Therapy with natalizumab is associated with high JCV ... · the index values of JCV1 patients did...

Nicholas Schwab PhDTilman Schneider-

Hohendorf PhDBeacuteatrice Pignolet PhDJohanna Breuer PhDCatharina C Gross PhDKerstin Goumlbel MDDavid Brassat MD PhDHeinz Wiendl MD

Correspondence toProf Wiendlheinzwiendlukmuensterdeor Dr Schwabnicholasschwabukmuensterde

See editorial

Supplemental dataat Neurologyorgnn

Therapy with natalizumab is associatedwith high JCV seroconversion and risingJCV index values

ABSTRACT

Objective The aim of the study was to analyze John Cunningham virus (JCV) serology innatalizumab-treated patients over time and assess whether they are influenced by natalizumabtreatment

Methods German (n 5 1921 525 longitudinally) and French (n 5 1259 711 longitudinally)patients were assessed for JCV serology alongside their therapy with natalizumab

Results JCV serostatus changed in 69 of 525 longitudinally followed German patients (131)over 148 months Seroconversion according to serostatus was seen in 43 of 339 initially JCV2German patients (127 in 148 months 103 per year) and 41 of 243 initially JCV2 Frenchpatients (169 in 24months 85 per year) JCV index values could be reproduced (R2 5089)with the caveat of 8 of 50 samples (16) being set into different risk categories between 2assessments Index values of JCV1 patients rose over time (p5 0009) but not because of agingTreatment with natalizumab was associated with a 159 increase of value in JCV1 patients in148 months (129 per year)

Conclusions JCV seroconversion and index values may be influenced by treatment with natalizu-mab It is therefore important to monitor patientsrsquo JCV serology but also to incorporate additionalrisk factors into the progressive multifocal leukoencephalopathy risk stratification Neurol

Neuroimmunol Neuroinflamm 20163e195 doi 101212NXI0000000000000195

GLOSSARYJCV 5 John Cunningham virus MS 5 multiple sclerosis PML 5 progressive multifocal leukoencephalopathy RRMS 5relapsing-remitting multiple sclerosis

Natalizumab was approved for the treatment of active relapsing-remitting multiple sclerosis(RRMS) starting in 200612 The development of 566 cases of progressive multifocal leukoen-cephalopathy (PML) (June 20153) has been a significant problem of an otherwise successfultreatment regimen Patients are currently stratified using 3 measures prior immunosuppressantuse duration of natalizumab treatment and presence of antibodies against the PML-inducingJohn Cunningham virus (JCV)4 Recently the JCV serology biomarker has been extended toinclude the level of anti-JCV antibody titers represented as a JCV index value5 In April 2015the European Medicines Agency initiated a re-review of the drug after data from an interimreport of the STRATIFY-2 trial suggested that the JCV seroconversion during natalizumabtherapy might be higher than previously assumed We have been assessing JCV serostatus andindex values in 2 large cohorts of German and French patients with multiple sclerosis (MS)treated with natalizumab This study shows how JCV serology (status and index) is influencedby treatment with natalizumab in addition to the known JCV serostatus change by aging6 from a

These authors contributed equally to this work

From the Department of Neurology (NS TS-H JB CCG KG HW) University of Muumlnster Germany and Pole des NeurosciencesCentre Hospitalier Universitaire Toulouse (BP DB) CPTP INSERM UMR 1043 et Universiteacute de Toulouse UPS Toulouse France DB alsorepresents the BioNAT Study Group

Funding information and disclosures are provided at the end of the article Go to Neurologyorgnn for full disclosure forms The Article ProcessingCharge was paid by the University of Muumlnster

BioNAT Study Group coinvestigators are listed at Neurologyorgnn

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CCBY-NC-ND) which permits downloading and sharing the work provided it is properly cited The work cannot be changed in any way or usedcommercially

Neurologyorgnn copy 2016 American Academy of Neurology 1

ordf 2016 American Academy of Neurology Unauthorized reproduction of this article is prohibited

study in which patients were not treated withnatalizumab but with many other disease-modifying treatments

METHODS Patients and biomaterials Serum samples of

1921 patients (Germany) and 1259 patients (France BioNAT)

with RRMS alongside natalizumab therapy were processed as pub-

lished previously7 The patient cohorts and their seroprevalence are

shown in a flow diagram in figure e-1 at Neurologyorgnn

Standard protocol approvals registrations and patientconsents The study was approved by the local ethics committee

(University of Muenster Ethik-Kommission der Aumlrztekammer

Westfalen-Lippe und der Medizinischen Fakultaumlt der

Westfaumllischen Wilhelms-Universitaumlt registration number 2010-

245-f-S Comite ethique du sud ouest et outre mer II 2-09-02)

and informed written consent was obtained from all participants

This study was performed according to the Declaration of Helsinki

Anti-JCV antibody status and index value Sera samples

were processed and analyzed by Unilabs (Copenhagen Denmark)

with the second-generation ELISA kit STRATIFY JCVDxSelect8

(EL1950 Focus Diagnostics Cypress CA) according to the

manufacturerrsquos instructions5

Statistics Continuous variables such as JCV index are character-

ized by means Categorical variables such as JCV serostatus are

described by absolute and relative frequencies Univariate correla-

tions are estimated by Spearman correlation coefficient Data are

visualized as scatterplots and supplemented by linear regression

lines Significance of longitudinal JCV index value changes was

calculated using Wilcoxon matched-pairs signed rank test The

p values were considered significant at 005 No adjustment

for multiplicity was performed Statistical analyses were

conducted using Prism (version 5 GraphPad San Diego CA)

RESULTS JCV serostatus Overall JCV serostatusassessment set 1052 of 1921 German patients(547) under treatment with natalizumab as JCV1(figure 1A) Longitudinally 525 of these patients wereaccompanied for a mean period of observation of 1486 (SD) 82 months Two hundred ninety-six of these525 patients (564) were JCV2 during the completeperiod of observation and 171 were JCV1 (326)Forty-three patients changed from being JCV2 toJCV1 (82) and 15 patients changed from beingJCV1 to JCV2 (29) Overall there were 11patients (21) who transiently changed serostatusduring the period of observation but ended up withtheir initial serostatus (figure 1B) If JCV serostatus wasused to determine seroconversion the longitudinalassessment started out with 339 initially JCV2patients (initial seroprevalence of the longitudinalcohort 354) The serostatus of 43 of these initiallyJCV2 patients changed to JCV1 which is 127 in148 months (1030 per year or 086 per monthultimate seroprevalence of the longitudinal cohort408) (figure 1C) In the French BioNAT cohortof 1259 patients (seroprevalence of the wholecohort before start of treatment was 604 of 1020patients 5 59 and of the longitudinally followed

cohort 468 of 711 patients 5 658) of theinitially 243 JCV2 patients of 711 patients whereall 3 longitudinal time points were available theserostatus of 20 changed to JCV1 in their first yearof treatment (82) and 21 in their second year oftreatment (86 of 243 initially JCV2 patients or94 of the 223 patients who were still JCV2 after1 year of treatment) Taken together the serostatus of41 of 243 patients (169) changed to JCV1 in thefirst 2 years of natalizumab treatment (85 per year or070 per month ultimate seroprevalence of theFrench longitudinal cohort after 2 years 716)(figure 1D)

JCV index value The JCV serostatus was recentlyextended to incorporate the level of anti-JCVantibodies in serum normalized to a ldquoJCV indexrdquovalue To assess the reproducibility of this value 50patient serum samples were measured twice byUnilabs Overall reproducibility was very good withan R2 of 089 However 8 of these 50 patients(16) would have been set in different riskcategories between the 2 measurements (thresholdsfrom Plavina et al5 2014 JCV21 09 12 15)with one sample actually being set as JCV1 in the firstand JCV2 in the second measurement and 2 samplesbeing either measured at very high PML risk (JCVindex 15) or very low risk (JCV index 09)(figure 2A) in the 2 measurements The index valueschanged in 525 patients in the observation period Theproportion of patients with an index value 04 wasreduced by 20 patients (from 651 to 613) andthe group of patients with low risk (between 04 and09) was reduced by one patient to 78 The patientgroups with medium (09ndash15) and high risk (15)grew by 7 patients from 46 to 59 and by 14patients from 223 to 25 respectively (figure2B) Overall this was a reflection of the change inserostatus (figure 1C) but also suggested that patientswho changed serostatus directly presented with highanti-JCV antibody titers afterward as the groups oflow and medium risk did not grow substantially overtime This trend was also clearly visible in the completecohort of patients (figure 2C) where up to 7longitudinal samples were assessed from each patientand the seroconversion mostly led to JCV index valuesof 15 (figure 2D)

Change in JCV index values in JCV1 patients Whilemost of the changes in JCV index values could beattributed to the seroconversion of initially JCV2patients it was also important to see whether patientspresented with stable index values once they con-verted to JCV seropositivity Two hundred oneJCV1 patients were therefore followed over timeand it became clear that the biomarker showedchanges over time with higher values after the period

2 Neurology Neuroimmunology amp Neuroinflammation


of observation (mean 2046 vs 2158 p 5 0009)(figure 3A) That age had a role in these fluctuationswas ruled out because the age of patients contributedto the overall rise in index values (Spearmanr 20113 p 00001) due to seroconversion butthe index values of JCV1 patients did not changewith age (Spearman r 00001 p 5 0996) (figure3B) this was also true for patients who seroconvertedduring therapy with natalizumab The index correla-tion with age in the whole population (JCV2 andJCV1) is attributable to seroconversion but if only

JCV1 patients are considered their index values donot differ with age they only differ with prolongedtreatment duration

One hundred sixty-one of 201 JCV1 patients(80) presented with stable JCV index values overtime (630 in the period of observation) Howeverthe remaining 40 patients (20) presented with fluc-tuations of more than 30 in 148 months Only 6of these patients (3) presented with decreasingindex values but 34 (17) presented with increasingindex values (mean 2008) Taken together the

Figure 1 JCV serostatus

(A) Serostatus of 1921 natalizumab-treated patients with multiple sclerosis with JCV2 patients shown in black and JCV1 patients in red A total of 1052of these 1921 patients were set as JCV1 (5476) (B) Serostatus of a longitudinally assessed cohort of 525 patients during 148 months of natalizumabtreatment (C) Serostatus of the German cohort of 339 initially JCV2 patients (of 525 patients) before and after the period of observation (148 months ofnatalizumab treatment) Forty-three of these 339 patients (127) were set as JCV1 after the period of observation (D) Serostatus of the French cohort(n 5 1259 711 longitudinally BioNAT) of the 243 initially JCV2 patients of 711 where all 3 time points were available during their first 2 years oftreatment Twenty of these patients (82) were set as JCV1 after the first year and an additional 21 (86) after the second year of treatment resulting in41 of 243 patients (169) changing their serostatus to JCV1 in the first 2 years of treatment JCV 5 John Cunningham virus

Neurology Neuroimmunology amp Neuroinflammation 3


Figure 2 JCV index

(A) Fifty samples of natalizumab-treated patients with multiple sclerosis were assessed twice for their anti-JCV antibodyindex (JCV index) with an R2 of 089 Eight of these 50 samples (16) showed 2 different risk associations with thethresholds JCV21 JCV index 04 09 12 and 15 in the 2 assessments (B) JCV index value distributions of a longitudinalcohort of 525 patients at the start and end of the period of observation of 148 months (C) Serial assessment of JCV indexvalues of 525 patients alongside their natalizumab therapy (2ndash7 JCV serology assessments) Red lines indicate the thresh-olds 04 09 and 15 (D) Serial assessment of JCV index values Only patients whose index values changed by more than30 over time and whose PML risk group changed are shown (0ndash04 [green] 04ndash09 [yellow] 09ndash15 [orange]15 [red])JCV 5 John Cunningham virus



index value of all JCV1 patients increased by 159on average in 148 months (129 per year or 011per month) (figure 3C)

DISCUSSION There has been strong debate aboutwhether the underlying JCV seroconversion rate by

aging is influenced by treatment with natalizu-mab9ndash11 The high seroconversion which has alreadybeen suggested (Plavina et al5 2014 n 5 553045ndash072 per month depending on the defini-tion of seroconversion) and the data of seroconver-sion rates in longitudinally monitored JCV2 patients

Figure 3 JCV index changes in JCV1 patients

(A) Increasing JCV index values of 201 JCV1 natalizumab-treated patients with multiple sclerosis at the beginning and endof a period of observation of 148 months (mean 2046 vs 2158 p 5 0009) (B) Age (in years) and JCV index value of1921 natalizumab-treated patients with multiple sclerosis Overall there is a strong rise in index values by age (Spearmanr 0113 p 00001) but no rise in the index values of JCV1 patients (Spearman r 00001 p 5 0996) or patients whoconverted to JCV seropositivity during treatment with natalizumab (Spearman r 00257 p 5 0907) (C) Change of JCVindex values of 201 JCV1 patients during natalizumab treatment in (index value at the end of the period of observationindex value at the beginning of the period of observation3 100) Mean rise in index values was 159 in 148 months Onehundred sixty-one of 201 patients (80) presented with stable index values (630) 34 patients (17) presented withincreasing index values and 6 patients (3) with decreasing index values JCV 5 John Cunningham virus



in our study (103 and 85 per year) clearly sup-port the facilitation by treatment with natalizumabIt is important to distinguish between seroconver-sion (a JCV2 patient converting to JCV1) and anincrease in seroprevalence (the percentage of JCV1patients within a cohort) The published rise in se-roprevalence by age is 05 per year612 whichtranslates into a JCV seroconversion of approxi-mately 1 per year in the 40 to 45 of JCV2patients within these cohorts However in both cal-culations our observed seroconversion of 8 to10 per year and the rise in seroprevalence of 5to 6 in 15 to 24 months is at least 8 to 10 times asmuch as would be expected by age This datasetsuggests that not every patient with MS is suscepti-ble to JCV seroconversion by treatment but natali-zumab might facilitate it in patients who aresusceptible There has recently been an extensivestudy of 7724 patients and their JCV serostatus ina group of control patients6 The authors clearlyshow that when adjusted for age sex and countryof origin the duration of MS treatment has no influ-ence on JCV seroprevalence leaving treatment withnatalizumab as the only factor in our study as sexand country of origin do not change in longitudinalcohorts Because as yet there are no studies on theinfluence of other treatments on JCV index valuesand despite a very recent study also supporting thishypothesis13 we cannot be certain that it was thetreatment with natalizumab that led to the risingindex values in our study However because therewas no correlation with age in JCV1 patients andthese patients have certainly been treated longerwith disease-modifying drugs the older they are itcan be speculated that it is specifically the treatmentwith natalizumab that induces rising JCV indexvalues (and therefore anti-JCV titers) The highseroconversion (putatively induced by higher JCVactivity) is also in agreement with the publishedlower CD62L values induced by natalizumabtreatment7 as both are associated with higherPML risk Because previous hypotheses concerningJCV titers suggested that higher titers are the resultof a higher replication rate of the virus14ndash16 it isconceivable that a higher replication rate isattributable to the fact that the compromisedimmune cells of natalizumab-treated patients areless capable of suppressing the viral activity ofJCV As long as these biological backgrounds arenot fully elucidated it seems prudent to includethe theory of (re)infection with JCV as a sourcefor seroconversion However since there arepatients who shed the virus in their urine withoutbeing antibody seropositive17 it seems unlikely thatthe process leading to seropositivity is solely linkedto (re)exposure to JCV

One drawback of our study is the fact that JCV2patients naturally tend to reassess their JCV serologymore often than JCV1 patients for a potential sero-conversion Therefore our prospective longitudinalGerman cohort in which patients could decide forthemselves how often to assess their serostatus has amuch lower seroprevalence than the overall patientpopulation This might overexaggerate the percentageof seroconverters in the entire natalizumab-treatedMS collective which is why it is important to calcu-late the seroprevalence in addition to the seroconver-sion Apart from this it was recently shown that IVimmunoglobulin treatment may cause transientlyhigh anti-JCV antibody titers and thereby transientfalse-positive JCV serostatus results However we canrule this out for our seroconverting patients as well asfor the complete cohort because IV immunoglobu-lins are usually not applied during natalizumab treat-ment in Europe18

Both observations of this studymdashthe high sero-conversion and the rising index values in JCV1patientsmdashhave implications for PML risk stratifica-tion using JCV serology It is important to regularlycheck patientsrsquo JCV serology (status and index) foran accurate assessment of their PML risk accordingto this biomarker Unfortunately with a JCV indexvalue mean of more than 2 most JCV1 patients areset into the highest PML risk category with veryfew of these patients ultimately developing PMLJCV serology should not be the only PML risk bio-marker used in the stratification of patients treatedwith natalizumab The exploration and potentialapplication of additional biomarkers such asCD62L in peripheral blood719 or IgM bands inCSF20 is needed to accurately inform patients oftheir PML risk and ultimately help in reducingPML incidences

If the hypothesis that treatment with natalizu-mab is associated with enhanced JCV seroconver-sion and higher index values is proven it wouldalso be important to determine whether cessationof natalizumab therapy (or perhaps prolonged infu-sion intervals) could lead to lower JCV index valuesas well This remains to be seen in the studiescurrently under way regarding switching to othertherapies or prolonged infusion intervals Howeverfrom a risk stratification point of view this wouldnot influence patients because they should alwaysassume the highest measured risk to be on the safeside and even lowered JCV index values shouldnot suggest that a patientrsquos risk has diminishedThese further and larger clinical studies with a strictstudy protocol should be performed to assess inwhich capacity natalizumab influences JCV serocon-version and whether this is influenced by treatmentdosageintervals



Taken together JCV serology is a sensitive bio-marker for PML risk but it is very dynamic andshould be regarded as such JCV2 patients shouldreassess their status regularly and JCV1 patientsshould check their JCV index values until they havereached the highest risk category after which JCVserology loses some of its usefulness The fact thattreatment with natalizumab is associated with avery high rate of seroconversion and rising indexvalues does not diminish its clinical efficacy butcalls for more elaborate strategies for PML riskstratification according to current scientific devel-opments also regarding patients with prior use ofimmunosuppressants where the JCV index is nothelpful5

AUTHOR CONTRIBUTIONSNS designed and performed research collected data analyzed data and

generated funding TS-H designed and performed research collected

and analyzed data BP performed research collected and analyzed data

JB KG and CCG performed research collected and analyzed data

DB performed research collected and analyzed data and generated

funding HW designed research analyzed data and generated funding

All authors wrote the manuscript HW and NS had full access to all of

the data in the study and take responsibility for the integrity of the data

and the accuracy of the data analysis

ACKNOWLEDGMENTThe authors thank Barbara Wrobel Petra Babucke and Verena Schuumltte

for excellent technical assistance and the patients for their participation in

the study

STUDY FUNDINGThis study was funded by Deutsche Forschungsgesellschaft (DFG) grant

CRC128 Project B1 to NS and HW Project Z2 to HW the PML

consortium to NS and HW the Kompetenznetz Multiple Sklerose

(Competence Network for Multiple Sclerosis) funded by the Federal

Ministry of Education and Research (FKZ 01GI1308B 01GI0907) to

HW French Ministry of Health (PHRC 2008-005906-38) ARSEP

(French MS Society grant 2009 and 2011) to DB and the EU

(BEST-MS FP7 305477) to DB and HW

DISCLOSUREN Schwab received travel funding from Biogen speaker honoraria from

Novartis holds a patent for usage of L-selectin as a predictive marker for

PML and received research support from DFG University Munster T

Schneider-Hohendorf received travel funding from Biogen holds a patent

for usage of L-selectin as a predictive marker for the risk to develop PML

B Pignolet and J Breuer report no disclosures CC Gross received

speaker honoraria andor travel funding from Genzyme Novartis Pharma

GmbH and Bayer Health Care is a review editor for Frontiers in Immu-

nology and received research support from the German Research Foun-

dation University of Munster K Goumlbel reports no disclosures D

Brassat receives travel funding andor speaker honoraria from Biogen

Sanofi-Genzyme Teva Merck Serono Bayer and Almirall received

research support from the French Ministry of Health French Multiple

Sclerosis Society and the European Union H Wiendl is on the scientific

advisory board for Bayer Healthcare Biogen Idec Sanofi-Genzyme

Merck Serono Novartis Roche and Teva received travel funding and

or speaker honoraria from Bayer Vital GmbH Bayer Schering AG Bio-

gen CSL Behring EMD Serono Fresenius Medical Care Sanofi-

Genzyme Merck Serono OmniaMed Novartis and Teva is on the

editorial board for Journal of Clinical Practice Journal of Neuroinflamma-

tion and PLoS One has consulted for Biogen Idec Merck Serono No-

vartis OmniaMed Roche and Sanofi-Genzyme received research

support from Bayer Healthcare Bayer Vital Biogen Idec Merck Serono

Novartis Sanofi-Genzyme Sanofi US Teva Pharma German Ministry

for Education and Research Deutsche Forschungsgesellschaft European

Union Else Kroner Fresenius Foundation Fresenius Foundation Hertie

Foundation NRW Ministry of Education and Research Interdisciplinary

Center for Clinical Studies Muenster RE Childrenrsquos Foundation and

Else Kroner Fresenius Foundation Go to Neurologyorgnn for full dis-

closure forms

Received July 29 2015 Accepted in final form November 10 2015

REFERENCES1 Polman CH OrsquoConnor PW Havrdova E et al A ran-

domized placebo-controlled trial of natalizumab for

relapsing multiple sclerosis N Engl J Med 2006354

899ndash910

2 Miller DH Khan OA Sheremata WA et al A controlled

trial of natalizumab for relapsing multiple sclerosis N Engl

J Med 200334815ndash23

3 Biogen medical information Available at httpsmedinfo

biogenideccom Accessed July 28 2015

4 Bloomgren G Richman S Hotermans C et al Risk of

natalizumab-associated progressive multifocal leukoen-

cephalopathy N Engl J Med 20123661870ndash1880

5 Plavina T Subramanyam M Bloomgren G et al Anti-

JC virus antibody levels in serum or plasma further

define risk of natalizumab-associated progressive multi-

focal leukoencephalopathy Ann Neurol 201476

802ndash812

6 Bozic C Subramanyam M Richman S Plavina T

Zhang A Ticho B Anti-JC virus (JCV) antibody preva-

lence in the JCV Epidemiology in MS (JEMS) trial Eur J

Neurol 201421299ndash304

7 Schwab N Schneider-Hohendorf T Posevitz V et al

L-selectin is a possible biomarker for individual PML risk

in natalizumab-treated MS patients Neurology 201381

865ndash871

8 Lee P Plavina T Castro A et al A second-generation

ELISA (STRATIFY JCVtrade DxSelecttrade) for detection of

JC virus antibodies in human serum and plasma to sup-

port progressive multifocal leukoencephalopathy risk strat-

ification J Clin Virol 201357141ndash146

9 Outteryck O Ongagna JC Duhamel A et al Anti-

JCV antibody prevalence in a French cohort of MS

patients under natalizumab therapy J Neurol 2012

2592293ndash2298

10 Olsson T Achiron A Alfredsson L et al Anti-JC virus

antibody prevalence in a multinational multiple sclerosis

cohort Mult Scler 2013191533ndash1538

11 Trampe AK Hemmelmann C Stroet A et al Anti-JC

virus antibodies in a large German natalizumab-treated

multiple sclerosis cohort Neurology 2012781736ndash1742

12 Gorelik L Lerner M Bixler S et al Anti-JC virus anti-

bodies implications for PML risk stratification Ann Neu-

rol 201068295ndash303

13 Raffel J Gafson AR Malik O Nicholas R Anti-JC virus

antibody titres increase over time with natalizumab treat-

ment Mult Scler 2015211833ndash1838

14 Bohl DL Brennan DC Ryschkewitsch C Gaudreault-

Keener M Major EO Storch GA BK virus antibody titers

and intensity of infections after renal transplantation

J Clin Virol 200843184ndash189

15 Pastrana DV Brennan DC Cuburu N et al Neutral-

ization serotyping of BK polyomavirus infection in kid-

ney transplant recipients PLoS Pathog 20128

e1002650



16 Pastrana DV Wieland U Silling S Buck CB Pfister H

Positive correlation between Merkel cell polyomavirus viral

load and capsid-specific antibody titer Med Microbiol

Immunol 201220117ndash23

17 Berger JR Houff SA Gurwell J Vega N Miller CS

Danaher RJ JC virus antibody status underestimates infec-

tion rates Ann Neurol 20137484ndash90

18 Kister I Kuesters G Chamot E et al IV immunoglobulin

confounds JC virus antibody serostatus determination

Neurol Neuroimmunol Neuroinflamm 20141e29

19 Schneider-Hohendorf T Philipp K Husstedt IW

Wiendl H Schwab N Specific loss of cellular L-selectin

on CD4(1) T cells is associated with progressive multifo-

cal leukoencephalopathy development during HIV infec-

tion AIDS 201428793ndash795

20 Villar LM Costa-Frossard L Masterman T et al Lipid-

specific immunoglobulin M bands in cerebrospinal fluid

are associated with a reduced risk of developing progressive

multifocal leukoencephalopathy during treatment with

natalizumab Ann Neurol 201577447ndash457



DOI 101212NXI000000000000019520163 Neurol Neuroimmunol Neuroinflamm

Nicholas Schwab Tilman Schneider-Hohendorf Beacuteatrice Pignolet et al index values

Therapy with natalizumab is associated with high JCV seroconversion and rising JCV

This information is current as of January 27 2016

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httpnnneurologyorgcontentsuppl2016030131e195DC2 httpnnneurologyorgcontentsuppl2016012731e195DC1

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References httpnnneurologyorgcontent31e195fullhtmlref-list-1

This article cites 19 articles 0 of which you can access for free at

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

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2016 American Academy of Neurology All rights reserved Online ISSN 2332-7812Published since April 2014 it is an open-access online-only continuous publication journal Copyright copy

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

study in which patients were not treated withnatalizumab but with many other disease-modifying treatments

METHODS Patients and biomaterials Serum samples of

1921 patients (Germany) and 1259 patients (France BioNAT)

with RRMS alongside natalizumab therapy were processed as pub-

lished previously7 The patient cohorts and their seroprevalence are

shown in a flow diagram in figure e-1 at Neurologyorgnn

Standard protocol approvals registrations and patientconsents The study was approved by the local ethics committee

(University of Muenster Ethik-Kommission der Aumlrztekammer

Westfalen-Lippe und der Medizinischen Fakultaumlt der

Westfaumllischen Wilhelms-Universitaumlt registration number 2010-

245-f-S Comite ethique du sud ouest et outre mer II 2-09-02)

and informed written consent was obtained from all participants

This study was performed according to the Declaration of Helsinki

Anti-JCV antibody status and index value Sera samples

were processed and analyzed by Unilabs (Copenhagen Denmark)

with the second-generation ELISA kit STRATIFY JCVDxSelect8

(EL1950 Focus Diagnostics Cypress CA) according to the

manufacturerrsquos instructions5

Statistics Continuous variables such as JCV index are character-

ized by means Categorical variables such as JCV serostatus are

described by absolute and relative frequencies Univariate correla-

tions are estimated by Spearman correlation coefficient Data are

visualized as scatterplots and supplemented by linear regression

lines Significance of longitudinal JCV index value changes was

calculated using Wilcoxon matched-pairs signed rank test The

p values were considered significant at 005 No adjustment

for multiplicity was performed Statistical analyses were

conducted using Prism (version 5 GraphPad San Diego CA)

RESULTS JCV serostatus Overall JCV serostatusassessment set 1052 of 1921 German patients(547) under treatment with natalizumab as JCV1(figure 1A) Longitudinally 525 of these patients wereaccompanied for a mean period of observation of 1486 (SD) 82 months Two hundred ninety-six of these525 patients (564) were JCV2 during the completeperiod of observation and 171 were JCV1 (326)Forty-three patients changed from being JCV2 toJCV1 (82) and 15 patients changed from beingJCV1 to JCV2 (29) Overall there were 11patients (21) who transiently changed serostatusduring the period of observation but ended up withtheir initial serostatus (figure 1B) If JCV serostatus wasused to determine seroconversion the longitudinalassessment started out with 339 initially JCV2patients (initial seroprevalence of the longitudinalcohort 354) The serostatus of 43 of these initiallyJCV2 patients changed to JCV1 which is 127 in148 months (1030 per year or 086 per monthultimate seroprevalence of the longitudinal cohort408) (figure 1C) In the French BioNAT cohortof 1259 patients (seroprevalence of the wholecohort before start of treatment was 604 of 1020patients 5 59 and of the longitudinally followed

cohort 468 of 711 patients 5 658) of theinitially 243 JCV2 patients of 711 patients whereall 3 longitudinal time points were available theserostatus of 20 changed to JCV1 in their first yearof treatment (82) and 21 in their second year oftreatment (86 of 243 initially JCV2 patients or94 of the 223 patients who were still JCV2 after1 year of treatment) Taken together the serostatus of41 of 243 patients (169) changed to JCV1 in thefirst 2 years of natalizumab treatment (85 per year or070 per month ultimate seroprevalence of theFrench longitudinal cohort after 2 years 716)(figure 1D)

JCV index value The JCV serostatus was recentlyextended to incorporate the level of anti-JCVantibodies in serum normalized to a ldquoJCV indexrdquovalue To assess the reproducibility of this value 50patient serum samples were measured twice byUnilabs Overall reproducibility was very good withan R2 of 089 However 8 of these 50 patients(16) would have been set in different riskcategories between the 2 measurements (thresholdsfrom Plavina et al5 2014 JCV21 09 12 15)with one sample actually being set as JCV1 in the firstand JCV2 in the second measurement and 2 samplesbeing either measured at very high PML risk (JCVindex 15) or very low risk (JCV index 09)(figure 2A) in the 2 measurements The index valueschanged in 525 patients in the observation period Theproportion of patients with an index value 04 wasreduced by 20 patients (from 651 to 613) andthe group of patients with low risk (between 04 and09) was reduced by one patient to 78 The patientgroups with medium (09ndash15) and high risk (15)grew by 7 patients from 46 to 59 and by 14patients from 223 to 25 respectively (figure2B) Overall this was a reflection of the change inserostatus (figure 1C) but also suggested that patientswho changed serostatus directly presented with highanti-JCV antibody titers afterward as the groups oflow and medium risk did not grow substantially overtime This trend was also clearly visible in the completecohort of patients (figure 2C) where up to 7longitudinal samples were assessed from each patientand the seroconversion mostly led to JCV index valuesof 15 (figure 2D)

Change in JCV index values in JCV1 patients Whilemost of the changes in JCV index values could beattributed to the seroconversion of initially JCV2patients it was also important to see whether patientspresented with stable index values once they con-verted to JCV seropositivity Two hundred oneJCV1 patients were therefore followed over timeand it became clear that the biomarker showedchanges over time with higher values after the period










Figure 2 JCV index





























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Figure 2 JCV index





























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closure forms





899ndash910













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Reprints




Therapy with natalizumab is associated with high JCV ... · the index values of JCV1 patients did...

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