ARTICLE OPEN ACCESS CLASS OF EVIDENCE

High serum neurofilament light chain normalizesafter hematopoietic stem cell transplantationfor MSSimon Thebault MD Daniel R Tessier PhD Hyunwoo Lee PhD Marjorie Bowman MScN Amit Bar-Or MD

Douglas L Arnold MD Harold L Atkins MD Vincent Tabard-Cossa PhD and Mark S Freedman MD

Neurol Neuroimmunol Neuroinflamm 20196e598 doi101212NXI0000000000000598

Correspondence

Dr Thebault

sthebaulttohca

AbstractObjectiveTo evaluate neurofilament light chain (NfL) levels in serum and CSF of patients with aggressiveMS pre- and post-treatment with immunoablation followed by autologous hematopoietic stemcell transplantation (IAHSCT) and examine associations with clinical and MRI outcomes

MethodsPaired serum and CSF in addition to MRI and clinical measures were collected on 23 patientswith MS at baseline and 1 and 3 years post-IAHSCT An additional 33 sera and CSF pairs weretaken from noninflammatory neurologic controls NfL levels were quantitated using the Simoaplatform (Quanterix)

ResultsBaseline MS NfL levels were significantly elevated relative to controls in serum (p = 0001) andCSF (p = 0001) Following IAHSCT high pretreatment NfL levels significantly reduced inserum (p = 00023) and CSF (p = 00068) and were not significantly different from controlsSerum and CSF NfL levels highly correlated (r = 081 p lt 00001) Baseline NfL levels wereassociated with worse pretreatment disease measures (Expanded Disability Status Scale[EDSS] relapses MRI lesions and MR spectroscopy (MRS) N-acetylaspartatecreatine)Elevated baseline NfL levels were associated with persistently worse indices of disease burdenpost-IAHSCT (sustained EDSS progression cognition quality of life T1 and T2 lesion vol-umes MRS and brain atrophy)

ConclusionThese data substantiate that serum and CSF NfL levels reflect disease severity and treatmentresponse in patients with MS and may therefore be a useful biomarker Baseline serum levelsassociated with markers of pretreatment disease severity and post-treatment outcomes

Classification of evidenceThis study provides Class II evidence that for patients with aggressive MS serumNfL levels areassociated with disease severity

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EditorialBlood neurofilament lightchain at the doorstep ofclinical application

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From the The University of Ottawa and Ottawa Hospital Research Institute (ST MB HLA MSF) Department of Physics (DRT VT-C) University of Ottawa MontrealNeurological Institute (HL DLA) and Perelman School of Medicine and University of Pennsylvania (AB-O)

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 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

MS is known for its diverse clinical presentation with somepatients presenting early with minimal disease and otherspresenting late with considerable damage from silent disease1

There are now many therapeutic options with differingmechanisms of action efficacy and safety profiles An annualMRI scan is recommended as a ldquostandard of carerdquo in the as-sessment of ongoing disease activity2 but this is a costly time-consuming test and is impractical in areas where access to MRIis limited MRI is also semiquantitative with significant inter-scan variability on routine scanning It has limited sensitivity forpredicting disease progression34 and is not recommended formonitoring treatment response2 Routine MRI scanning alsoignoresmost of the spinal cord (inmany cases the cervical cordis examined) spinal cord disease more closely correlates withExpanded Disability Status Scale (EDSS) progression espe-cially as the disease advances5 As such there is an unmet needfor alternative biomarkers to help clinicians prognosticatemonitor for disease activity and assess treatment responseSerum neurofilament light chain (NfL) a neuroaxonal in-termediate protein is a promising biomarker candidateAdvances in highly sensitive detection methodologies using

single-molecule counting tomeasure biomarker concentrationsdown to the femtomolar levels have enabledNfL quantificationin plasma or serum67 Serum NfL has been closely correlatedwithCSF levels Elevated serum levels in patients withMSdropwhen MS activity seems controlled by a variety of MS treat-ments8 and may even be of prognostic value9ndash11 Many hopethat the precise quantification of this abundant CSF biomarkerin the serum could become a convenient objective mainstay ofMS monitoring and prognostication

Immunoablation followed by autologous hematopoietic stemcell transplantation or IAHSCT (previously referred to asautologous bone marrow transplantation) is an intensive yetefficacious therapy reserved for particularly aggressive cases ofMS The rigorous regimen adopted in Canada involvesmyeloablative doses of chemotherapy and antithymocyteglobulin followed by autologous hematopoietic stem celltransplants Using this approach we have reported thatpatients with MS experience a robust treatment response(stabilization or improvement in most) in the absence ofongoing disease-modifying treatments (DMTs) following

GlossaryDMT = disease-modifying treatment EDSS = Expanded Disability Status Scale FIS = Fatigue Impact Score IAHSCT =immunoablation followed by autologous hematopoietic stem cell transplantation MRS = MR spectroscopy MSQOL = MSQuality of Life NAACr = N-acetylaspartatecreatine NfL = neurofilament light chain RRMS = relapsing-remitting MSSPMS = secondary progressive MS VOI = volume of interest

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 NeurologyorgNN

IAHSCT12 These patients had aggressive disease as definedby frequent disabling relapses and early rapid progressiondespite reasonable trials of DMT Following IAHSCT theyhad complete suppression of focal white matter inflammatorydisease activity as measured by MRI and no clinical relapsesafter more than a decade of follow-up Despite the lack ofongoing inflammatory disease some patients continued toshow disability progression andMRI atrophy We consideredtherefore that this unique cohort of patients might be ofinterest to study the prognostic and therapeutic utility ofmeasuring NfL levels as a baseline marker of disease severityas a marker of treatment response and possibly a predictor ofpost-treatment outcomes

MethodsPatients with aggressive MS undergoingIAHSCTThe study has been previously described in detail12 but brieflywas a phase II nonrandomized control study of IAHSCT (n =24) vs best available treatment (n = 8) performed at 3 hos-pitals in Canada in patients with aggressive MS who hada poor predicted 10-year prognosis1314 (ClinicalTrialsgovNCT01099930) Detailed follow-up of the initial study in-cluded predefined and scheduled clinical laboratory andMRIexaminations starting 5 months before the IAHSCT andcontinuing for at least 3 years The study was then extendedfor monitoring alone for an additional 5 years and beyond

Available data included demographics HLA risk allele sub-type longitudinal clinical measures (number of clinicalrelapses EDSSMSQuality of Life [MSQOL-54] physical andmental scores cognitive Paced Auditory Serial Addition Testscore and Fatigue Impact Score [FIS]) longitudinal serologicmeasures (oligoclonal bands immunoglobulin G index andimmunoglobulin G-albumin ratio) and longitudinal MRImeasures (T1-weighted sequences pre- and post-gadolinium[Gd] T2-weighted sequences MR spectroscopy [MRS]N-acetylaspartatecreatine [NAACr] ratio and brain volumemetrics) Two pretreatment MRI scans were performed witha minimum interval of 2 months at 5 months and 3 monthsbefore AHSCT Post-treatment MRI time points were 1 2 46 9 and 12 months then every 6 months until 3 yearsthereafter every 12 months maximum duration 105 years

Paired sera and CSF were available for 23 patients at baseline(at the point of enrollment into the study) 2023 patients at 1year and 1823 at 3 years after treatment Samples had beenstored and aliquoted at minus80degC to avoid freeze-thaw cyclesComplete serum-CSF paired samples were available in 2323patients at baseline

Noninflammatory controlsThirty-three patients were identified with noninflammatoryillnesses with available serum and CSF obtained with appro-priate consent

Research questions (level II evidence)Primary research question In this cohort of patients withaggressive MS treated with IAHSCT do levels of serum NfLreduce following treatment

Secondary research question Are pre- and post-treatmentserum NfL levels associated with clinical and MRI indicatorsof disease severity and treatment response

Neurofilament quantificationNfL levels in each serumCSF were quantified in duplicate inaccordance with manufacturersrsquo instructions with appropriatestandards and internal controls Quanterix commerciallyavailable NfL immunoassay kits (cat103186) were run in thefully automated ultrasensitive Simoa HD-1 Analyzer(Quanterix)

MRI analyses

T1 and T2 lesion volumesMRI lesion-based measurements included number and vol-ume of T1-weighted lesions (precontrast) number andvolume of gadolinium-enhancing (Gd) lesions (T1 post-contrast) number of new or enlarging T2-weighted lesionsand volume of T2-weighted lesions

Atrophy (whole brain gray matter and white matter)Longitudinal brain volume changes (atrophy) were calculatedin individual patients using pairs of precontrast T1-weightedimages All measurements were made with respect to thebaseline MRI scans Percentage whole-brain volume changeswere calculated using FSL-Structural Image Evaluation usingNormalisation of Atrophy15 Gray and white matter volumechanges were separately calculated using Jacobian integrationwhich is a nonlinear registration-based method16

MRS NAACrMRS examinations were obtained using a Philips 15TGyroscan ACS II scanner that was later changed to a Siemens15T Sonata scanner The spectroscopic volume of interest(VOI) consisted of a 15-mm thick slab centered on the corpuscallosum and angulated parallel to the callosal line The meanNAACr was calculated within the spectroscopic VOIs

Statistical analysesData analysis was performed using Graphpad Prism 5 soft-ware For all analyses a 95 significance level was used andp values were 2 tailed Complete serum-CSF paired sampleswere available in 2323 patients at baseline 2023 at 1 yearand 1823 at 3 years The EDSS data set was complete exceptfor 2 patients who were lost to follow-up at 24 months and36 months post-IAHSCT respectively Complete MRI datawere available on 2323 patients at baseline 2023 by12 months and 1823 by 36 months The last-observation-carried-forward method was used to impute the missing EDSSand MRI data To compare NfL levels at baseline 1 and 3years 1-way analysis of variance was used with Bonferronimultiple comparison test For comparisons between 2 groups

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 3

paired and unpaired T tests were used as appropriate Toassess for correlations of baseline serum NfL levels and othervariables Pearson (parametric) and Spearman (non-parametric) correlation were selected as indicated by theDrsquoAgostino-Pearson omnibus K2 test and linear regressionused to generate a best-fit line To allow binary comparisonsof patients with ldquohighrdquo compared with ldquolowrdquo baseline NfLlevels and outcomes the median baseline serumNfL level wasselected as a cutoff patients with an NfL level greater than themedian were considered ldquohighrdquo and patients with a baselineserum NfL less than the median were considered ldquolowrdquoSubsequent comparisons of ldquohighrdquo vs ldquolowrdquo NfL and theirinteraction with clinical and MRI variables over time wereperformed using 2-way repeat-measures (mixed model)analysis of variance or survival curves and applying the log-rank (Mantel-Cox) test All statistics were performed in du-plicate for consistency

Registrations patient consents and approvalsEthics and protocols in this study had been approved byRegional Ethics Boards of participating institutions as part ofthe original study ClinicalTrialsgov Identifier NCT0109993012

All participants had given informed written consent

Data availabilityIndividual deidentified participant data from this study will beshared on written request Requests to access the data will beconsidered on request to the corresponding author

ResultsBaseline characteristics of IAHSCTpatients andnoninflammatory controlsBaseline characteristics of patients with aggressive MS areoutlined in table 1

Of the 33 noninflammatory controls 24 were women and 9were men The mean age at the time of sampling was 375years SD 109 years Following exclusion of inflammatorydegenerative neurologic illnesses by neurologist assessmentMRI and CSF analysis primary diagnoses were as followssomatization (9) migraine (7) chronic fatigue syndrome (6)fibromyalgia (5) anxiety (3) labyrinthitis (2) and Bell palsy(commonly known as Bellrsquos palsy) (1)

Serum and CSF NfL levels following IAHSCTSerum NfL levels (figure 1A) were high in the patients withaggressive MS at baseline (mean 3106 pgmL median 2824pgmL SD 2501 pgmL) relative to noninflammatory con-trols (mean 820 pgmL SD 390 pgmL) The differenceswere significant (p = 00001 95 CI for the difference1222ndash3349 pgmL) Levels reduced significantly at 12months (mean 1439 pgmL SD 594 pgmL p = 00023 CI519ndash2814 pgmL) and remained low at 36 months (mean1092 pgmL SD 463 pgmL) following IAHSCT Levels at12 and 36 months did not differ significantly from eachother and were not significantly different from non-inflammatory controls These results were similar to those

seen in the CSF albeit the latter showed much higher levels(figure 1B mean for baseline group 2248 pgmL SD 1897pgmL p value for difference between baseline and controls= 00331 p value for difference between baseline and 12months = 00068) Correspondingly serum and CSF NfLlevels correlated (figure 1C Spearman r = 076 [056ndash089]p = 00001) Despite the significant reduction in serum NfLlevels post-IAHSCT baseline serum levels correlatedweakly with post-treatment serum levels at 12 months (r =05 p = 0015) and 36 months (r = 060 p = 00083) (datanot shown)

Baseline serum NfL levels compared withpre-IAHSCT clinical and MRI findingsThe mean number of days between baseline serumCSFsampling and the IAHSCT induction was 129 SD 121 daysThe mean number of days between baseline serologic sam-pling and comparator MRI was 41 SD 255 days

Although patients were all considered to have aggressiveMS at baseline for inclusion in the study patients withEDSS scores ge6 had higher levels of NfL (n = 11 mean4319 SD 2498 pgmL) compared with those with anEDSS of lt6 (n = 12 mean 1994 SD 2004 pgmL) (figure2A p = 0022) In addition patients who had experienceda clinical relapse in the 12 months before baseline samplingalso had had higher NfL levels (n = 9 mean 4462 SD3368 pgmL) compared with those who had not hada clinical relapse (n = 13 mean 2324 SD 123) (figure 2Bp = 0047)

Spearman correlations between baseline serum NfL and MRImeasures are summarized in table 2

Increased serum NfL levels correlated with increased lesion-basedMRI markers including T2 lesion volume (figure e-1AlinkslwwcomCPJA114 p = 00001 r = 074) the number

Table 1 Canadian IAHSCT cohort

Sex male female 914

MS subtype RRMS SPMS 11 12

Age at onset mean (SD) 26 (7)

Age at diagnosis mean (SD) 27 (6)

Onset to EDSS score 3 mean mo (SD) 48 (39)

Onset to IAHSCT mean mo (SD) 89 (46)

Last EDSS score before IAHSCT median (range)

All 5 (3ndash65)

RRMS 4 (3ndash65)

SPMS 6 (4ndash6)

Abbreviations SPMS = secondary progressive MS RRMS = relapsing-re-mitting MS EDSS = Expanded Disability Status Scale IAHSCT = autologoushematopoietic stem cell transplantation

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 NeurologyorgNN

of newenlarging T2 lesions (p = 00189 r = 060 figuree-1B linkslwwcomCPJA114) and T1 lesion volumes (p= 00247 r = 050 figure e-1C linkslwwcomCPJA114)There was a negative correlation between serumNfL and theMRS marker of health tissue NAACr ratio (figure e-1ClinkslwwcomCPJA114 p = 00111 r = minus06614) Therewas no significant correlation between baseline NfL levelsand sex age at onset of MS clinical subtype (relapsing-remitting MS [RRMS] vs secondary progressive MS

[SPMS]) and baseline number and volume of Gd-enhancing lesions

Baseline serumNfL levels comparedwith post-IAHSCT clinical and MRI findingsTo enable a binary comparison of NfL levels the baselinegroup was divided into ldquohighrdquo and ldquolowrdquo levels using themedian to establish a cutoff (2824 pgmL) Baselinesamples with NfL gt2824 were considered ldquohighrdquo (n = 11)

Figure 1 Serum and CSF neurofilament levels following Immunoablative Autologous Haematopoietic StemCell Transplant

Baseline NfL levels (n = 23) were significantly ele-vated relative to controls (n=33) in (A) serum (p =00001 95 CI 1333-3238) and (B) CSF (p = 0000195 CI 1122-2638 ) Following IAHSCT levels signif-icantly reduced in both compartments (serump=00023 95 CI 639-2694 CSF p = 00068 95 CI5220-2141) Post-ASCT NfL levels (12 and 36m) inboth serum and CSF were not significantly differentfrom controls (p ge 005) Serum and CSF NfL levelswere c correlated (p = 00001 Spearman r = 076)12m 12 months post IAHSCT 36m 36 months postIAHSCT Error bars represent the standard error ofthe mean CI = confidence interval IAHSCT =immunoablation followed by autologous hemato-poietic stem cell transplantation NfL = neurofila-ment light chain

Figure 2 Baseline clinical status of patients and serum NfL levels

Patients who had a baseline NfL greater than the median value(gt2824 pgml-1 red line n = 11) had rapidly reached EDSS score of6 prior to IAHSCT (A) median duration 691 years compared topatients with lower levels (le2824 pgml-1 blue line n = 12) Nineof the 23 patients had clinical relapses in the 12 months prior toIAHSCT On average these patients had higher baseline serumNfL compared to the 13 patients who had not (p = 0047) Errorbars represent the standard error of themean EDSS = ExpandedDisability Status Scale IAHSCT = immunoablation followed byautologous hematopoietic stem cell transplantation NfL = neu-rofilament light chain

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 5

and samples with le 2824 pgmL were considered low(n = 12)

Following IAHSCT none of the 23 patients with previouslyaggressive MS had further clinical relapses or newMRI lesionsAlthough EDSS scores in most patients remained stable or

even improved following treatment 723 patients experiencedsustained accumulation of additional disability Sustained pro-gression was defined as an increase in the EDSS score observedon at least 2 occasions more than 6months apart in the absenceof new relapse or new MRI lesions In these 7 patients sus-tained progression occurred in the first 2 years followingtreatment Deteriorations were nonspecific and by functionalsystem included visual (n = 1) brainstem (n = 5) pyramidal(n = 4) cerebellar (n = 3) sensory (n = 3) bowel and bladder(n = 4) and mental (n = 2) Six of 7 patients with sustainedprogression had high baseline serum NfL levels (gt2824 pgmL the median baseline value) Conversely 112 patients withlow baseline NfL levels experienced EDSS progression (figure3A p = 0013 log-rank Mantel-Cox test)

Using the total scores from the Paced Auditory Serial Addi-tion test as a surrogate for cognition we found that patientswith high NfL consistently had worse cognitive scores both atbaseline and following treatment (figure 3B p = 00009) Theinteraction of cognitive scores over time between high andlow NfL groups was not significant indicating that the ob-served differences post-treatment are attributable to baselinedifferences post-treatment cognitive scores between thegroups paralleled each other Similarly patients with baseline

Table 2 Spearman correlation between baseline serumNfL and baseline MRI findings

Correlation coefficient(95 CI) p Value

T2 lesion volume 074 (044 to 089) lt00001

MRI spectroscopy NAACrratio

minus069 (minus090ndash to minus023) 00065

No of newenlarging T2 060 (008 to 078) 00189

T1 lesion volume 050 (007 to 078) 00247

Gd lesion number 034 (minus010 to 069) 01109 (ns)

Gd lesion volume 026 (minus019 to 062) 01490 (ns)

Abbreviations NAACr = N-acetylaspartatecreatine NfL = neurofilamentlight chain

Figure 3 Baseline NfL levels and post IAHSCT clinical outcomes

A baseline serum Nf-L greater than the median value (gt2824 pgml-1 red line n = 11) compared to less than the median (lt2824 pgml-1 blue line n = 12) wasassociated with (A) a greater chance of experiencing sustained progression (p = 0029 Kaplan-Meir Log-rank test) (B) worse cognitive outcomes (p = 0009)and (C) worse quality of life scores (p = 0001) after IAHSCT There were no differences in (D) fatigue scores following AHSCT All interactions were non-significant indicating that differences where seen were attributable to baseline differences between the high and low NfL groups Error bars represent thestandard error of themean IAHSCT = immunoablation followed by autologous hematopoietic stem cell transplantation NfL = neurofilament light chain ns =non-significant

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 NeurologyorgNN

NfL had persistently worse quality of life as indicated by theMSQOL-54 in physical (figure 3C p = 00001) but notmental score (data not shown) Again the interaction was notsignificant indicating that post-treatment differences arelikely related to baseline differences Although patients withhigh NfL at baseline had significantly more fatigue (measuredby the Fatigue Impact Scale) at baseline compared with thosewith low NfL levels (p = 0048 data not shown) followingIAHSCT fatigue dropped to similar levels in patients withhigh and low baseline NfL levels and there were no significantdifferences seen between the lines (figure 3D)

High NfL at baseline was associated with increased T1lesion volumes (p = 00017 figure 4A) T2 lesion volumes(p = 00009 figure 4B) and a lower MRS NAACr ratio (p= 00038 figure 4C) after IAHSCT The interaction overtime of post-IAHSCT T1 lesion volumes between high andlow NfL groups was significant (p = 00060 figure 4A)ie patients with high NfL at baseline had more ongoing T1lesion volume accrual post-IAHSCT compared with thosewho had low NfL levels Although significant this in-teraction was mild accounting for 09 of the variationHigher baseline NfL was also associated with and increasedwhole-brain atrophy (p value for high vs low NfL = 0029figure 5A) which was sustained at a higher rate aftertreatment (p value for the interaction 00008 accounting

for 351 of the variation) This atrophy was greatest inwhite matter (NfL p = 00043 interaction p = 00001variation 548 figure 5B) compared with the gray matteratrophy where differences were accounted for baselinedifferences (NfL p = 0021 insignificant interactionfigure 5C)

Post-IAHSCT serum NfL levels compared withpost-IAHSCT clinical and MRI findingsFollowing IAHSCT none of the patients experienced anynew relapses or evidence of ongoing MRI lesion activityNonetheless we found that 12-month NfL levels were higherin the 7 patients who experienced sustained progression(mean 186 pgmL SD 66) compared with those whoremained stable or improved (figure e-2A linkslwwcomCPJA114 mean 126 pgmL SD 48 p = 0029) In additionthere was also a positive correlation of 12-month serum NfLand 12-month white matter atrophy (figure e-2B linkslwwcomCPJA114 p = 0017 Spearman r = 0494) and a weakerassociation with whole-brain and gray matter atrophy whichdid not reach significance There was no correlation with 12-month NfL and T1T2 lesion volumes MRI spectroscopyNAACr or any of the clinical indices including FIS PacesAuditory Serial Addition Test or the MSQOL InventoryThere was no association with 36-month serum NfL levelsand clinical or MRI outcomes

Figure 4 Baseline NfL levels and post IAHSCT MRI outcomes

A baseline serum Nf-L gt2824 pgml-1 (red lines n = 11) com-pared to lt2824 pgml-1 (blue lines n = 12) was associated with(A) greater T1 lesion volumes (p = 0017) (B) greater T2 lesionvolumes (p = 00009) and (C) reduced MRI spectroscopy NAACr ratio (p = 00038) The interaction of NfL over time was onlysignificant for T1 accounting for 09 of the variabilitypatients with high baseline NfL had slightly more T1 lesionvolume accrual post-treatment Error bars represent thestandard error NfL = neurofilament light chain

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 7

DiscussionIn this small study of patients with aggressive MS treated withIAHSCT we show that elevated baseline levels of both serumand CSF NfL reduce quickly following treatment and remainlow for at least 3 years of follow-up Serum and CSF levelscorrelate closely Raised baseline serum NfL levels also asso-ciated with poorer clinical outcomes following IAHSCT andmore extensive MRI disease compared with those with lowerlevels

Pre- and post-treatment samples from this IAHSCT cohortrepresent the extremes of MS disease severity with aggressivedisease at baseline followed by durable suppression of in-flammatory activity following treatment They thereforeprovided a valuable opportunity to investigate NfL as a bio-marker of treatment response

Before IAHSCT mean NfL levels in our group of patients(311 pgmL) were much higher than levels recently reportedin a large population of patients with RRMS using the sameassay platform (169 pgmL although we note that this wasquantified using an in-house rather than commercially availablekit as in our study)17 Even so at baseline there was significantvariability in the observed levels although 4 patients had levelsgt70 pgmL some patients with clinically aggressive disease hadserum NfL levels similar to those of the controls This likelyreflects a spectrum of disease activity even within this groupwhere we found associations between pretreatment NfL levelsand established clinical and MRI measures of MS activity

similar to other studies5613 Before treatment high baselineNfL levels were associated with more rapid EDSS progressionand higher EDSS scores more relapses in the preceding yeargreater MRI lesion burden and an unfavorable MRI spec-troscopy profile Further chart review revealed that of the 3 ofthe 4 patients with the highest levels had clinically relapsed inthe 3 months before IAHSCT and none had received in-duction therapies Conversely 0 of 6 patients with the lowestlevels had relapsed in the 6 months before IAHSCT and 4 hadreceived mitoxantrone therapy in the preceding 2 years tostabilize their condition These observations substantiate therole of NfL as a potential marker of disease activity

Following IAHSCT all of the patientsrsquo NfL levels remainedsuppressed in both serum and CSF and were not significantlydifferent from a non-MS control group However thereremained a weakly significant correlation of pre- and post-treatment levels at both 12 and 36 months The meanreduction from 3106 pgmL to 1439 pgmL by 1 year post-treatment is the most dramatic reduction in serum NfL levelsfollowing an MS treatment reported to date8101819 For ex-ample using the same assay in a study of 240 patients startingfingolimod19 mean serum NfL reduced from 204 pgmL to135 pgmL after 1 year of treatment (although again we notethat this study used an in-house methodology perhaps lim-iting comparability with the commercially available kit weused) Our study is unique in that NfL levels were consistentlysuppressed in all of the patients following treatment in theabsence of other DMTrsquos in all of the previous studies

Figure 5 Baseline NfL levels and post IAHSCT MRI brain atrophy

High baseline NfL (gt2824 pgml-1 redlines n = 11) was associated with (A)more brain atrophy following AHSCT (p =0029) compared to low baseline NfL(lt2824 pgml-1 blue lines n = 12) More-over the high NfL group continued toendure a greater rate of on-going brainatrophy post IAHSCT signified by the in-teraction of NfL over time between thegroups (p = 00008 351 of total varia-tion) This was more attributable to (B)white matter (p = 00043) rather than (C)grey matter volume loss (p = 0037) Thegreatest differential rate of brain atrophywas seen in the white matter which per-sisted at 36 months post IAHSCT (p lt00001) Error bars represent the stan-dard error IAHSCT = immunoablationfollowed by autologous hematopoieticstem cell transplantation NfL = neuro-filament light chain

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 NeurologyorgNN

although there was a correlation of NfL reduction withtreatment response there were individual patients who ex-perienced significant increases in NfL associated with treat-ment failure We have shown previously that IAHSCTproduces a long-term suppression of ongoing inflammatorydisease activity12 and here we show that this is associatedwith a substantial sustained suppression of NfL levelsNonetheless the observation that pretreatment NfL levelsstill correlated with those post-treatment likely indicates thatinflammatory disease activity is not the only driver of NfLrelease in patients with MS Secondary neurodegenerativeprocesses may be also be contributory and continue irre-spective of ongoing inflammation

Higher baseline NfL levels were associated with a worse post-treatment clinical prognosis (sustained EDSS progressioncognitive markers and quality of life indices) and worse MRIoutcomes (T1 and T2 lesion burden MRS and brain atro-phy) Most of these post-treatment associations seem to beattributable to baseline differences between these groupsmore severe patients fared the worst post-IAHSCT largelydue to more progressed disease to begin with However post-IAHSCT brain atrophy continued to accumulate at a fasterrate in patients who had high pretreatment NfL levels Thesedifferences were greatest in the white matter as opposed to thegray matter and interestingly even the 12-month post-treatment NfL levels weakly correlated with white matteratrophy Although others have shown that serum NfL ispredictive of future MRI brain atrophy920 we saw this in ourstudy even in the absence of ongoing inflammatory diseaseactivity a possible confounder Furthermore this is the firststudy to correlate NfL levels with differential measures ofwhite and gray matter atrophy in MS The finding that NfL ismost strongly associated with future white matter atrophy wassurprising as NfL is thought to represent neuroaxonal loss

The association of serum NfL with the MRS NAACr isnovel but makes sense as both NfL and NAACr are markersof neuroaxonal damage It is not clear why there was a sus-tained rate of T1 lesion accrual post-IAHSCT in patients withhigh NfL (as none of these patients had any new lesions)However the magnitude of this interaction was mild (ac-counting for only 09 of variation) and may represent nat-ural evolution of existing lesions

The substrate of post-treatment clinical (EDSS) progressionseen in 723 of the IAHSCTpatients is unclear Remarkably thepatients whowent on to develop sustained progression generallyhad high pretreatment NfL levels (figure 3A) A similar obser-vation was made by Petzold et al21 who observed that highserum neurofilament heavy chain levels (a close relative of NfL)were associated with sustained disability progression on theEDSS following a similar bone marrow transplant

In our study these associations remained weakly significant inthe 12-month neurofilament levels Together the positiveassociations of pre- and post-treatment NfL levels with

sustained EDSS progression and MRI atrophy perhaps in-dicate ongoing neurodegeneration more evident in somepatients which persists irrespective of inflammatory diseasemore evident in patients with more active and advancedpretreatment disease This is important if we are to un-derstand the use of NfL in progressive MS trials

We found associations with highNfL and poorer outcomes onwell-validated functional indices of cognition (PASAT22) andquality of life (MSQOL-5423 physical composite) There wasno association with the mental composite of the MSQOL-54or fatigue although there was a 36 reduction followingIAHSCT in this cohort as reported elsewhere24 The observeddifferences in cognition and quality of life are attributable tobaseline differences between the groups

Although this study was limited by its size this was dictated bythe small number of patients who underwent IAHSCT foraggressive MS as part of the Canadian Bone Marrow Trans-plant Study Other limitations include the retrospective studydesign and the wide intervals between sampling A separatestudy to investigate more frequent time points in the first yearpost-IAHSCT is currently underway This will help delineatethe timeline of the NfL reduction compared MRI observa-tions As shown in figure 5 there was a particularly largeincrease in brain volume loss in the first month followingIAHSCT This has also been observed following induction ofother treatments25 Likely contributory explanations includepseudoatrophy related to brain shrinkage as the inflammationsubsides25 or perhaps more concerning in the case ofIAHSCT neuronal loss related to chemotherapy neurotox-icity This hypothesis is substantiated by increases in neuro-filament heavy chain in the CSF in the months following bonemarrow transplant for MS21 Serial quantification of serumNfL and other markers of neuronal injury at early post-treatment time points will be informative

This study provides group-level data supporting the role ofNfL as a quantitative marker of MS disease severity andtreatment response This convenient measure may one dayform part of routine MS monitoring However furtherstudies are required before clinicians can depend on it toinform treatment choices for their patients it is not clearhow changes in NfL translate to individual patients andtreatment decisions Ongoing prospective studies will helpto address this Further basic research is also necessary toclarify the mechanisms underlying NfL release CSF-serumclearance and how these factors might differ betweenpatients independently of neuroaxonal loss

AcknowledgmentThe authors thank Iva Stonebridge for technical and logisticalsupport Dr Tim Ramsey for assistance reviewing statisticalmethods and analyses and Dr Lisa Walker and Dr GauruvBose for sharing data related to fatigue They acknowledge thegrant support from the MS Society of Canada and thecontribution of Quanterix

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 9

Study fundingThis study was funded by the MS Foundation of Canada

DisclosureS Thebault DR Tessier H Lee and M Bowman report nodisclosures A Bar-Or has participated as a speaker in meet-ings sponsored by and received consulting fees andor grantsupport from Actelion Atara Biotherapeutics Biogen IdecCelgeneReceptos GenentechRoche MAPI MedImmuneMerckEMD Serono Novartis and Sanofi Genzyme andreceives research funding from the Canadian Institutes ofHealth Research Multiple Sclerosis Society of Canada Mul-tiple Sclerosis Scientific Foundation National Multiple Scle-rosis Society NIH Melissa and Paul Anderson Chair inNeuroinflammation D Arnold reports consultant fees andor grants from Acorda Adelphi Alkermes Biogen CelgeneFrequency Therapeutics Genentech Genzyme Hoffmann-La Roche Immune Tolerance Network ImmunotecMedDay Merck Serono Novartis Pfizer Receptos RocheSanofi-Aventis Canadian Institutes of Health Research MSSociety of Canada International Progressive MS Alliance andan equity interest in NeuroRx Research HL Atkins reportsno disclosures V Tabard-Cossa is funded by NIH1-R21EB024120-01 Genome Canada the Province of Ontarioand the Natural Sciences and Engineering Research Councilof Canada He also received research support from Abbottand royalty payments from Abbott and Multerra Bio MSFreedman received a research grant from Genzyme Canadahonoraria from Actelion Bayer Healthcare Biogen IdecCelgene Canada Chugai EMD Canada Genzyme MerckSerono Novartis Hoffmann-La Roche Sanofi-Aventis andTeva Canada Innovation He serves on the advisory boards ofActelion (now Johnson and Johnson) Bayer HealthcareBiogen Idec Celgene Clene Nanomedicine Hoffmann-LaRoche Merck SeronoMedDay Novartis and Sanofi-AventisHe participates in a company sponsored speakerrsquos bureau forSanofi Genzyme Go to NeurologyorgNN for fulldisclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationJanuary 11 2019 Accepted in final form April 29 2019

References1 Freedman MS Induction vs escalation of therapy for relapsing multiple sclerosis the

evidence Neurol Sci 200829250ndash2522 Wattjes MP Steenwijk MD Stangel M MRI in the diagnosis and monitoring of

multiple sclerosis an update Clin Neuroradiol 201525157ndash1653 Rovira A Wattjes MP Tintore M et al Evidence-based guidelines MAGNIMS

consensus guidelines on the use of MRI in multiple sclerosismdashclinical implementa-tion in the diagnostic process Nat Rev Neurol 201511471ndash482

4 Nielsen S Nagelhus EA Amiry-Moghaddam M Specialized membrane domains forwater transport in glial cells high-resolution immunogold cytochemistry ofaquaporin-4 in rat brain J Neurosci 199717171ndash180

5 Kearney H Miller DH Ciccarelli O Spinal cord MRI in multiple sclerosis-diagnosticprognostic and clinical value Nat Rev Neurol 201511327ndash338

6 Rissin DM Kan CW Campbell TG et al Single-molecule enzyme-linked immuno-sorbent assay detects serum proteins at subfemtomolar concentrations Nat Bio-technol 201028595ndash599

7 Kuhle J Barro C Andreasson U et al Comparison of three analytical platforms forquantification of the neurofilament light chain in blood samples ELISA electro-chemiluminescence immunoassay and Simoa Clin Chem Lab Med 2016541655ndash1661

8 Disanto G Barro C Benkert P et al Serum neurofilament light A biomarker ofneuronal damage in multiple sclerosis Ann Neurol 201781857ndash870

9 Chitnis T Gonzalez C Healy BC et al Neurofilament light chain serum levelscorrelate with 10-year MRI outcomes in multiple sclerosis Ann Clin Transl Neurol20181478ndash1491

10 Varhaug KN Barro C Bjoslashrnevik K et al Neurofilament light chain predicts diseaseactivity in relapsing-remitting MS Neurol Neuroimmunol NeuroInflamm 20185e422 doi101212NXI0000000000000422

11 Barro C Benkert P Disanto G et al Serum neurofilament as a predictor of diseaseworsening and brain and spinal cord atrophy in multiple sclerosis Brain 20181412382ndash2391

12 Atkins HL BowmanM Allan D et al Immunoablation and autologous haemopoieticstem-cell transplantation for aggressive multiple sclerosis a multicentre single-groupphase 2 trial Lancet 2016388576ndash585

13 Weinshenker BG Rice GPA Noseworthy JH Carriere W Baskerville J Ebers GCThe natural history of multiple sclerosis a geographically based study Brain 19911141045ndash1056

14 Menon S Shirani A Zhao Y et al Characterising aggressive multiple sclerosisJ Neurol Neurosurg Psychiatry 2013841192ndash8

15 Smith SM Zhang Y Jenkinson M et al Accurate robust and automated longitudinaland cross-sectional brain change analysis Neuroimage 200217479ndash489

16 Nakamura K Guizard N Fonov VS Narayanan S Collins DL Arnold DL Jacobianintegration method increases the statistical power to measure gray matter atrophy inmultiple sclerosis NeuroImage Clin 2014410ndash7

Appendix Authors

Name Location Role Contribution

SimonThebault MD

University ofOttawa

Author Designed andconceptualized the studydata collection analyzedthe data and drafted themanuscript for intellectualcontent

Hyunwoo LeePhD

MontrealNeurologicalInstitute

Author Major role in analysis ofMRIdata

Daniel TessierPhD

University ofOttawa

Author Major role in NfL dataacquisition

Appendix (continued)

Name Location Role Contribution

MarjorieBowmanMScN

University ofOttawa

Author Conducted original IAHSCTstudy and revised themanuscript for intellectualcontent

Amit Bar-OrMD

University ofPennsylvania

Author Conducted original IAHSCTstudy and revised themanuscript for intellectualcontent

VincentTabard-CossaPhD

University ofOttawa

Author NfL data acquisition andrevised the manuscript forintellectual content

Harold AtkinsMD

University ofOttawa

Author Conducted original IAHSCTstudy and revised themanuscript for intellectualcontent

Douglas LArnold MD

MontrealNeurologicalInstitute

Author Conducted original IAHSCTstudy and revised themanuscript for intellectualcontent

Mark SFreedmanMD

University ofOttawa

Author Conducted original IAHSCTstudy study design andconceptualisation andrevised the manuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 NeurologyorgNN

17 Novakova L Zetterberg H Sundstrom P et al Monitoring disease activity in multiplesclerosis using serum neurofilament light protein Neurology 2017892230ndash2237

18 Novakova L Axelsson M Khademi M et al Cerebrospinal fluid biomarkers asa measure of disease activity and treatment efficacy in relapsing-remitting multiplesclerosis J Neurochem 2017141296ndash304

19 Piehl F Kockum I Khademi M et al Plasma neurofilament light chain levels inpatients withMS switching from injectable therapies to fingolimod Mult Scler J 2018241046ndash1054

20 Kuhle J Nourbakhsh B Grant D et al Serum neurofilament is associated withprogression of brain atrophy and disability in early MS Neurology 201788826ndash831

21 Petzold A Mondria T Kuhle J et al Evidence for acute neurotoxicity after chemo-therapy Ann Neurol 201068806ndash815

22 Tombaugh TN A comprehensive review of the Paced Auditory Serial Addition Test(PASAT) Arch Clin Neuropsychol 20062153ndash76

23 Vickrey BG Hays RD Harooni R Myers LW Ellison GW A health-related quality oflife measure for multiple sclerosis Qual Life Res 19954187ndash206

24 Bose G Atkins HL Bowman M Freedman MS Autologous hematopoietic stem celltransplantation improves fatigue in multiple sclerosis Mult Scler J Epub 2018 Sep 25

25 Stefano N De Arnold DL Towards a better understanding of pseudoatrophy in thebrain of multiple sclerosis patients Mult Scler 201521675ndash676

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 5 | September 2019 11

DOI 101212NXI000000000000059820196 Neurol Neuroimmunol Neuroinflamm

Simon Thebault Daniel R Tessier Hyunwoo Lee et al transplantation for MS

High serum neurofilament light chain normalizes after hematopoietic stem cell

This information is current as of August 9 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

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

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Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

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dy_design_httpnnneurologyorgcgicollectionclinical_trials_methodology_stuClinical trials Methodologystudy design

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

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

ServicesUpdated Information amp

httpnnneurologyorgcontent65e598fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent65e598fullhtmlref-list-1

This article cites 23 articles 2 of which you can access for free at

Citations httpnnneurologyorgcontent65e598fullhtmlotherarticles

This article has been cited by 2 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

dy_cohort_case_controlhttpnnneurologyorgcgicollectionclinical_trials_observational_stuClinical trials Observational study (Cohort Case control)

dy_design_httpnnneurologyorgcgicollectionclinical_trials_methodology_stuClinical trials Methodologystudy design

httpnnneurologyorgcgicollectionclass_iiClass IIfollowing collection(s) This article along with others on similar topics appears in the

Permissions amp Licensing

httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

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