Plasma Biomarkers Discriminate Clinical Forms of Multiple Sclerosis

22 Pages·2017·1.36 MB·English

Plasma Biomarkers Discriminate Clinical Forms of
Multiple Sclerosis
Marta Tejera-Alhambra, Armanda Casrouge, Clara de Andrés, Ansgar
Seyfferth, Rocío Ramos-Medina, Bárbara Alonso, Janet Vega, Lidia
Fernández-Paredes, Matthew L. Albert, Silvia Sánchez-Ramón
To cite this version:
Marta Tejera-Alhambra, Armanda Casrouge, Clara de Andrés, Ansgar Seyfferth, Rocío Ramos-
Medina, et al.. Plasma Biomarkers Discriminate Clinical Forms of Multiple Sclerosis. PLoS ONE,
2015, 10 (6), pp.e0128952. ￿10.1371/journal.pone.0128952￿. ￿pasteur-01380997￿
HAL Id: pasteur-01380997
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RESEARCHARTICLE
Plasma Biomarkers Discriminate Clinical
Forms of Multiple Sclerosis
MartaTejera-Alhambra1,ArmandaCasrouge2,3,ClaradeAndrés4,AnsgarSeyfferth5,
RocíoRamos-Medina1¤a,BárbaraAlonso1,JanetVega6,LidiaFernández-Paredes7,
MatthewL.Albert2,3,SilviaSánchez-Ramón1¤b*
1 DepartmentofImmunology,HospitalGeneralUniversitarioGregorioMarañón,Madrid,Spain,
2 DepartmentofImmunology,CenterforHumanImmunology,InstitutPasteur,Paris,France,3 Department
ofImmunology,INSERMU818,InstitutPasteur,Paris,France,4 DepartmentofNeurology,HospitalGeneral
UniversitarioGregorioMarañón,Madrid,Spain,5 STAT-UPStatisticalConsulting&Services,Madrid,Spain,
6 CenterAliciaKoplowitzforMultipleSclerosisoftheCommunityofMadrid,Madrid,Spain,7 Departmentof
ClinicalImmunology,HospitalClínicoSanCarlos,Madrid,Spain
¤a Currentaddress:DepartmentofClinicalOncology,LaboratoryofTranslationalOncology,Hospital
GeneralUniversitarioGregorioMarañón,Madrid,Spain
¤b Currentaddress:DepartmentofClinicalImmunology,HospitalClínicoSanCarlos,Madrid,Spain
* [email protected]
Abstract
OPENACCESS
Citation:Tejera-AlhambraM,CasrougeA,deAndrés
C,SeyfferthA,Ramos-MedinaR,AlonsoB,etal. Multiplesclerosis,themostcommoncauseofneurologicaldisabilityinyoungpopulation
(2015)PlasmaBiomarkersDiscriminateClinical aftertrauma,representsasignificantpublichealthburden.Currentchallengesassociated
FormsofMultipleSclerosis.PLoSONE10(6):
withmanagementofmultiplesclerosis(MS)patientsstemfromthelackofbiomarkersthat
e0128952.doi:10.1371/journal.pone.0128952
mightenablestratificationofthedifferentclinicalformsofMSandthusprompttreatmentfor
AcademicEditor:OscarArias-Carrion,Hospital
thosepatientswithprogressiveMS,forwhomthereiscurrentlynotherapyavailable.Inthe
GeneralDr.ManuelGeaGonzález,MEXICO
presentworkweanalyzedasetofthirtydifferentplasmacytokines,chemokinesandgrowth
Received:October21,2014
factorspresentincirculationof129MSpatientswithdifferentclinicalforms(relapsingremit-
Accepted:April30,2015 ting,secondaryprogressiveandprimaryprogressiveMS)and53healthycontrols,across
Published:June3,2015 twoindependentcohorts.ThesetofplasmaanalyteswasquantifiedwithLuminexxMAP
technologyandtheirpredictivepowerregardingclinicaloutcomewasevaluatedbothindivid-
Copyright:©2015Tejera-Alhambraetal.Thisisan
openaccessarticledistributedunderthetermsofthe uallyusingROCcurvesandincombinationusinglogisticregressionanalysis.Ourresults
CreativeCommonsAttributionLicense,whichpermits fromtwoindependentcohortsofMSpatientsdemonstratethatthedivergentclinicalandhis-
unrestricteduse,distribution,andreproductioninany
tology-basedMSformsareassociatedwithdistinctprofilesofcirculatingplasmaproteinbio-
medium,providedtheoriginalauthorandsourceare
markers,withdistinctsignaturesbeingcomposedofchemokinesandgrowth/angiogenic
credited.
factors.Withthiswork,weproposethatanevaluationofasetof4circulatingbiomarkers
DataAvailabilityStatement:Allrelevantdataare
(HGF,Eotaxin/CCL11,EGFandMIP-1β/CCL4)inMSpatientsmightserveasaneffective
withinthepaper.
toolinthediagnosisandmorepersonalizedtherapeutictargetingofMSpatients.
Funding:ThisstudywasfundedbyFundación
Mapfre(http://www.fundacionmapfre.org),Fundación
Salud2000(http://www.fundacionsalud2000.com),
FondodeInvestigaciónSanitaria(FIS#12/2759),and
theEuropeanResearchCouncilStartingAward
(http://erc.europa.eu).MartaTejera-Alhambra
receivedanEFISgrantin2011forherstayatInstitut Introduction
PasteurinParis.AnsgarSeyfferthisanindependent
Multiplesclerosis(MS),themostcommoncauseofneurologicaldisabilityinyoungpopulation
statisticalconsultantforSTAT-UPStatistical
Consulting&Services.Thefundershadnorolein aftertrauma,representsasignificantpersonal,socialandeconomicpublichealthburden.MS
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 1/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
studydesign,datacollectionandanalysis,decisionto isachronicautoimmuneinflammatorydisorderofthecentralnervoussystem(CNS)charac-
publish,orpreparationofthemanuscript. terizedbymultipledemyelinationlesions,axonaldegenerationandoligodendrocyteandneu-
CompetingInterests:AuthorAnsgarSeyfferthisa ronalloss.ThepreciseetiologyofMSremainsunknown,althoughitiswidelyheldthatMSisa
freelancestatisticalconsultantforSTAT-UPStatistical Th1/Th17autoimmunedisease,whereself-reactiveeffectorTcellsinitiatetheinflammatory
Consulting&Services,anindependentbiostatistics cascade.TheclinicalcourseofMSgoesfromanearlyinflammatoryphaseofthediseasewith
company.Thisdoesnotaltertheauthors'adherence
relapsesandremissions,wherepatientsmayrespondtoimmunomodulatorydrugs,toapro-
toPLOSONEpoliciesonsharingdataandmaterials.
gressiveandneurodegenerativephasethatisunresponsivetoanycurrentlyavailabletreatment.
Between60–70%ofpatientswitharelapsing-remittingMS(RR-MS)formevolvetoasecond-
ary-progressiveformofMS(SP-MS).About20%ofpatientssufferfromaprogressiveonsetof
thediseasewithoutremissionsofinfaustprognosis,knownasprimaryprogressiveMS
(PP-MS)[1].Ineithercase,itremainsplausiblethatthevariousclinicalMSformsmayrepre-
sentdivergentetiologies,giventhedistinctpathologicalpatternsandtheclinicalcharacteristics
theyexhibit.AlthoughCNSmagneticresonanceimaging(MRI)andthepresenceofoligoclo-
nalbandsinthecerebrospinalfluid(CSF)havehelpedinthediagnosisofMS,theydonotdis-
criminatebetweentheinflammatoryandprogressiveformsatMS.
ManycytokinesandtheirreceptorshavebeenpredominantlydetectedinMSlesionsand
theyarethoughttoplayaroleinMSpathogenesisviaimmunesystemactivationaswellasvia
damagingneuronalcells.Proinflammatorycytokineshavebeenextensivelystudied.CSFlevels
ofproinflammatorycytokinesareoftenelevatedinMSpatients[2–8].
ThecurrentchallengesinthemanagementofMSpatientsarelinkedtothelackofbiomark-
erscapableofstratifyingthedifferentclinicalformsofMS.Thisisahighpriorityduetothe
needtodefinethosepatientsthatmayevolvetoprogressiveforms.Indeed,thedevelopmentof
minimallyinvasivebiomarkersrepresentsanimportantavenuefordiscriminatingamongthe
differentformsofthediseaseandforpredictingtreatmentresponse.Theycanalsohelptoshed
lightonMSpathogenesis.
InourcohortofMSpatients,weevaluatedplasmaprofilesofcytokines,chemokinesand
growthfactorsbothindividuallyusingROCcurvesandincombinationusinglogisticregres-
sionanalysisfortheirpredictivepowerregardingtheclinicaloutcome.Wehavefoundasetof
potentialbiomarkersdifferentlyexpressedintherelapsing-remittingMSpatientscomparedto
MSpatientsintheprogressivephaseofthediseasethatmightserveasaneffectivetoolforstrat-
ifyingMSpatientsandbettertargetpersonalizedtherapiesforthiscomplexdisease.
MaterialsandMethods
Subjects
Atotalof182subjectswerestudiedinthiswork.Amongthem,129patientswithdefiniteMS
diagnosisaccordingtoMcDonald’scriteria[9]wereconsecutivelyrecruitedfromNovember
2010toJune2012,attheUnitofMultipleSclerosisoftheUniversityGeneralHospitalGregorio
MarañónandfromCenterAliciaKoplowitzforMultipleSclerosisoftheCommunityofMa-
drid,Spain.Agroupof53age-matchedhealthycontrols(HC)(29women/24men;age:37
years(31–43)medianvalue(IQR1-3))fromtheBloodDonorBankoftheUniversityHospital
GregorioMarañónwasalsoincluded.Patientswereconsideredandanalyzedastwoindepen-
dentcohorts:thetestcohort(cohort1)wasrecruitedfromNovember2010toFebruary2011
andconsistedof65MSpatients(47womenand18men)and16HC(9womenand7men);
thevalidationcohort(cohort2)wasrecruitedfromJune2011toJune2012andconsistedof64
MSpatients(38womenand26men)and37HC(20womenand17men).
MSpatientswerecharacterizedintotwomaingroups,as“inflammatoryMSorRR-MS”and
“progressiveMS”.The“inflammatoryMS/RR-MS”groupconsistedofpatientsdiagnosedas
Relapsing-Remittingformthatwereintheinflammatoryphaseofthediseaseandwas
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 2/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
Table1. Epidemiologicalandclinicalcharacteristicsofmultiplesclerosispatientsincludedinthestudy.
InflammatoryMS/RR-MS ProgressiveMS
Characteristics CD-MS RR-MS RR-MS RELAPSES RESPONDERS NON SP-MS PP-MS
Remission Active RESPONDERS
No.ofpatientsa 129 23 13 11 20 13 31 18
GenderF/Ma 85/44 17/6 8/5 8/3 10/10 11/2 22/9 9/9
Age(years)b 42(33–53) 38(32–46) 29(26–37) 30(26–42) 37(33–41) 38(29–43) 53(46– 58(45–64)
58)
Diseaseduration 11(3– 9(5–16) 1(1–4.5) 1(0–9) 9(1.8–16.3) 11(5–15.5) 19(13– 20.5(8.8–
(yrs)b 17.5) 27) 26.3)
Ageatonset(yrs)b 28(24–36) 28(22–32) 26(25–35) 28(25–40) 26(23–35) 26(24–38) 32(26– 40(33–50)
41)
EDSSb 2.5(1.0– 0.0(0.0–1.5) 2.5(1.0– 3.0(2.0– 1.0(0.0–2.0) 3.5(2.3–4.5) 8.0(7.5– 8.0(7.3–
5.5) 3.0) 4.0) 8.5) 8.0)
ProgressionIndexc 0.3(0.1– 0.0(0.0–0.1) 1.5(0.2– 0.8(0.2– 0.1(0.0–0.3) 0.3(0.2–0.5) 0.4(0.3– 0.4(0.3–
0.6) 2.9) 5.4) 0.6) 0.9)
No.ofrelapsesd
0 73 17 0 6 2 0 30 18
1 18 4 3 3 4 3 1 0
2–5 38 2 10 2 14 10 0 0
Treatment,IFNβa 20 _ _ _ 20 _ _ _
Treatmentduration 18(6–24) _ _ _ 18(6–24) _ _ _
(mo)b
CD-MSclinicallydefiniteMSgroup,RR-MSRemission:relapsingremittingMSinremission,RR-MSActive:relapsingremittingMSwithactivedisease,
RESPONDERS:RR-MSundertreatmentwithIFNβ,NON-RESPONDERS:relapsingremittingMSthatdidnotrespondtoInterferon-β,SP-MS:secondary
progressiveMS,PP-MS:primaryprogressiveMS.
EDSSexpandeddisabilitystatusscale,IFNβ:interferon-β.
aNumberofpatients;
bMedian(25th-75thpercentiles)
cEDSS/Diseaseduration(years);
dNumberofrelapsesduringpreceding2years.
doi:10.1371/journal.pone.0128952.t001
composedofthefollowingsubgroups:Recurrent-RemittingMSpatientsinremission(RR-MS
Remission),RR-MSpatientswithactivedisease(RR-MSActive),patientsatclinicalexacerba-
tionorrelapse(RELAPSES),RR-MSpatientsundertreatmentwithIFN-βforaminimumof6
monthswhohadagoodclinicalresponsetoIFN-βandweredefinedaslong-termIFN-βre-
sponders(RESPONDERS),RR-MSpatientsthathadnotrespondedtoIFN-β(NON-RE-
SPONDERS)andweregoingtostartotherdisease-modifyingtherapy(DMT).The
“progressiveMS”groupincludedpatientsdiagnosedasSecondary-ProgressiveMS(SP-MS)or
Primary-ProgressiveMS(PP-MS).Exceptforthelong-termIFN-βRESPONDERSgroup,the
restofthepatientshadnotreceivedanyglucocorticoidtreatment,immunosuppressiveor
DMTbeforebloodsamplinginthethreemonthspriortostudyentry.Thedifferentgroupsare
depictedinTable1.InthegroupofRR-MSRemission,therewere17benignformsofMSthat
hadbeensymptom-freeforyears.ApatientwithRR-MSwithactivedisease(RR-MSActive)
wasdefinedasapatientwithclinicalandparaclinicalactivityofthedisease,thatwasstudiedat
least1monthaftertheendofaclinicalexacerbationandwaseligibletostartaDMT.Relapse
wasdefinedastheappearanceorreappearanceofoneormoreneurologicalabnormalitiesthat
persistedforatleast24handwhichhadbeenprecededbyatleast30daysofstableorimproved
neurologicalstate,withoutanyunderlyinginfectiousdisease;bloodsamplesweredrawnfrom
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 3/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
relapsepatientsbeforeinitiationofglucocorticoidtreatment.Clinicaldiseaseseverityanddis-
abilitywasratedaccordingtotheKurtzkeExpandedDisabilityStatusScale(EDSS)[10].For
detailedpatientcharacteristics,seeTable1.ThisstudywasapprovedbyUniversityHospital
GregorioMarañónEthicsCommitteeinMadridandallsubjectsenrolledinthestudygave
writteninformedconsent.
Multiplexedbiomarkersdetection
PeripheralbloodsamplesweretakenbyvenouspunctureandcollectedinsterileEDTAVacu-
tainers,between8:00and10:00a.m.,theywereprocessedimmediatelyorwithin2hoursafter
extraction.Plasmasampleswereobtainedafterhighspeedcentrifugationfor10minutesat
3,500–4,000rpmandimmediatelyaliquotedandfrozenat-80°Cforitsconservation.
Plasmasampleswereclarifiedbyhigh-speedcentrifugationandanalyzedusingLuminex
xMAPtechnologyplatform.Thecurrentinvestigationrequiredtheassemblyofanextensive
multiplexarrayconsistingofcytokines,chemokines,solublereceptors,growthandangiogenic
factors,whichwereevaluatedusingbead-basedimmunoassays.Theselectedarraywasthe
Humancytokine30-Plexpanel(Invitrogen)withthefollowinganalytes:IL-1β,IL-1RA,IL-2,
IL-2R,IL-4,IL-5,IL-6,IL-7,IL-8,IL-10,IL-12,IL-13,IL-15,IL-17,TNF-α,IFN-α,IFN-γ,
GM-CSF,MIP-1α,MIP-1β/CCL4,IP-10,MIG,Eotaxin/CCL11,Rantes/CCL5,MCP-1/CCL2,
VEGF,G-CSF,EGF,FGF-basic,andHGF.
Statisticalanalysis
Descriptivedataarepresentedasmean±standarddeviation(SD)andmedian(interquartile
range).Wecomparedcategoricalbythechi-squareχ2test.Whenmultiplegroupswithcontin-
uousoutcomeswerecompared,thenonparametricKruskal-Wallisranksumtestwasused,fol-
lowedbypairwiseMann-Whitneytestsiftheformerindicatedsignificantdifferences.
CorrelationswereassessedusingSpearmancorrelation(r)coefficients.Receiveroperating
s
characteristic(ROC)curveswereusedtoselecttheoptimalcut-offvaluesofsignificantvari-
ablesforconsideringnegativeorpositivepredictorsofthedevelopmentofanygivenclinical
conditionstudied.Logisticregressionandtheareaunderthereceiveroperatingcurves
(AUROC)wereusedtodeterminethediagnosticaccuracyofamodelthatincludedbiomarkers
abletodiscriminatebetweenpatientsandhealthycontrols.DatawereanalyzedwithSPSS(Chi-
cago,Illinois)andGraphPadPrismsoftware(CA,USA).Ap-valuelessthan0.05wasconsid-
eredasstatisticallysignificant.TheBonferronicorrectionwasappliedtocompensatethe
multiplicityofthetests.
Results
DemographicandclinicalcharacteristicsofMSpatients
InourMSstudypopulation,80outof129(62%)MSpatientswereclassifiedintheinflamma-
toryphaseofthediseaseandwerediagnosedasrelapsing-remittingMS:RR-MSRemission
n=26,RR-MSActiven=13,RESPONDERSn=20,NONRESPONDERSn=13andRE-
LAPSESn=11(for6outof11itwastheirfirstclinicalepisode(CIS),andtheywerelaterdiag-
nosedasRR-MS).Therewere49patientsintheprogressivephaseofthedisease,31were
diagnosedasSP-MSand18asPP-MS.
RR-MSpatientsandpatientsatrelapsehadsimilarages,whileprogressivepatientsSP-MS
andPP-MSwerearound20yearsolder(Table1).Thelowestdiseaseduration(median1year)
wasinthegroupsofRR-MSwithactivediseaseandpatientsatrelapse.InRR-MSatremission
andrespondersandnon-responderstoIFNβ,meandiseasedurationwasaboutadecadeandin
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 4/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
theprogressiveforms(SP-MSandPP-MS)twodecades.SP-MSandPP-MShadthehighest
EDSScomparedtotheothergroups.EDSSinRR-MSRemissionandinIFNβRESPONDERS
werethelowest(median,0and1,respectively),asexpected.Theprogressionindex(EDSS/Dis-
easeduration(years)wasthehighestinRR-MSActivepatients,astheyhadbeenrecentlydiag-
nosedandpresentedwithconsiderabledisability.AsdepictedinTable1,thepatientswith
highestnumberofrelapsesintheprecedingtwoyearswherethoseundertreatmentwithIFNβ
(RESPONDERS)orabouttostarttherapywithaDMT(RR-MSActiveandNON
RESPONDERS).
PanelofPlasmaBiomarkersthatDiscriminatebetweenRelapsing-
RemittingandProgressiveClinicalFormsofMultipleSclerosis
Weproceededfromthehypothesisthatbiomarkerspresentinthecirculationofpatientsdiag-
nosedwithdifferentclinicalformsofMScanprovideclinicallyrelevantinformationpertaining
totheprogressionofthediseaseandalsoabasisforthediscriminationbetweenthethreecon-
ditions(RR-MS,SP-MSandPP-MS).Theevaluationofthishypothesiswasconductedaccord-
ingtothefollowingobjectives:firstly,biomarkerslevelspresentintheplasmaofseveral
distinctclinicalformsofMSwereexaminedinordertoidentifyalterationsassociatedwithspe-
cificdiseaseforms.Secondly,wefocusedontheuseofacombinationofbiomarkersusinglo-
gisticregressionfortheirpredictivepowerachievingsuperiorsensitivityandspecificity.
GiventheintrinsicheterogeneityofMS,thetwocohortswerepooledtoincreasethenumber
ofcasesandcontrols.WithalltheMSpatientsgloballyconsidered,the30analytesweretested
aspredictorsforthediscriminationbetweenRelapsing-RemittingandProgressiveClinical
formsofMSbyROCcurves.Forsixofthem(HGF,Eotaxin/CCL11,MCP-1/CCL2,Rantes/
CCL5,EGFandMIP-1β/CCL4)ap-Value<0.05wasfound,whichinallsixcaseswas
also<0.05/30(Table2).HencetheseresultsarestillsignificantevenwhenapplyingtheBon-
ferronicorrectiontothesignificancethreshold,themostconservativemethodtocompensate
themultiplicityofthetests.
HGF,Eotaxin/CCL11,MCP-1/CCL2andRantes/CCL5arepresentin
lowerconcentrationsintheplasmaofRelapsing-Remittingthanin
ProgressiveMultipleSclerosispatients
TheanalytesHGF,Eotaxin/CCL11,MCP-1/CCL2andRantes/CCL5werepresentatsignifi-
cantlylowercirculatinglevelsintheRR-MSpatientsthanintheprogressiveclinicalformsof
MSpatients(SP-MSandPP-MS).
TheanalysisofthetestcohortrevealedthatHGF,Eotaxin/CCL11,MCP-1/CCL2,Rantes/
CCL5werepresentinsignificantlylowerconcentrationsintheinflammatory(RR-MS)phase
Table2. BiomarkersthatdiscriminatebetweenRelapsing-RemittingandProgressiveClinicalformsofMSbyROCcurves.
RR-MSvsProgressiveMS AUC (95%CI) pvalue Cutoff(pg/ml)
HGF 0.702 (0.604–0.799) <0.0002 387
Eotaxin/CCL11 0.755 (0.662–0.849) <0.0001 132
MCP-1/CCL2 0.721 (0.626–0.816) <0.0001 550
Rantes/CCL5 0.792 (0.707–0.876) <0.0001 5536
EGF 0.689 (0.592–0.786) 0.0005 66
MIP-1β/CCL4 0.702 (0.607–0.798) <0.0002 109
Theareasunderthecurve(AUC),95%ConfidenceInterval(95%CI)andpvalueforeachanalytearedepictedinTable2.
doi:10.1371/journal.pone.0128952.t002
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PlasmaBiomarkersintheDifferentClinicalFormsofMS
Table3. HGF,Eotaxin/CCL11,MCP-1/CCL2,Rantes/CCL5,EGFandMIP-1β/CCL4comparisoninthe
testandvalidationcohorts.
RR-MS ProgressiveMS
Testcohort
HGF[pg/mL] 117(67–351)** 406(188–508)
Eotaxin/CCL11[pg/mL] 69(44–99)** 119±65
MCP-1/CCL2[pg/mL] 325(265–519)# 447(337–561)
Rantes/CCL5[pg/mL] 2,359(1,105–6,066)*** 7,791(5,548–7,791)
EGF[pg/mL] 55(8–89)*** 8(8–8)
MIP-1β/CCL4[pg/mL] 135(88–183)*** 51(37–93)
Validationcohort
HGF[pg/mL] 334(239–430)** 536(344–805)
Eotaxin/CCL11[pg/mL] 88(60–122)*** 230(121–381)
MCP-1/CCL2[pg/mL] 357(205–461)*** 680(391–1,005)
Rantes/CCL5[pg/mL] 2,641(1,649–4,841)*** 23,644(4,029–23,644)
EGF[pg/mL] 106(63–155)# 64(46–106)
MIP-1β/CCL4[pg/mL] 131(95–189)# 94(79–129)
Mann—Whitneystatisticaltestwasusedforcalculationofthereportedp-valuebetweenRR-MSvs
ProgressiveMS;RR-MS=Relapsing-RemittingMSpatients.NotethattheRelapsing-remittinggroup
comprisestheclinicalgroups:RR-MSRemission,RR-MSActive,RESPONDERS,NONRESPONDERS
andRELAPSES.TheProgressivegroupcomprisesSecondary(SP-MS)andPrimary-progressivepatients.
ResultsaregivenasMedianvalue(IQR1–3).Statisticalsignificanceismarkedas:
*p<0.05;
**p<0.01;
***p<0.001.
#p=0.06(trend).
doi:10.1371/journal.pone.0128952.t003
withrespecttotheprogressiveMSclinicalforms(SP-MSandPP-MS),findingsthatwerecon-
firmedinthevalidationcohort(Table3).HGF,Eotaxin/CCL11andRantes/CCL5differences
betweenthesetwogroupswerestatisticallysignificantinbothcohortsanalyzedindependently
(Table3).PlasmaMCP-1/CCL2levelsinRR-MSpatientsshowedatrendtobelowerinthe
testcohort(p=0.06),whileinthevalidationcohort,thisdifferencewasstatisticallysignificant
(p=0.0002).
Thetwocohortswerepooledtoincreasethenumberofcasesandcontrols.Again,inMSpa-
tientsgloballyconsidered,wefoundthatplasmalevelsofHGF,Eotaxin/CCL11,MCP-1/CCL2
andRantes/CCL5weresignificantlylowerintheinflammatory(RR-MSpatients)thaninthe
progressiveforms(SP-MSandPP-MS).InFig1theresultsfromRR-MSarecomparedtothose
oftheprogressiveMSpatients:(HGF:294±221vs484±310,p=0.0002;Eotaxin/CCL11:87±56
vs187±143,p<0.0001;MCP-1/CCL2:375±227vs588±309,p<0.0001;Rantes/CCL5:4,173
±4,855vs11,371±8,800,p<0.0001).Interestingly,thelevelsofHGF,MCP-1/CCL2and
Eotaxin/CCL11intheprogressiveMSpatientsweresimilartothoseobservedinhealthycon-
trols(HGF:484±310vs476±652;Eotaxin/CCL11:187±143vs147±138;MCP-1/CCL2:588
±309vs538±315),whileRantes/CCL5wasmoreelevatedintheprogressiveformswithrespect
tohealthycontrols(Rantes/CCL5:11,371±8,800vs5,518±6,796).RR-MSpatientshadsignifi-
cantlylowerlevelsofEotaxin/CCL11,MCP-1/CCL2andRantes/CCL5thanhealthycontrols
(Eotaxin/CCL11:87±56vs147±138,p=0.001,MCP-1/CCL2:375±227vs538±315,p=0.001
andRantes/CCL5:4,173±4,855vs5,518±6,796,p=0.0002).
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PlasmaBiomarkersintheDifferentClinicalFormsofMS
Fig1.DifferentplasmalevelsofHGF,Eotaxin/CCL11,MCP-1/CCL2andRantes/CCL5inMSclinicalforms.PlasmalevelsofHGF,Eotaxin/CCL11,
MCP-1/CCL2andRantes/CCL5werelowerinrelapsing-remittingMSthanintheprogressiveMSpatients.ComparisonoftheplasmalevelsofHGF,Eotaxin/
CCL11(topdotplots),MCP-1/CCL2andRantes/CCL5(lowerdotplots)inrelapsing-remitting(RR-MS,n=80)andinthegroupofprogressiveMS(n=49)
patients.Mann—Whitneystatisticaltestwasusedforcalculationofthereportedp-value;medianvaluesarerepresentedbyagraybar;individualdots
indicatesingledonorvalues.NotethattheRelapsing-remittinggroupcomprisestheclinicalgroups:RR-MSRemission,RR-MSActive,RESPONDERS,
NONRESPONDERSandRELAPSES.TheProgressivegroupcomprisesSecondary(SP-MS)andPrimary-progressivepatients.
doi:10.1371/journal.pone.0128952.g001
TheROCanalysesindicatedagoodperformanceofplasmaHGF,Eotaxin/CCL11,MCP-1/
CCL2andRantes/CCL5forthediscriminationbetweenRR-MSfromprogressiveclinical
forms(Table2).
HGFcorrelatedstronglywithMCP-1/CCL2bothinMSpatientsandinhealthycontrols(r
s
=0.4674,p<0.0001),whilstHGFcorrelatedwithEotaxin/CCL11(r =0.5574,p<0.0001)and
s
Rantes/CCL5(r =0.3317,p=0.0002)onlyinMSpatients.
s
HGF,MCP-1/CCL2,Eotaxin/CCL11andRantes/CCL5correlatedalsowithneurological
disabilitymeasuredbytheEDSS:HGF(r =0.3254,p=0.0003),MCP-1/CCL2(r =0.2714,
s s
p=0.003),Eotaxin/CCL11(r =0.3610,p<0.0001),Rantes/CCL5(r =0.4386,p<0.0001).
s s
EGFandMIP-1β/CCL4arediminishedinProgressiveMSclinicalforms
Bothinthetestcohortandinthevalidationcohort,EGFandMIP-1β/CCL4plasmalevelswere
lowerintheprogressiveMSforms(SP-MSandPP-MS)withrespecttoRR-MSpatients.Inthe
testcohort,plasmaEGFandMIP-1β/CCL4statisticallydifferedinbothclassificationgroups
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 7/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
Fig2.DifferentplasmalevelsofEGFandMIP-1β/CCL4inMSclinicalforms.PlasmalevelsofEGFandMIP-1β/CCL4werelowerintheprogressiveMS
thaninrelapsing-remittingMSpatients.ComparisonoftheplasmalevelsofEGFandMIP-1β/CCL4betweenpatientswithrelapsing-remitting(RR-MS,
n=80)andinprogressiveMSpatients(n=49).Mann—Whitneystatisticaltestwasusedforcalculationofthereportedp-value;medianvaluesare
representedbyagraybar;individualdotsindicatesingledonorvalues.NotethattheRelapsing-remittinggroupcomprisestheclinicalgroups:RR-MS
Remission,RR-MSActive,RESPONDERS,NONRESPONDERSandRELAPSES.TheProgressivegroupcomprisesSecondary(SP-MS)andPrimary-
progressivepatients.
doi:10.1371/journal.pone.0128952.g002
(p<0.0001andp=0.0002,respectively);inthevalidationcohort,bothanalytesshowedatrend
(p=0.06)tobeatlowerconcentrationsintheplasmaofprogressivepatients(SP-MSand
PP-MS)thaninRR-MSpatients(Table3).
Thepoolofbothcohortsdisplayedthesamedistributionwithsignificantlydiminishedcir-
culatinglevelsofEGFandofthechemokineMIP-1β/CCL4inpatientswithprogressiveforms
ofMSthaninRR-MSpatients(EGF:87±71vs50±54,p=0.0005;MIP-1β/CCL4:141±79vs93
±67,p=0.0002)(Fig2).RR-MSpatientsandhealthycontrolsshowedsimilarcirculatinglevels
ofEGFandMIP-1β/CCL4(EGF:87±71vs115±92;MIP-1β/CCL4:141±79vs170±116)and
progressivepatientshadsignificantlylowerlevelsofEGFandMIP-1β/CLL4thanHC(EGF:50
±54vs115±92,p<0.0001andMIP-1β/CCL4:93±67vs170±116,p=0.0002,respectively).The
ROCcurvesofplasmaEGFandMIP-1β/CCL4wereusedtoassessthepotentialidentification
betweenRR-MSandprogressive(SP-MSorPP-MS)clinicalforms(Table2).
Asexpected,EGFandMIP-1β/CCL4correlatednegativelywiththedisabilityscoreEDSS:
EGF(r =-0.2906,p<0.002,MIP-1β/CCL4(r =-0.355,p<0.0001).
s s
Logisticregression
Wecarriedoutthemultivariatelogisticregressionanalysisincludingallthesesixanalytes
(HGF,MCP-1/CCL2,Eotaxin/CCL11,Rantes/CCL5,EGFandMIP-1β/CCL4)asindependent
variablesandtheclinicalMS-forms(Relapsing-RemittingvsProgressive)asthedichotomous
targetvariable.Weobtainedamodelthatincludedthe4analytesHGF,EGF,Eotaxin/CCL11
andMIP-1β/CCL4)(Table4)aspredictorsfortheMS-clinicalform,whileRantes/CCL5and
MCP-1/CCL2didnotreachstatisticalsignificancetobeincluded.
Withthiscombinationmodel,wewereabletogivetheoddsforaspecificpatienttobediag-
nosedasprogressiveMS,i.e.theprobability—P—ofbeingclassifiedasprogressiveclinicalform
ofMSdividedbytheprobabilityofdevelopingarelapsing-remittingform1-P,accordingtothe
modelaregivenbytheproductof0.4021andthefouroddsratioseachraisedbytherespective
predictors’values(inpg/mL)measuredinanindividualpatient.Eachanalytehasanoddsratio
(O.R.)whichisraisedtothepowerofitsrespectivevalue[pg/mL]inapatient.Forinstance,for
apatientwithvalues[pg/mL]ofEotaxin/CCL11:200;HGF:323;EGF:17;MIP-1β/CCL4:54,
PLOSONE|DOI:10.1371/journal.pone.0128952 June3,2015 8/21
PlasmaBiomarkersintheDifferentClinicalFormsofMS
Table4. LogisticregressionwithHGF,Eotaxin/CCL11,EGFandMIP-1β/CCL4asindependentvari-
ablesandtheclinicalMS-forms(Relapsing-RemittingvsProgressiveMS)asthedichotomoustarget
variable.
RR-MSvsProgressiveMS Pvalue O.R. C.I.(95%)
HGF[pg/mL] 0.0085 1.0038 1.0010–1.0066
Eotaxin/CCL11[pg/mL] 0.0004 1.0147 1.0066–1.0229
EGF[pg/mL] 0.0056 0.9808 0.9675–0.9944
MIP-1β/CCL4[pg/mL] 0.0171 0.9873 0.9769–0.9977
Constant 0.1049 0.4021
O.R.(oddsratio);C.I.(95%)Intervalofconfidence.
doi:10.1371/journal.pone.0128952.t004
theoddsare0.4021(cid:1)1.0147200(cid:1)1.0038323(cid:1)0.980817(cid:1)0.987354=9.14.Hence,themodelassigns
tothispatientaprobabilityofhavingaprogressiveformof9.14/(1+9.14)=90.1%.
HGFandEotaxin/CCL11withO.R.>1(Table4)areriskfactorsfordevelopingaprogres-
siveclinicalformofMS,whileEGFandMIP-1β/CCL4withO.R.<1areprotectivefactorsfor
developingaprogressiveclinicalformofMS.
Consideringodds>/<1,i.e.agreater/lessprobabilityofhavingaprogressiveversus
RR-MSclinicalformaccordingtothemodel,asapositive/negativeprognosis,themodelgives
asensitivityof71.7%,aspecificityof89.9%,apositivepredictivevalue(PPV)of82.5%,anega-
tivepredictivevalueof82.7%andanaccuracyof82.6%forourcohortofMSpatients.
Eachoftheanalytesincludedinthecombinationmodelbymultivariatelogisticregression
analysiswasindependentlyanalyzedbyunivariateregressionanalysis(ROCAnalysis)forits
predictiveabilitytodiscriminatebetweenaProgressiveandaRR-MSpatient.Foreachana-
lyte,anspecificcut-offvalue:HGF>387,Eotaxin/CCL11>132,EGF<66andMIP-1β/CCL4
<109pg/mlcoulddiscriminatewithdifferentsensitivity,specificity,positivepredictivevalue
(PPV),negativepredictivevalue(NPV)andaccuracybetweenaProgressiveandaRR-MSpa-
tient.ThecombinationofthefourplasmalevelsofHGF,Eotaxin/CCL11,EGFandMIP-1β/
CCL4withmultivariatelogisticregressionanalysesgaveahighersensitivityandspecificity
(Table5).
FGFbcandiscriminatebetweenPrimaryandSecondaryProgressive
patientsandbetweenPrimaryProgressivepatientsandRelapsing-
Remittingpatientsundergoingclinicalrelapse
PlasmaFGFbwasmarkedlydiminishedinPP-MSpatientswhencomparedtoSP-MS(11±10
vs26±27,p=0.01),patientsundergoingaclinicalrelapse(11±10vs21±11,p=0.01),or
Table5. ClinicalutilityofHGF,Eotaxin/CCL11,EGFandMIP-1β/CCL4asMSbiomarkersandclinicalutlityoftheircombinationinamodelbymulti-
variatelogisticregressionanalysis.
RR-MSvsProgressiveMS Sensitivity Specificity PPV NPV Accuracy
HGF 59.6% 72.6% 58.3% 73.6% 67.5%
Eotaxin/CCL11 53.2% 90.8% 78.1% 75.8% 76.4%
EGF 74.5% 59.7% 54.7% 78.2% 65.5%
MIP-1β/CCL4 70.2% 68.4% 57.9% 78.8% 69.1%
Modelbylogisticregressionanalysis 71.7% 89.9% 82.5% 82.7% 82.6%
PPV:positivepredictivevalue,NPV:negativepredictivevalue.
doi:10.1371/journal.pone.0128952.t005
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