assessing neurofilaments as biomarkers of neuroprotection

ARTICLE OPEN ACCESS

Assessing Neurofilaments as Biomarkers ofNeuroprotection in Progressive Multiple SclerosisFrom the MS-STAT Randomized Controlled Trial

Thomas E Williams BA MB BChir MRCP Katherine P Holdsworth MSc Jennifer M Nicholas PhD

Arman Eshaghi PhD Theodora Katsanouli MSc Henrietta Wellington PhD FRCP Amanda Heslegrave PhD

Henrik Zetterberg PhD Chris Frost PhD and Jeremy Chataway PhD FRCP

Neurol Neuroimmunol Neuroinflamm 20229e1130 doi101212NXI0000000000001130

Correspondence

Dr Williams

thomasewilliamsuclacuk

AbstractBackground and ObjectivesImproved biomarkers of neuroprotective treatment are needed in progressive multiple sclerosis(PMS) to facilitate more efficient phase 2 trial design The MS-STAT randomized controlledtrial supported the neuroprotective potential of high-dose simvastatin in secondary progressiveMS (SPMS) Here we analyze serum from the MS-STAT trial to assess the extent to whichneurofilament light (NfL) and neurofilament heavy (NfH) both promising biomarkers ofneuroaxonal injury may act as biomarkers of simvastatin treatment in SPMS

MethodsThe MS-STAT trial randomized patients to 80 mg simvastatin or placebo Serum was ana-lyzed for NfL and NfH using Simoa technology We used linear mixed models to investigatethe treatment effects of simvastatin compared with placebo on NfL and NfH Additionalmodels examined the relationships between neurofilaments andMRI and clinical measures ofdisease severity

ResultsA total of 140 patients with SPMS were included There was no evidence for a simvastatintreatment effect on NfL or NfH compared with placebo NfL was 12 lower (95 CI 106lower to 92 higher p = 0820) and NfH was 04 lower (95 CI 184 lower to 216higher p = 0969) in the simvastatin treatment group Secondary analyses suggested that higherNfL was associated with greater subsequent whole brain atrophy higher T2 lesion volume andmore newenlarging T2 lesions in the previous 12 months as well as greater physical disabilityThere were no significant associations between NfH and MRI or clinical variables

DiscussionWe found no evidence of a simvastatin treatment effect on serum neurofilaments Whileconfirmation of the neuroprotective benefits of simvastatin is awaited from the ongoing phase 3study (NCT03387670) our results suggest that treatments capable of slowing the rate of wholebrain atrophy in SPMS such as simvastatin may act via mechanisms largely independent ofneuroaxonal injury as quantified by NfL This has important implications for the design offuture phase 2 clinical trials in PMS

Trial Registration InformationMS-STAT NCT00647348

MORE ONLINE

Class of EvidenceCriteria for ratingtherapeutic and diagnosticstudies

NPuborgcoe

From the Queen Square Multiple Sclerosis Centre (TEW AE JC) Department of Neuroinflammation UCL Queen Square Institute of Neurology Faculty of Brain SciencesUniversity College London National Hospital of Neurology and Neurosurgery (TEW JC) London London School of Hygiene and Tropical Medicine (KPH JMN TK CF) and UKDementia Research Institute at UCL (HW AH HZ) United Kingdom

Go to NeurologyorgNN 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 40 (CC BY-NC-ND) which permits downloadingand 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 copy 2022 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Classification of EvidenceThis study provides class I evidence that simvastatin treatment does not have a large impact on either serum NfL or NfH asquantified in this study in SPMS

Few treatments are available for progressive multiple sclerosis(PMS) with most restricted to those with active inflammatorydisease Treatments for nonactive PMS1 may require an alter-native approach utilizing neuroprotective mechanisms alone orin combination with immunomodulation One such candidateneuroprotective treatment is simvastatin which in the MS-STAT trial demonstrated a 43 reduction in annualized brainatrophy in addition to benefits on secondary physical and cog-nitive outcomes No differences were noted on a panel of im-munologic markers supporting a neuroprotective rather thanimmunomodulatory mechanism23 The phase 3 MS-STAT2study is ongoing (NCT03387670)

Blood neurofilament light (NfL) is a biomarker of neuroaxonalinjury and an appealing surrogate outcome measure for trials inrelapsing-remitting MS4 It is less well studied in PMS5 Initia-tion of immunosuppressive disease-modifying treatment is as-sociated with a reduction in blood NfL in PMS cohorts but animportant question remains over the ability of NfL to capturethe treatment effect of purportedly neuroprotective therapies6-9

Clarifying this is an important goal of the progressive MS alli-ance with implications on future phase 2 trial design10 Data forblood neurofilament heavy (NfH) are more limited but it mayhave utility as a biomarker of neuroprotection1112

Here we analyze serumNfL andNfH from theMS-STAT trialWe use the known positive effect of simvastatin on whole brainatrophy to interrogate our primary research question whetherserum NfL and NfH may act as biomarkers of neuroprotectivetreatment with simvastatin in secondary progressive MS(SPMS) We additionally perform further analyses to examinethe relationships between serum neurofilaments and estab-lished MRI and clinical variables

MethodsMS-STAT Trial and Sample ProcessingThe MS-STAT study protocol has been outlined previously2

Briefly patients with SPMS aged 18ndash65 years with ExpandedDisability Status Scale (EDSS) score 40ndash65 were eligible Keyexclusion criteria included primary progressive MS relapse orsteroid use within 3 months and the use of immunosuppressiveor disease-modifying therapy within the last 6 months A total of

140 patients were randomized 11 to simvastatin 80 mg orplacebo Baseline characteristics are shown in Table 1 Bloodsamples were acquired at baseline and months 6 12 and 24Serum was separated and stored at minus80degC until time of analysis

Neurofilament QuantificationNfL and NfH are components of a neurone-specific in-termediate filament differing in their C-terminal domain andphosphorylation state Both are released following neuro-axonal injury into the CSF and blood where they may bequantified Serum NfL and NfH were measured by Simoatechnology on a HD-1 analyzer according to the manufac-turerrsquos instructions (Quanterix Billerica MA) The SimoaNF-Light Advantage and Simoa pNF-heavy Discovery Kits(Quanterix) were used Briefly serum samples were thawed at21degC vortexed and centrifuged at 10000 RCF for 5 minutesat 21degC On-board the HD-1 samples were diluted 14 withsample diluent and bound to paramagnetic beads coated witha capture antibody specific for human NfL or NfH Antibody-coated beads were incubated with a biotinylated anti-NfL oranti-NfH detection antibodies that in turn were labeled with astreptavidin-β-galactosidase complex Following the additionof the β-galactosidase substrate resorufin β-D-galactopyrano-side a fluorescent signal proportional to the concentration ofneurofilament present in the sample was generated in theantigen-containing microwells of the Simoa plates

Duplicate measurements were taken of each sample Sampleconcentrations were extrapolated from a standard curve fittedusing a 4-parameter logistic algorithm The lower limit ofquantification (LLoQ) for NfL is 0174 pgmL and for NfH is288 pgmL1314 Values below the LLoQ were assigned thevalue of half the LLoQ The coefficient of variation (CoV)between sample replicates tends to be higher for lower valueresults To avoid bias all data were therefore included in theprimary statistical analysis regardless of the CoV Each assaywas run in the same or consecutive batches by the sameoperator who was blinded to treatment allocation

MRI ProcessingThe imaging data have been previously published and wereacquired as previously described215 Briefly 3D T1-weighteddouble-echo proton density and T2-weighted MRI wasobtained at baseline month 12 and month 25 Whole brain

Glossary9HPT = 9-hole peg test 25FW = 25-foot timed walkCoV = coefficient of variation EDSS = Expanded Disability Status ScaleIQR = interquartile range LLoQ = lower limit of quantificationMS = multiple sclerosis NfH = neurofilament heavy NfL =neurofilament light PMS = progressive MS SPMS = secondary progressive MS T2LV = T2 lesion volume

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 9 Number 2 | March 2022 NeurologyorgNN

atrophy was determined using the boundary shift integralmethod and expressed as percentage change in whole brainvolume T2 newenlarging lesions were expressed as a countand T2 lesion volume (T2LV) in milliliters

Statistical AnalysisThe prespecified primary analysis was to examine the effect ofsimvastatin (80 mg) vs placebo on levels of serum NfL at 24months The primary analysis was conducted on the intention totreat population regardless of treatment adherence An exploratoryanalysis was undertaken to examine the treatment effect using a

per-protocol data set which included patients who complied withtreatment and completed follow-up to 25 months Participantswere considered compliantwith treatment if they reported takingon average at least 90 of their tablets at the protocol dose of 2tablets per day

The prespecified secondary analysis examined the relationshipbetween serum NfL and whole brain atrophy rate Furtheranalyses of the association of NfL with other MRI and clinicalvariables and all analyses of NfH data were exploratory Neu-rofilament data were skewed and hence all analyses were

Table 1 Characteristics of theMS-STAT Trial Cohort and Descriptive Statistics for SerumNfL and SerumNfH Across TimePoints

Placebo (n = 70) Simvastatin (n = 70) All (N = 140)

Baseline characteristics

Sex n () female 48 (69) 49 (70) 97 (69)

Ethnicity n () White 63 (90) 69 (99) 132 (94)

Relapse in last 24 mo n () 18 (26) 8 (11) 26 (19)

Age y mean (SD) 511 (68) 515 (70) 513 (69)

MS duration y mean (SD) 203 (88) 221 (83) 212 (86)

SPMS duration y mean (SD) 71 (48) 73 (56) 72 (52)

EDSS score median (IQR) 6 (55ndash65) 6 (55ndash65) 6 (55ndash65)

Previous use of interferon n () 12 (17) 10 (14) 22 (16)

Serum NfL pgmL

Baseline median (IQR) 153 (102ndash222) 139 (109ndash197) 146 (108ndash202)

N 63 65 128

Month 6 median (IQR) 148 (120ndash230) 150 (115ndash218) 148 (117ndash226)

N 53 59 112


N 53 59 112


N 48 64 112

Serum NfH pgmL


N 63 62 125


N 49 53 102


N 52 57 109


N 48 64 112

Abbreviations EDSS = Expanded Disability Status Scale IQR = interquartile range MS = multiple sclerosis NfH = neurofilament heavy NfL = neurofilamentlight SPMS = secondary progressive MS

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 9 Number 2 | March 2022 3

performed following log2 transformation Clinical data in-cluded EDSS scores 25-foot timed walk (25FW) expressed asspeed (inverse of completion time in seconds) and 9-hole pegtest (9HPT) expressed as a speed (1000 times inverse of com-pletion time in seconds) Analyses were conducted in Stata 151or later

Analysis of Simvastatin Treatment Effect onNfL and NfHA mixed effect model was used to estimate the simvastatintreatment effect on serum neurofilaments at 6 12 and 24months for NfL and NfH A single model was used to esti-mate a separate treatment effect at each visit (6 12 and 24months) by including a categorical variable for visit and aninteraction between visit and treatment group Baseline neu-rofilament data were included as an additional end point butwith the treatment effect here constrained to be 0 This isessentially equivalent to adjusting for the baseline neurofila-ment level16 The model included an unstructured residualcovariance matrix for the residuals (hence allowing a differentvariance at each visit and different covariances between eachpair of measurements on the same participant) The followingbaseline variables which were used as minimization factors inthe randomization were adjusted for by including them andtheir interactions with visit as fixed effects age sex di-chotomized EDSS scores (40ndash55 60ndash65) and study siteThe mean of the estimated treatment effects across all follow-up visits is also presented In addition an exploratory analysiswas conducted which adjusted for the minimization factors inthe randomization and the following baseline variables T2LV

and number of relapses in the previous 24 months In thesemodels we did not perform adjustments for covariates mea-sured after randomization as they may be influenced by thetreatment allocation and hence introduce bias into the anal-ysis of treatment effect17

Analysis Relating NfL and NfH to MRI andClinical VariablesThis analysis used data on baseline neurofilament levels andthe rates of change in neurofilament levels during the trial aspredictors of MRI and clinical outcomes A 2-stage analysiswas performed

In the first stage a summary measure for rate of change in NfLand NfH for each participant was calculated as the slope froma simple linear regression model relating each participantrsquosrepeated measures of log NfL and separately log NfH to timefrom baseline using ordinary least squares18 In the secondstage each participantrsquos estimated rate of change in log NfL orlog NfH was used as a predictor variable along with baseline-centered log NfL or log NfH in a series of separate models forMRI and clinical outcomes For each outcome a separatemodel was fitted for log NfL and log NfH

The model for whole brain atrophy was an extension of apreviously described linear mixedmodel for directly measuredchange between each pair of MRI visits (baseline to 12baseline to 25 and 12ndash25 months) as the outcome19 It in-cluded participant-level random slopes for time between scansand random effects for visit All predictor variables were in-cluded as interactions with time between scans in order tomodel the associations with atrophy rate The model includedbaseline neurofilaments and change in neurofilaments as wellas the following baseline variables age sex MRI site SPMSduration relapse in the previous 24 months previous use ofinterferon and baseline treatments for fatigue depressionneuropathic pain spasticity and bladder urgency

The model for T2LV was a linear mixed model with T2LV ateach MRI visit (baseline 12 months and 25 months) as theoutcome Analysis of 25FW and 9HPT used a linear mixedmodel with speed at each visit (baseline 12 months and 24months) as the outcome These models included participantlevel random slopes for change over time and random inter-cepts allowing for correlation between these random effectsIn each model baseline log neurofilament was included as apredictor along with its interaction with time since baselineAnalysis of T2LV additionally included change in log neuro-filament and its interaction with time The models for T2LVincluded the same variables that were adjusted for in thewhole brain atrophy model on their own as well as an in-teraction with time The models for clinical variables includedthese same adjustments with the exception of MRI site AsT2LV and clinical variables violated normality assumptionsinference from these models is based on nonparametric bias-corrected and accelerated 95 and 99 CIs calculated from10000 bootstrap replications clustered on participant p

Table 2 Effect of High-Dose Simvastatin on Serum NfLand Serum NfH

Difference ingeometric mean ()a 95 CI p Value

Serum NfL (pgmL)

Month 6 minus329 minus162 to 116 0646

Month 12 535 minus74 to 199 0429


Mean treatmenteffect

minus116 minus106 to 92 0820

Serum NfH (pgmL)


Month 12 474 minus151 to 293 0666

Month 24 169 minus184 to 267 0881



Abbreviations EDSS = Expanded Disability Status Scale IQR = interquartilerange NfH = neurofilament heavy NfL = neurofilament lighta Percentage difference in geometric mean serum neurofilaments simvas-tatin vs placebo adjusted for age sex dichotomized EDSS scores (40ndash5560ndash65) and study site Differences are shown for each follow-up visit andalso the average treatment effect across all follow-up visits


values are therefore not calculated for these models but theranges in which they lie can be inferred from the CIs

For the EDSS score the linear mixed model did not convergeInstead a linear regression model for score at each visit(baseline 12 months and 24 months) was used To allow forthe nonindependence of measures from the same participantnonparametric bias-corrected and accelerated CIs clusteredon participant were used as described above The models forthe EDSS score included the same adjustment variables as forthe other clinical outcomes

Although T2LV may reflect overall disease burden the identifi-cation of active lesions (either gadolinium-enhancing lesions on asingle scan or newenlarging T2 lesions when comparing 2 timepoints) is the keyMRImeasure of ongoing neuroinflammation20

The MS-STAT cohort did not include gadolinium-enhancedimaging and newenlarging T2 lesions cannot be determined atbaseline To further explore the known relationship betweenserum NfL and neuroinflammation in this SPMS cohort wetherefore performed additional exploratory linear regressionmodeling usingmonth 24 logNfL as the dependent variable Thisallowed inclusion of recent active lesions (newenlarging T2 le-sions during month 0 to month 12 and month 12 to month 25)and concurrent T2LV (month 25) as predictors Models werefitted including each of the MRI variables on their own and thentogether in a mutually adjusted multivariable model Thesemodels included the same covariates as the T2LV models

Standard Protocol Approvals Registrationsand Patient ConsentsThe study was conducted in accordance with Good ClinicalPractice and the Declaration of Helsinki21 The MS-STATprotocol was approved by each study sitersquos institutional reviewboard and a national ethics committee all patients gavewritten informed consent before entering the study andethical approval for the retrospective analysis of serum

samples was received The MS-STAT clinical trial identifica-tion number is NCT00647348

Data AvailabilityAnonymized NfL and NfH data are provided as a supple-mentary data file (linkslwwcomNXIA679)

ResultsNfL and NfH DataData on NfL were available from at least 1 visit for 138 pa-tients (69 in each treatment group) with 128 patients havingNfL data from at least 1 follow-up visit (61 placebo 67 sim-vastatin) For NfH data were available from 137 patients (69placebo 68 simvastatin) with 127 patients having NfH datafrom at least 1 follow-up visit (60 placebo 67 simvastatin) NoNfL measures were below the LLoQ and all sample replicateshad a CoV lt20 For NfH 8 samples (18) were below theLLoQ and 39 samples (86) had a CoV gt20 At baselinemedian NfL was 146 pgmL (interquartile range [IQR]108ndash202 pgmL) and median NfH was 655 pgmL (IQR240ndash1185 pgmL) (Table 1) Characteristics of the per-protocol dataset are included in eTable 1 (linkslwwcomNXIA679)

Analysis of Simvastatin Treatment Effect onNfL and NfHThere was no evidence of a simvastatin treatment effect oneither NfL or NfH at any time point (Table 2) with adjustedmarginal mean NfL and NfH levels being similar in the 2treatment groups at each follow-up visit (Figure 1) Takingthe mean of the treatment effects across all follow-up timepoints the geometric mean NfL was 12 lower in the sim-vastatin group than in the placebo group (95 CI 106lower to 92 higher p = 0820) whereas the geometric meanNfH was 04 lower in the simvastatin group than on placebo

Figure 1 Estimated Mean Serum NfL (A) and Serum NfH (B) in Simvastatin and Placebo Treatment Groups

Values are marginal adjusted geometric means from linear mixed models with treatment effect at baseline constrained to 0 There was no evidence for atreatment effect on either NfL or NfH NfH = neurofilament heavy NfL = neurofilament light


(95 CI 184 lower to 216 higher p = 0969) The resultsfrom the exploratory per-protocol analysis were similar tothose found for the intention-to-treat analysis with no evi-dence of a simvastatin treatment effect on either NfL or NfHat any time point (eTable 2 linkslwwcomNXIA679)

Sensitivity analyses found that the results were not materiallyaltered following the exclusion of 2 individuals with outlyingneurofilament values In addition an exploratory analysisfound that results were essentially unchanged with adjustmentfor baseline lesion volume and relapses within the last 24months (eTable 3 linkslwwcomNXIA679)

Association of NfL and NfH With MRI VariablesThe relationships between both NfL and NfH and each ofwhole brain atrophy and T2LV are shown in Table 3 andFigure 2 There was evidence for an association betweenhigher baseline NfL and faster whole brain atrophy rate and

between higher baseline NfL and greater T2LV a twofoldincrease in baseline NfL was associated with a 021yearincrease in brain atrophy and with a 77 mL higher baselineT2LV The rate of change in NfL was not associated with therate of change in T2LV or the rate of brain atrophy Patientswith a greater increase in NfL from baseline to month 24however tended to have a higher T2LV at baseline andfollow-up time points As there was little effect of change inNfL on T2LV the effect was similar across all visits an in-crease of 1 extra doubling per year in NfL was associated witha 151 mL higher baseline T2LV and 159 mL higher month25 T2LV There was no evidence for an association betweenNfH and any MRI variables (Table 3 Figure 2)

NfL at month 24 was associated with concurrent T2LV andnewenlarging T2 lesions between baseline and month 12and between months 12 and 25 when examined in separatemodels (Table 4) When these were combined together as

Table 3 Relationship Between Serum NfL NfH and Imaging Variables

Parameter estimate 95 CI p Value

Whole brain atrophy

Relationship with rate of whole brain atrophy ( per year)

Predictor variable

Baseline NfL (per doubling) 0207 0072 to 0341 0003

Rate of change in NfL (doublings per yeara) 0093 minus0201 to 0387 0535

Baseline NfH (per doubling) 0048 minus0005 to 0102 0078

Rate of change in NfH (doublings per yeara) 0016 minus0105 to 0136 0795

T2 lesion volume

Relationship with baseline T2 lesion volume (mL)

Predictor variable

Baseline NfL (per doubling) 768 366 to 1248 lt001

Rate of change in NfL (doublings per yeara) 1508 667 to 2640 lt001

Baseline NfH (per doubling) 0736 minus0921 to 2631 gt005

Rate of change in NfH (doublings per yeara) 0413 minus6696 to 8001 gt005

Relationship with change in T2 lesion volume (mLy)

Predictor variable

Baseline NfL (per doubling) 029 minus014 to 063 gt005

Rate of change in NfL (doublings per yeara) 039 minus060 to 102 gt005

Baseline NfH (per doubling) minus0070 minus0218 to 0051 gt005

Rate of change in NfH (doublings per yeara) minus0032 minus0535 to 0541 gt005

Abbreviations NfH = neurofilament heavy NfL = neurofilament lightThe results of 4 separatemodels are presented withwhole brain atrophy or T2 lesion volume as the dependent variables andNfL orNfHdata as the predictorvariables In all analyses neurofilament data were log2 transformed Results are from covariate adjusted models as indicated in the Methods section For T2lesion volume the p value bounds (ltgt005 and ltgt001) can only be inferred from the 95 and 99 bias-corrected and accelerated cluster bootstrap (10000replications) CIsa A 1-unit increase in the number of doublings per year corresponds to a change from stable levels to a doubling per year or from doubling once every year todoubling every 6 months (2 doublings per year)


predictors in the same mutually adjusted model both con-current T2LV and month 12ndash25 newenlarging T2 lesionsremained independently associated with month 24 NfLwhereas the association with new and enlarging lesions be-tween baseline and month 12 was lost In the final modelmonth 24 NfL was increased by 57 for each newenlargingT2 lesion between months 12 and 25 and by 089 for eachmilliliter increase in month 25 T2LV

Association of NfL and NfH WithClinical VariablesHigher baseline NfL was significantly associated withhigher baseline EDSS score but not with the rate of changein the EDSS score from baseline to 2 years (Table 5)Higher baseline NfL was also associated with worse base-line 9HPT performance and with a greater rate of wors-ening in the 25FW speed from baseline to 2 years BaselineNfH was not materially associated with any clinical vari-ables Sensitivity analyses demonstrated that the resultswere not materially changed following the exclusion of 2neurofilament outliers

Classification of EvidenceThis study assessed the ability of serum NfL and NfH to act asbiomarkers of a neuroprotective treatment response with high-dose simvastatin compared with placebo in patients withSPMS It provides Class I evidence that these biomarkers asquantified in this study do not act as biomarkers of neuro-protection with simvastatin

DiscussionOur results demonstrate that despite simvastatin reducing theannualizedwhole brain atrophy rate by 43per year comparedwith placebo we did not find evidence to support a simvastatintreatment effect on serum NfL or NfH We also replicatepreviously observed findings demonstrating that higher NfL isassociated with a greater subsequent rate of whole brain atro-phy and that recent inflammatory activity (newenlarging T2lesions) as well as T2LV is associated with higher NfL

The existing literature suggests that in MS the degree ofneuroaxonal injury reflected by serum NfL is predominantly

Figure 2 Relationship Between Each of Baseline SerumNfL andNfH and Each ofWhole Brain Atrophy Rate and Baseline T2Lesion Volume

Points represent individual patient data Whole brain atrophy rate is reported as yearly change frombaseline tomonth 25 and baseline T2 lesion volume inmilliliters (A) Baseline NfL and baseline to month 25 whole brain atrophy rate (B) Baseline NfH and baseline to month 25 whole brain atrophy rate (C)Baseline NfL and baseline T2 lesion volume (D) Baseline NfH and baseline T2 lesion volume NfH = neurofilament heavy NfL = neurofilament light


related to ongoing neuroinflammation Such neuroinflammationmay be detected by conventional MRI measures such as recentT1 gadolinium-enhancing lesions or new T2 lesions or by ad-vancedMRImeasures of chronic neuroinflammation such as theidentification of chronic active lesions via their paramagneticrims522 Our previous findings suggested that simvastatin treat-ment was not systemically immunomodulating in this cohorthence providing 1 possible rational for the absence of a treatmenteffect on NfL2

Many of the pathophysiologic mechanisms contributing to pro-gressive MS ultimately converge on neuroaxonal damage whichmay be reflected by increased NfL23 The dissociation betweenthe previously observed benefits of simvastatin (on whole brainatrophy and disability measures) and the absence of a treatmenteffect on NfL however does also highlight the potential im-portance of additional processes independent of neuroaxonalinjury in the pathophysiology of SPMS Comorbidities particu-larly cardiovascular are prevalent within the MS population andare known to have an impact on future disability and brainatrophy24-26 MRI measures of brain atrophy have been validatedagainst clinical treatment effects long-term disability outcomesand measures of neuroaxonal loss27-30 Brain atrophy is howevernot specific to neuroaxonal injury and may be influenced byvolume changes in other CNS tissue compartments Themechanism of action of simvastatin in progressive MS is thesubject of an ongoing mechanistic vascular perfusion study(OPT-MS NCT03896217) and the ultimate confirmation ofthe efficacy of simvastatin in SPMS awaits the results of theongoing phase 3 MS-STAT2 trial (NCT03387670) Our datatherefore suggest that caution is required when considering NfLas an outcome measure for treatments in progressive MS if themechanism of action is not known to directly affect neuroaxonalinjury such as with simvastatin

Although our results are not necessarily generalizable to otherneuroprotective treatments in PMS they are supported bydata from ibudilast The SPRINT-MS study demonstrated asignificant 48 reduction in the rate of brain atrophy in PMS

with ibudilast compared with placebo31 There was howeverno significant difference between the treatment groups in eitherserum or CSF NfL9 Although ibudilast is likely to have pleo-tropic effects (such as modulation of CNS innate immunitythrough inhibition of phosphodiesterases macrophage migra-tion inhibitory factor and Toll-like receptor 4) like simvastatinit is not thought to be systemically immunomodulatory32

NfL has shown utility as a biomarker of treatment with fin-golimod siponimod natalizumab and ocrelizumab in PMScohorts6-8 These treatments all share a predominantly im-munomodulatory mechanism of action and their ability toreduce NfL is therefore entirely in keeping with the knownassociation between NfL neuroaxonal injury and markers ofinflammatory activity in PMS5 Supporting this subgroupanalyses suggest that there may be a greater treatment effect ofnatalizumab siponimod and ocrelizumab on NfL in patientswith recent inflammatory activity6-8

The estimated treatment effects of simvastatin onNfL andNfHwere small and not statistically significant with the 95 CIsufficiently narrow to exclude an important treatment effect ineither direction Exploratory analyses did find that the month24 serum NfL level increased by 6 (95 CI 2 to 9) foreach newenlarging T2 lesion in the preceding year and by09 (95 CI 03 to 15) for each milliliter increase inconcurrent T2LV further supporting the known relationshipbetween NfL and neuroinflammation The key question of thisstudy however was to determine the extent to which serumneurofilaments may act as biomarkers of a neuroprotectivetreatment that does not appear to have direct effects on neu-roinflammation using simvastatin as our example Indeed NfLhas shown utility as a biomarker of noninflammatory neuro-degeneration and neuroprotection in other neurologicconditions3334 Our results however together with those fromSPRINT-MS suggest that either these treatments producebenefits on the rate of whole brain atrophy by mechanismsindependent of neuroaxonal injury or that the degree ofneuroprotection induced is insufficient to produce material

Table 4 MRI Predictors of Month 24 Serum NfL

Predictor variable

Separate model for each T2 lesion variable Combined mutually adjusted model

Increase in 24 mo NfL perunit increase in predictor 95 CI p Value

increase in 24 mo NfL perunit increase in predictor 95 CI p Value

Month 0ndash12 T2 newenlarging lesions (count)

323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001

Month 25 T2 lesionvolume (mL)

110 051 to 167 lt0001 089 029 to 149 0004

Abbreviation NfL = neurofilament lightResults are presented from the 3 separate models and then from the combined linear regression model with month 24 NfL out the outcome and T2 lesionvariables as predictors Results are from covariate adjusted models as indicated in the Methods section As previously NfL was log2 transformed T2 newenlarging lesions are reported as a count and T2 lesion volume in milliliters Coefficients are expressed as increase in 24month NfL per unit increase in T2lesion variables


changes in serum NfL We speculate that the latter may bedue to the association between neuroinflammation and NfLpersisting independent of such neuroprotective treatmentFuture work should focus on replicating this NfL analysis insamples from the ongoing phase 3 MS-STAT2 clinical trialonce the effects of simvastatin on clinical disability pro-gression are confirmed and also on developing novel CNSbiomarkers capable of capturing neuroprotective treatmenteffects independent of neuroinflammation

In 2 studies assessing the neuroprotective potential of sodiumchannel blockade (with phenytoin in acute optic neuritis andlamotrigine in SPMS) NfH has shown promise as a bio-marker of neuroprotective treatment1112 Our data howeverfound no evidence of a simvastatin treatment effect on NfH orany associations of NfH with MRI or clinical measures ofdisease severity Although previous studies have shown strongand consistent correlations between serum and CSF NfL35

inconsistent results have been found for correlation between

Table 5 Relationship Between Baseline Serum NfL and Serum NfH and Clinical Variables


EDSS score

Relationship with baseline EDSS score

Predictor variable



Relationship with change in the EDSS score (units per year)

Predictor variable



25FW

Relationship with baseline 25FW (1s)

Predictor variable

Baseline NfL (per doubling) minus0159 minus0390 to 0056 gt005


Relationship with change in 25FW (1s per year)

Predictor variable

Baseline NfL (per doubling) minus0183 minus0312 to minus0055 lt001


9HPT

Relationship with baseline 9HPT (1000s)

Predictor variable



Relationship with change in 9HPT (1000s per year)

Predictor variable



Abbreviations 9HPT = 9-hole peg test 25FW = timed 25-foot walk EDSS = Expanded Disability Status Scale NfH = neurofilament heavy NfL = neurofilamentlightThe results of 6 separate models are presented with EDSS 25FW or 9HPT as the dependent variables and NfL or NfH data as the predictor variables EDSSscore is reported as the score both 9HPT and25FWare reported as a speed (9HPT as 1000 times sminus1 and 25FWas sminus1) In all analyses neurofilament datawere log2transformed Results are from covariate-adjustedmodels as indicated in theMethods section pValue bounds (ltgt005 and ltgt001) can only be inferred fromthe 95 and 99 bias-corrected and accelerated cluster bootstrap (10000 replications) CIs


serum and CSF NfH36-38 This suggests that NfH instabilitybetween CSF and serum compartments may have limited itspotential as a biomarker in our cohort The Quanterix SimoaNF-Light Advantage assay has been widely used and validatedagainst clinical and MRI outcomes The Simoa pNF-heavyDiscovery assay however has been less widely used Onestudy has used this assay to demonstrate modest associationsbetween serum NfH and T2LV in a mixed MS cohort38

Although Simoa digital ELISA platforms tend to improvesensitivity and accuracy over traditional ELISA techniquesthe limited data from this NfH assay therefore suggest that theNfH data should be interpreted with caution

In conclusion our results show that despite simvastatin treatmentbeing associated with a significant reduction in whole brain at-rophy and benefits in secondary outcomes our results are mostcompatible with no important effect on serum NfL or NfH inSPMS Although higher NfL is associated with greater diseaseseverity and faster progression our results together with thosefrom ibudilast suggest that candidate nonimmunomodulatoryneuroprotective treatments in PMS may act via mechanisms in-dependent of the main determinants of serum neurofilamentconcentrations While confirmation of the neuroprotective effi-cacy of simvastatin in SPMS is awaited from the ongoing phase 3study our results together with those of others suggest that theutility of serum neurofilaments as biomarkers of treatment re-sponse in progressiveMSmay be limited to interventions that areeither known to suppress acute or chronic neuroinflammatoryactivity or to otherwise directly affect neuroaxonal injury

Study FundingNo targeted funding reported

DisclosureTE Williams has received honorarium for educational talksfrom Novartis and Merck KP Holdsworth JM Nicholas AEshaghi T Katsanouli H Wellington and A Heslegravereport no disclosures relevant to the manuscript H Zetter-berg has served at scientific advisory boards for Denali RocheDiagnostics Wave Samumed Siemens Healthineers PinteonTherapeutics Nervgen AZTherapies and CogRx has givenlectures in symposia sponsored by Cellectricon FujirebioAlzecure and Biogen and is a cofounder of Brain BiomarkerSolutions in Gothenburg AB (BBS) which is a part of the GUVentures Incubator Program C Frost reports no disclosuresJ Chataway has received support from the Efficacy andMechanism Evaluation Programme and Health TechnologyAssessment Programme (NIHR) UK Multiple SclerosisSociety the National Multiple Sclerosis Society and theRosetrees Trust He is supported in part by the NationalInstitute for Health Research University College LondonHospitals Biomedical Research Centre London UK He hasbeen a local principal investigator for a trial in MS funded bythe Canadian MS society a local principal investigator forcommercial trials funded by Actelion Biogen Novartis andRoche has received an investigator grant from Novartis andhas taken part in advisory boardsconsultancy for Azadyne

Biogen Celgene Janssen MedDay Merck NervGenNovartis and Roche Go to NeurologyorgNN for fulldisclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJune 16 2021 Accepted in final form November 23 2021

References1 Lublin FD Reingold SC Cohen JA et al Defining the clinical course of multiple

sclerosis Neurology 201483(3)278-2862 Chataway J Schuerer N Alsanousi A et al Effect of high-dose simvastatin on

brain atrophy and disability in secondary progressive multiple sclerosis (MS-STAT) a randomised placebo-controlled phase 2 trial Lancet 2014383(9936)2213-2221

3 Chan D Binks S Nicholas JM et al Effect of high-dose simvastatin on cognitiveneuropsychiatric and health-related quality-of-life measures in secondary progressivemultiple sclerosis secondary analyses from the MS-STAT randomised placebo-controlled trial Lancet Neurol 201716(8)591-600

Appendix Authors

Name Location Contribution

Thomas EWilliams BAMB BChirMRCP

Queen Square MultipleSclerosis Centre UCLQueen Square Institute ofNeurology UnitedKingdom

Acquisition of patientsamples neurofilamentlaboratory analysis dataanalysis and interpretationand drafting of themanuscript and revisions

Katherine PHoldsworthMSc

London School of Hygieneand Tropical MedicineUnited Kingdom

Data analysis andinterpretation andmanuscript revisions

Jennifer MNicholas PhD



ArmanEshaghi PhD


MRI analysis andmanuscript revisions

TheodoraKatsanouliMSc



HenriettaWellingtonPhD FRCP

UK Dementia ResearchInstitute at UCL

Supervision ofneurofilament laboratoryanalysis and manuscriptrevisions

AmandaHeslegravePhD



HenrikZetterbergPhD


Oversight of neurofilamentlaboratory analysis dataanalysis and interpretationand manuscript revisions

Chris FrostPhD


Supervision of data analysisand interpretation andmanuscript revisions

JeremyChatawayPhD FRCP


Chief investigator of the MS-STAT clinical trial studydesign and conception datainterpretation andmanuscript revisions


4 Sormani MP Haering DA Kropshofer H et al Blood neurofilament light as a po-tential endpoint in Phase 2 studies in MS Ann Clin Transl Neurol 20196(6)1081-1089

5 Williams T Zetterberg H Chataway J Neurofilaments in progressive multiple scle-rosis a systematic review J Neurol 2021268(9)3212-3222

6 Bar-Or A Thanei GA Harp CT et al Blood neurofilament light levels are lowered toa healthy donor range in patients with RMS and PPMS following ocrelizumabtreatment Presented at the 35th Congress of ECTRIMS 2019 Stockholm

7 Kapoor R Sellebjerg F Hartung HP et al Natalizumab reduced serum levels ofneurofilament light chain in secondary progressive multiple sclerosis patients fromthe phase 3 ASCEND study Presented at the 34th Congress of ECTRIMS 2018Berlin

8 Kuhle J Kropshofer H Haering D et al Neurofilament light levels in the blood ofpatients with secondary progressive MS are higher than in primary progressive MSand may predict brain atrophy in both MS subtypes Presented at the 34th Congressof ECTRIMS 2018 Berlin

9 Fox R Raska P Barro C et al Neurofilament light chain in a phase 2 clinical trial ofibudilast in progressive multiple sclerosis Mult Scler 202127(13)2014-2022

10 Kapoor R Smith KE Allegretta M et al Serum neurofilament light as a biomarker inprogressive multiple sclerosis Neurology 202095(10)436-444

11 Gnanapavan S Grant D Morant S et al Biomarker report from the phase II lamo-trigine trial in secondary progressive MSmdashneurofilament as a surrogate of diseaseprogression PLoS One 20138(8)e70019

12 Raftopoulos R Kuhle J Grant D et al Neurofilament results for the phase IIneuroprotection study of phenytoin in optic neuritis Eur J Neurol 202128(2)587-594

13 Quanterix Simoa NF-light Advantage Kit HD-1HD-X data sheet Epub 20171-214 Quanterix Simoa pNF-heavy Discovery Kit HD-1HD-X data sheet 20171-215 Eshaghi A Kievit RA Prados F et al Applying causal models to explore the mech-

anism of action of simvastatin in progressive multiple sclerosis Proc Natl Acad SciUSA 2019116(22)11020-11027

16 Liu G Lu K Mogg R Mallick M Mehrotra D Should baseline be a covariate ordependent variable in analyses of change from baseline in clinical trials Stat Med200928(20)2509-2530

17 European Medicines Agency Statistical principles for clinical trials Eur Med Agency19985(7)29

18 Matthews J Altman D Campbell M Royston P Analysis of serial measurements inmedical research Br Med J 1990300(6725)230-235

19 Frost C Kenward MG Fox NC The analysis of repeated ldquodirectrdquomeasures of changeillustrated with an application in longitudinal imaging Stat Med 200423(21)3275-3286

20 Barkhof F Simon JH Fazekas F et al MRI monitoring of immunomodulation inrelapse-onset multiple sclerosis trials Nat Rev Neurol 20128(1)13-21

21 World Medical Association Declaration of Helsinki ethical principles for medical re-search involving human subjects 2013 Accessed January 2021 wmaneten30publi-cations10policiesb317cpdf

22 Maggi P Kuhle J Schadelin S et al Chronic white matter inflammation and serumneurofilament levels in multiple sclerosis Neurology 202197(6)e543ndashe553

23 Ontaneda D Thompson AJ Fox RJ Cohen JA Progressive multiple sclerosisprospects for disease therapy repair and restoration of function Lancet 2017389(10076)1357-1366

24 Palladino R Marrie RA Majeed A Chataway J Evaluating the risk of macrovascularevents and mortality among people with multiple sclerosis in England JAMA Neurol202077(7)820-828

25 Marrie RA Rudick R Horwitz R et al Vascular comorbidity is associated with morerapid disability progression in multiple sclerosis Neurology 201074(13)1041-1047

26 Jakimovski D Gandhi S Paunkoski I et al Hypertension and heart disease areassociated with development of brain atrophy in multiple sclerosis a 5-year longi-tudinal study Eur J Neurol 201926(1)87-e8

27 Sormani MP Arnold DL De Stefano N Treatment effect on brain atrophy correlateswith treatment effect on disability in multiple sclerosisAnn Neurol 201475(1)43-49

28 Sastre-Garriga J Pareto D Battaglini M et al MAGNIMS consensus recommenda-tions on the use of brain and spinal cord atrophy measures in clinical practiceNat RevNeurol 202016(3)171-182

29 Cifelli A Arridge M Jezzard P Esiri MM Palace J Matthews PM Thalamic neuro-degeneration in multiple sclerosis Ann Neurol 200252(5)650-653

30 Popescu V Klaver R Voorn P et al What drives MRI-measured cortical atrophy inmultiple sclerosis Mult Scler 201521(10)1280-1290

31 Fox RJ Coffey CS Conwit R et al Phase 2 trial of ibudilast in progressive multiplesclerosis N Engl J Med 2018379(9)846-855

32 Barkhof FHulstHEDrulovic JUitdehaagBMJMatsudaK LandinR Ibudilast in relapsing-remitting multiple sclerosis a neuroprotectant Neurology 201074(13)1033-1040

33 Bridel C Van Wieringen WN Zetterberg H et al Diagnostic value of cerebrospinalfluid neurofilament light protein in neurology a systematic review and meta-analysisJAMA Neurol 201976(9)1035-1048

34 Olsson B Alberg L Cullen NC et al NFL is a marker of treatment response inchildren with SMA treated with nusinersen J Neurol 2019266(9)2129-2136

35 Novakova L Zetterberg H Sundstrom P et al Monitoring disease activity in multiplesclerosis using serum neurofilament light proteinNeurology 201789(22)2230-2237

36 Vorobyeva A Fominykh V Zakharova M Biochemical markers of neurodegenerationin multiple sclerosis Mult Scler 201319(11)369

37 Eikelenboom MJ Uitdehaag BMJ Petzold A Blood and CSF biomarker dynamics inmultiple sclerosis implications for data interpretation Mult Scler Int 201120111-6

38 Verberk I Koel-Simmelink M Twaalfhoven H et al Ultrasensitive immunoassayallows measurement of serumneurofilament heavy in multiple sclerosis Mult SclerRelat Disord 202150102840


DOI 101212NXI000000000000113020229 Neurol Neuroimmunol Neuroinflamm

Thomas E Williams Katherine P Holdsworth Jennifer M Nicholas et al Sclerosis From the MS-STAT Randomized Controlled Trial

Assessing Neurofilaments as Biomarkers of Neuroprotection in Progressive Multiple

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

Classification of EvidenceThis study provides class I evidence that simvastatin treatment does not have a large impact on either serum NfL or NfH asquantified in this study in SPMS

Few treatments are available for progressive multiple sclerosis(PMS) with most restricted to those with active inflammatorydisease Treatments for nonactive PMS1 may require an alter-native approach utilizing neuroprotective mechanisms alone orin combination with immunomodulation One such candidateneuroprotective treatment is simvastatin which in the MS-STAT trial demonstrated a 43 reduction in annualized brainatrophy in addition to benefits on secondary physical and cog-nitive outcomes No differences were noted on a panel of im-munologic markers supporting a neuroprotective rather thanimmunomodulatory mechanism23 The phase 3 MS-STAT2study is ongoing (NCT03387670)

Blood neurofilament light (NfL) is a biomarker of neuroaxonalinjury and an appealing surrogate outcome measure for trials inrelapsing-remitting MS4 It is less well studied in PMS5 Initia-tion of immunosuppressive disease-modifying treatment is as-sociated with a reduction in blood NfL in PMS cohorts but animportant question remains over the ability of NfL to capturethe treatment effect of purportedly neuroprotective therapies6-9

Clarifying this is an important goal of the progressive MS alli-ance with implications on future phase 2 trial design10 Data forblood neurofilament heavy (NfH) are more limited but it mayhave utility as a biomarker of neuroprotection1112

Here we analyze serumNfL andNfH from theMS-STAT trialWe use the known positive effect of simvastatin on whole brainatrophy to interrogate our primary research question whetherserum NfL and NfH may act as biomarkers of neuroprotectivetreatment with simvastatin in secondary progressive MS(SPMS) We additionally perform further analyses to examinethe relationships between serum neurofilaments and estab-lished MRI and clinical variables

MethodsMS-STAT Trial and Sample ProcessingThe MS-STAT study protocol has been outlined previously2

Briefly patients with SPMS aged 18ndash65 years with ExpandedDisability Status Scale (EDSS) score 40ndash65 were eligible Keyexclusion criteria included primary progressive MS relapse orsteroid use within 3 months and the use of immunosuppressiveor disease-modifying therapy within the last 6 months A total of

140 patients were randomized 11 to simvastatin 80 mg orplacebo Baseline characteristics are shown in Table 1 Bloodsamples were acquired at baseline and months 6 12 and 24Serum was separated and stored at minus80degC until time of analysis

Neurofilament QuantificationNfL and NfH are components of a neurone-specific in-termediate filament differing in their C-terminal domain andphosphorylation state Both are released following neuro-axonal injury into the CSF and blood where they may bequantified Serum NfL and NfH were measured by Simoatechnology on a HD-1 analyzer according to the manufac-turerrsquos instructions (Quanterix Billerica MA) The SimoaNF-Light Advantage and Simoa pNF-heavy Discovery Kits(Quanterix) were used Briefly serum samples were thawed at21degC vortexed and centrifuged at 10000 RCF for 5 minutesat 21degC On-board the HD-1 samples were diluted 14 withsample diluent and bound to paramagnetic beads coated witha capture antibody specific for human NfL or NfH Antibody-coated beads were incubated with a biotinylated anti-NfL oranti-NfH detection antibodies that in turn were labeled with astreptavidin-β-galactosidase complex Following the additionof the β-galactosidase substrate resorufin β-D-galactopyrano-side a fluorescent signal proportional to the concentration ofneurofilament present in the sample was generated in theantigen-containing microwells of the Simoa plates

Duplicate measurements were taken of each sample Sampleconcentrations were extrapolated from a standard curve fittedusing a 4-parameter logistic algorithm The lower limit ofquantification (LLoQ) for NfL is 0174 pgmL and for NfH is288 pgmL1314 Values below the LLoQ were assigned thevalue of half the LLoQ The coefficient of variation (CoV)between sample replicates tends to be higher for lower valueresults To avoid bias all data were therefore included in theprimary statistical analysis regardless of the CoV Each assaywas run in the same or consecutive batches by the sameoperator who was blinded to treatment allocation

MRI ProcessingThe imaging data have been previously published and wereacquired as previously described215 Briefly 3D T1-weighteddouble-echo proton density and T2-weighted MRI wasobtained at baseline month 12 and month 25 Whole brain

Glossary9HPT = 9-hole peg test 25FW = 25-foot timed walkCoV = coefficient of variation EDSS = Expanded Disability Status ScaleIQR = interquartile range LLoQ = lower limit of quantificationMS = multiple sclerosis NfH = neurofilament heavy NfL =neurofilament light PMS = progressive MS SPMS = secondary progressive MS T2LV = T2 lesion volume









Sex n () female 48 (69) 49 (70) 97 (69)



Age y mean (SD) 511 (68) 515 (70) 513 (69)





Serum NfL pgmL


N 63 65 128


N 53 59 112


N 53 59 112


N 48 64 112

Serum NfH pgmL


N 63 62 125


N 49 53 102


N 52 57 109


N 48 64 112












Serum NfL (pgmL)


Month 12 535 minus74 to 199 0429




Serum NfH (pgmL)


Month 12 474 minus151 to 293 0666

Month 24 169 minus184 to 267 0881
























Whole brain atrophy


Predictor variable





T2 lesion volume


Predictor variable






Predictor variable






















Predictor variable





323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001


110 051 to 167 lt0001 089 029 to 149 0004








EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints











Sex n () female 48 (69) 49 (70) 97 (69)



Age y mean (SD) 511 (68) 515 (70) 513 (69)





Serum NfL pgmL


N 63 65 128


N 53 59 112


N 53 59 112


N 48 64 112

Serum NfH pgmL


N 63 62 125


N 49 53 102


N 52 57 109


N 48 64 112












Serum NfL (pgmL)


Month 12 535 minus74 to 199 0429




Serum NfH (pgmL)


Month 12 474 minus151 to 293 0666

Month 24 169 minus184 to 267 0881
























Whole brain atrophy


Predictor variable





T2 lesion volume


Predictor variable






Predictor variable






















Predictor variable





323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001


110 051 to 167 lt0001 089 029 to 149 0004








EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints













Serum NfL (pgmL)


Month 12 535 minus74 to 199 0429




Serum NfH (pgmL)


Month 12 474 minus151 to 293 0666

Month 24 169 minus184 to 267 0881
























Whole brain atrophy


Predictor variable





T2 lesion volume


Predictor variable






Predictor variable






















Predictor variable





323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001


110 051 to 167 lt0001 089 029 to 149 0004








EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints























Whole brain atrophy


Predictor variable





T2 lesion volume


Predictor variable






Predictor variable






















Predictor variable





323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001


110 051 to 167 lt0001 089 029 to 149 0004








EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints



















Predictor variable





323 029 to 626 0031 minus015 minus308 to 286 0919


676 345 to 1018 lt0001 573 240 to 917 0001


110 051 to 167 lt0001 089 029 to 149 0004








EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints









EDSS score


Predictor variable




Predictor variable



25FW


Predictor variable




Predictor variable



9HPT


Predictor variable




Predictor variable
















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints















Appendix Authors











ArmanEshaghi PhD









AmandaHeslegravePhD



HenrikZetterbergPhD



Chris FrostPhD
























































Reprints





















































Reprints




assessing neurofilaments as biomarkers of neuroprotection

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