studies suggest that the concentration of neurofila- Quantification of Serum NFL Levels ment light chain (NFL) in peripheral blood and CSF is a Peripheral blood was collected by standard venipuncture and promising biomarker in MS.4,5 In a recent study, the serum NFL allowed to clot spontaneously for 30 minutes. Serum was levels measured with an electrochemiluminescence assay were obtained by centrifugation and stored frozen at −80°C until proposed as a biomarker for early identification of PML in pa- used. Levels of NFL were measured in serum samples using tients with MS under NTZ treatment.6 In the present study, we commercially available NFL immunoassay kits (Quanterix, aimed to expand on the potential for serum NFL levels measured cat#103186) run on the fully automated ultrasensitive Simoa with single molecule array (Simoa) to predict the risk of PML in a HD-1 Analyzer (Quanterix). Samples were run in duplicate in prospective cohort of NTZ-treated patients. We also aimed to accordance with manufacturers’ instructions with appropriate discriminate PML from MS relapses based on serum NFL levels. standards and internal controls. The intra-assay and interassay coefficients of variation were 5% and 9%, respectively. Methods Classification of Evidence Our primary research question was to ascertain whether se- Patient Cohorts rum NFL levels can identify NTZ-treated patients with MS Four different cohorts of patients with RRMS were included who will develop PML. The classification of evidence assigned in the study: to this question is Class I. 1. A multicentric prospective cohort of patients treated with Statistical Analyses NTZ (BIONAT cohort; ClinicalTrials.gov identifier: Statistical analysis was performed by using the IBM SPSS Sta- NCT00942214)7 was used to evaluate the association tistics version 22. The distribution of serum NFL levels was tested between serum NFL levels at baseline and at 1 and 2 years for normality with a Kolmogorov-Smirnov test. Afterward, paired of NTZ treatment and PML development. Patients and unpaired nonparametric tests were applied for comparisons belonging to this cohort were classified into 2 groups: of mean NFL levels among groups. When needed, analysis was patients who did not develop PML after a follow-up longer adjusted by age and disease duration. Quantitative data are pre- than 5 years (NTZ controls; NTZ-ctr) and patients who sented as mean values ± SD unless otherwise stated. Differences developed PML (pre-PML) under NTZ treatment. were considered statistically significant when p values were below 2. A cohort of patients whose blood was drawn during the 0.05. Receiver operating characteristic (ROC) curve analyses PML condition was included for comparison of NFL were used to determine the best cutoff values based on serum levels with the pre-PML group after 2 years of treatment NFL levels and the respective sensitivities and specificities. (during PML cohort). 3. An independent cohort of patients treated with NTZ Data Availability who did not develop PML after more than 5 years of All data analyzed during this study will be shared anonymized by follow-up was included to assess the reproducibility request of a qualified investigator to the corresponding author. between centers of serum NFL measurements after 2 years of NTZ treatment (replication cohort). 4. A cohort of patients whose blood was collected at the time Results of an acute relapse was included to investigate the potential Serum NFL Levels Are Elevated After 2 Years of for serum NFL levels to discriminate between the PML NTZ Treatment in Pre-PML Patients and condition and MS relapses (relapsing cohort). Twenty- During PML seven percent of these patients were receiving treatment At baseline, no significant differences were observed in serum with interferon-beta at the time of exacerbations. NFL levels between pre-PML and NTZ-ctr patients. At 1 and 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 4 | July 2021 Neurology.org/NN Table Demographic and Baseline Clinical Characteristics of Patients With RRMS Treated With NTZ BIONAT cohort Replication Baseline characteristics NTZ-ctrd Pre-PMLe During PML cohorta cohortb Relapsing cohortc n 37 17 13 29 30 Age (y) 36.2 (8.1) 38.6 (8.0) 37.6 (4.0) 37.1 (11.0) 32.3 (9.5) Female/male (% women) 29/8 (78.4) 13/4 (76.5) 10/3 (76.9) 19/10 (65.5) 23/7 (76.7) Duration of disease (y) 9.5 (6.4) 11.4 (6.1) 11.4 (6.4) 9.0 (6.6) 7.2 (6.2) EDSS score at baselinef 3.0 (2.0–4.2) 3.7 (2.6–5.4) 4.2 (1.6–5.4) 4.0 (2.5–5.2) 3.0 (2.4–3.6)g,h JCV status (+/2/unknown) at baseline 23/14/0 13/0/4 4/0/9 2/4/23 — i Duration of NTZ treatment/time to PML (y) 6.1 (1.6) 3.7 (1.4) 3.2 (0.7) 7.1 (3.7) — Serum NFL levels (pg/mL)f 8.9 (6.4–18.4) 16.0 (10.2–21.5) 171.1 (130.0–231.0) 6.2 (5.0–8.3) 13.9 (7.6–21.8) Abbreviations: EDSS = Expanded Disability Status Scale; JCV = JC virus; NFL = neurofilament light chain; NTZ = natalizumab; PML = progressive multifocal leukoencephalopathy; RRMS = relapsing-remitting multiple sclerosis. Data are expressed as mean (SD) unless otherwise stated. a During PML: refers to patients whose blood was collected during the PML condition (4 patients in this group are shared with the pre-PML group). In this group, information on the time between PML onset and blood collection was not available in for 4 patients. b Replication cohort: refers to an independent cohort of patients treated with NTZ who did not develop PML, and serum NFL levels were measured after 2 years of treatment. c Relapsing cohort: refers to a cohort of patients with MS whose blood was collected at the time of a clinical relapse. In this group, 8 patients (26.7%) were receiving treatment with interferon-beta during relapses, but none of the patients were treated with corticosteroids before blood extraction. d NTZ-ctr: refers to patients with MS treated with NTZ who did not develop PML. e pre-PML: refers to patients with MS treated with NTZ who developed PML. f Data are expressed as median (interquartile range). g Refers to EDSS score during relapse. h Information on EDSS score was not available in 10 patients. Duration of disease is calculated from disease onset to baseline. Age, percentage of women, duration of disease, and EDSS score at baseline did not statistically significantly differ between the NTZ-ctr group, pre-PML group, during PML cohort, and replication cohort. A trend for older age was observed in the during PML cohort compared with the relapsing cohort (p = 0.07). Percentage of women, duration of disease, and EDSS score were not significantly different between these 2 cohorts of patients. i Refers to mean time of NTZ treatment in the NTZ-ctr group and replication cohort and time to conversion to PML in the pre-PML group and during PML cohort. 2 years, serum NFL levels were significantly reduced by the effect As shown in figure 2B, the distribution of serum NFL values in of NTZ treatment both in the pre-PML and NTZ-ctr groups the replication cohort was similar to the NTZ-ctr group at 2 (figure 1). Comparisons of serum NFL levels at 1 and 2 years years of treatment, with mean NFL values of 6.9 ± 2.5 pg/mL between pre-PML and NTZ-ctr patients revealed higher NFL and 7.1 ± 2.5 pg/mL, respectively. Performance of NFL levels levels only in those patients with MS who will develop PML after in the replication cohort to predict PML was comparable to 2 years of NTZ treatment with mean values of 10.1 ± 5.9 pg/mL the NTZ-ctr cohort at 2 years, with an AUC of 71% (p = 0.03), and 7.1 ± 2.5 pg/mL, respectively (p = 0.03 both unadjusted and and a serum NFL level of 8.1 pg/ml as the best cutoff to after adjusting for age and disease duration; figure 1). classify pre-PML patients at 2 years and patients from the replication cohort, with a sensitivity and specificity of 67% and In the during PML cohort, blood was collected at a mean time 76%, respectively (figure 2C). from PML onset of 8.1 ± 16.3 days. Serum NFL levels during PML were higher compared with pre-PML patients after 2 years Serum NFL Levels Distinguish PML From of NTZ treatment with mean values of 163.6 ± 153.8 pg/mL MS Relapses and 10.1 ± 5.9 pg/mL, respectively (unadjusted, p = 7 × 10−5; Serum NFL levels in the relapsing cohort did not significantly after adjusting for age and disease duration, p = 0.02; figure 1), differ between untreated and interferon-beta–treated patients, which represents a 16-fold increase in serum NFL levels. and hence, this cohort was first analyzed as a whole. Comparison of serum NFL levels between PML and MS relapses revealed Serum NFL Levels Discriminate Between Pre- significantly higher NFL levels in patients during the PML con- PML and NTZ-ctr Patients After 2 Years of dition (p = 3 × 10−6; figure 3A), which represents a 8-fold in- NTZ Treatment crease in serum NFL levels compared with the relapsing cohort Figure 2A shows the prognostic performance of serum NFL (mean levels: 163.6 ± 153.8 pg/mL vs 20.8 ± 28.0 pg/mL, re- levels to predict PML development at 2 years. The area under spectively). Performance of serum NFL levels to differentiate the ROC curve (AUC) was 71% (p = 0.03), and a serum NFL between PML and MS relapses showed an AUC of 91% (p = 2 × value of 8.4 pg/mL resulted in the best cutoff to classify pre- 10−5), with an NFL value of 52.7 pg/mL as the best cutoff to PML and NTZ-ctr patients after 2 years of NTZ treatment, classify PML and MS relapses (figure 3B). Sensitivity and spec- with a sensitivity of 67% and specificity of 80%. ificity associated with this cutoff were 85% and 93%, respectively. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 4 | July 2021 3 at present routinely measured in clinical practice to estimate Figure 1 Increased NFL Levels in Serum After 2 Years of NTZ PML risk in patients with RRMS receiving NTZ. In a recent Treatment in Pre-PML Patients Compared With study, serum NFL levels measured with an electro- NTZ-ctr chemiluminescence assay were found to be 10-fold higher at PML onset compared with the pre-PML condition.6 Fur- thermore, serum NFL levels also demonstrated high perfor- mance to discriminate between patients with MS at PML onset and NTZ-treated patients who did not develop PML and treated patients with clinical or neuroradiologic evidence of disease activity 4 weeks before sample collection.6 In our study, serum NFL levels measured in a prospective cohort of NTZ-treated patients with the more sensitive Simoa assay11 were not predictive of PML development at baseline or after 1 year of treatment. However, despite a general sig- nificant decrease by the effect of treatment, serum NFL levels at 2 years were significantly increased in patients who will develop PML compared with NTZ-ctr patients, and NFL levels had good potential to discriminate between these 2 groups of patients in terms of PML development. Of interest, Graphs comparing serum NFL levels between pre-PML and NTZ-ctr patients performance of serum NFL levels to predict PML in an in- at baseline (T0; n = 16 for pre-PML and n = 36 for NTZ-ctr), after 1 (T1; n = 12 for pre-PML and n = 36 for NTZ-ctr) and 2 years (T2; n = 12 for pre-PML and n dependent cohort of NTZ-treated patients for 2 years was = 34 for NTZ-ctr) of treatment and during PML (n = 13). Each symbol rep- remarkably similar to the original cohort, results that support resents an individual, and horizontal bars indicate the median values and interquartile ranges. A y-axis segmentation was performed to represent the use of similar cutoff values between MS centers to esti- better high and low serum NFL levels. *Refers to p values <0.05. **Refers to mate PML risk in different cohorts of patients with MS after 2 p values <0.01. ***Refers to p values <0.001 in Mann-Whitney U tests (un- paired data) and Wilcoxon matched-paired test (paired data). NFL = neu- years of NTZ treatment. rofilament light chain; NTZ = natalizumab; PML = progressive multifocal leukoencephalopathy. In agreement with Dalla Costa et al.,6 serum NFL levels were far more elevated during PML compared with earlier stages of the disease. In our study, NFL levels were 16-fold higher A subanalysis in the untreated relapsing patients revealed during PML compared with pre-PML patients at 2 years of similar results to the whole cohort, with an AUC of 90% (p = 9 NTZ treatment and 8-fold higher compared with a relapsing × 10−5) and the same NFL value of 52.7 pg/mL as the best cohort. The latter may have implications in clinical practice to cutoff to classify patients. Sensitivity and specificity were 85% set a cutoff value of serum NFL levels that distinguish the and 91%, respectively. PML condition from clinical relapses in patients receiving NTZ treatment. A limitation in our study was the inclusion of a relapsing cohort either untreated or receiving interferon- Discussion beta, a treatment that was not associated with significant re- ductions in serum NFL levels. In this context, the inclusion of Few molecular biomarker studies have aimed to identify pa- a relapsing cohort of NTZ-treated patients would probably tients with MS at increased risk for PML under NTZ have been associated with greater differences in serum NFL treatment.8-10 However, none of the proposed biomarkers are levels between relapsing and PML patients, considering the Figure 2 Serum NFL Levels Differentiate Between Pre-PML and NTZ-ctr Patients After 2 Years of NTZ Treatment (A) Performance of serum NFL levels to dis- criminate between pre-PML and NTZ-ctr pa- tients after 2 years of treatment. (B) Distribution of serum NFL levels in the NTZ-ctr cohort at 2 years of treatment (n = 34), and in an independent cohort of patients with MS treated with NTZ for 2 years (replication cohort; n = 29). (C) Performance of the serum NFL levels to discriminate between pre-PML pa- tients at 2 years and patients from the repli- cation cohort after 2 years of treatment. NFL = neurofilament light chain; NTZ = natalizumab; PML = progressive multifocal leukoencephalopathy. 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 4 | July 2021 Neurology.org/NN Figure 3 NFL Levels in Serum Identify PML From MS Relapses (A) Distribution of serum NFL levels in the re- lapsing cohort (n = 30) and during PML (n = 13). A y-axis segmentation was performed to rep- resent better high and low serum NFL levels. *p = 3 × 10−6 In the relapsing cohort: untreated patients are represented in solid squares and interferon-beta–treated patients in solid trian- gles. (B) Performance of the serum NFL levels to discriminate between MS relapses and PML. Numbers in parentheses represent 95% CIs of the AUC. AUC = area under the ROC curve; MS = multiple sclerosis; NFL = neurofilament light chain; PML = progressive multifocal leu- koencephalopathy; Se = sensitivity; Sp = specificity. effect of NTZ reducing significantly serum NTL levels, as shown in our study. Appendix (continued) Name Location Contribution Based on our findings, in patients receiving NTZ treatment, we recommend to measure NFL levels longitudinally, and Beatrice CRC-SEP Neurosciences Acquisition and analysis Pignolet, Centre Hospitalier of the data and revised those patients having protein levels above the cut-offs calcu- PhD Universitaire Toulouse, the manuscript for lated in the study after 2 years of treatment should be mon- CPTP INSERM UMR 1043 intellectual content CNRS UMR 5282 et itored more closely for neurologic symptoms with additional Université de Toulouse III, NFL and MRI measures to rule out PML. UPS, France Jordi Rio, Servei de Neurologia- Analysis of the data and In conclusion, our results support the use of serum NFL levels MD, PhD Neuroimmunologia, Centre revised the manuscript d’Esclerosi Múltiple de for intellectual content in clinical practice to identify patients with RRMS at higher Catalunya (Cemcat), Hospital risk for PML based on protein levels at 2 years of NTZ Universitari Vall d’Hebron, Universitat Autònoma de treatment and to differentiate PML from clinical relapses in Barcelona, Spain patients receiving NTZ. Patrick Univ. Lille, Inserm U1172, Acquisition of the data Vermersch, CHU Lille, FHU Imminent, and revised the Study Funding MD, PhD France manuscript for No targeted funding reported. intellectual content Aurélie Université, Bordeaux, Acquisition of the data Disclosure Ruet, MD, France, CHU de Bordeaux, and revised the PhD INSERM-CHU CIC-P 0005, & manuscript for The authors report no disclosures relevant to the manuscript. Services de Neurologie, intellectual content Go to Neurology.org/NN for full disclosures. France, and Neurocentre Magendie, INSERM U1215 Bordeaux, France Publication History Jerome Department of Neurology, Acquisition of the data Received by Neurology: Neuroimmunology & Neuroinflammation deSèze, MD, Hôpital Civil, Strasbourg, and revised the November 23, 2020. Accepted in final form March 3, 2021. PhD France manuscript for intellectual content Pierre Department of Neurology, Acquisition of the data Appendix Authors Labauge, CHU Montpellier, France and revised the MD, PhD manuscript for Name Location Contribution intellectual content Nicolás Servei de Neurologia- Designed and Sandra Department of Neurology Acquisition of the data Fissolo, PhD Neuroimmunologia, Centre conceptualized the study; Vukusic, CHU Lyon, Bron, France and revised the d’Esclerosi Múltiple de acquisition and analysis MD, PhD manuscript for Catalunya (Cemcat), Institut of the data; and drafted intellectual content de Recerca Vall d’Hebron the manuscript for (VHIR), Hospital Universitari intellectual content Caroline Department of neurology, Acquisition of the data Vall d’Hebron, Universitat Papeix, MD Hôpital de la Salpétrière, and revised the Autònoma de Barcelona, Paris, France manuscript for Spain intellectual content Continued Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 8, Number 4 | July 2021 5 Appendix (continued) Appendix (continued) Name Location Contribution Name Location Contribution Laurent Chi Aix en Provence, France Acquisition of the data David CRC-SEP Neurosciences Acquisition of the data Martinez- and revised the Brassat, MD, Centre Hospitalier and revised the Almoyna, manuscript for PhD Universitaire Toulouse, manuscript for MD intellectual content CPTP INSERM UMR 1043 intellectual content CNRS UMR 5282 et Ayman Department of Neurology Acquisition of the data Université de Toulouse III, Tourbah, and Faculté de Médecine de and revised the UPS, France MD, PhD Reims, CHU de Reims, manuscript for URCA, France; LPN EA2027 intellectual content Manuel Servei de Neurologia- Designed and Université Paris VIII, Saint- Comabella, Neuroimmunologia, Centre conceptualized the study; Denis, Paris, France MD, PhD d’Esclerosi Múltiple de analyzed the data; and Catalunya (Cemcat), Institut drafted the manuscript Pierre Department of Neurology, Acquisition of the data de Recerca Vall d’Hebron for intellectual content Clavelou, CHRU Clermont Ferrand, and revised the (VHIR). Hospital Universitari MD, PhD France manuscript for Vall d’Hebron, Universitat intellectual content Autònoma de Barcelona, Spain Thibault Department of Neurology, Acquisition of the data Moreau, MD, CHU Dijon, France and revised the PhD manuscript for intellectual content References Jean Aix-Marseille Univ, APHM, Acquisition of the data 1. Polman CH, O’Connor PW, Havrdova E, et al. A randomized, placebo-controlled trial Pelletier, Hôpital de la Timone, Pôle and revised the of natalizumab for relapsing multiple sclerosis. N Engl J Med 2006;354(9):899–910. MD, PhD de Neurosciences Cliniques, manuscript for 2. Bloomgren G, Richman S, Hotermans C, et al. Risk of natalizumab-associated pro- Service de Neurologie, intellectual content gressive multifocal leukoencephalopathy. N Engl J Med 2012;366(20):1870–1880. CNRS, CRMBM UMR 7339, 3. Kappos L, Bates D, Edan G, et al. 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