BAFF/APRIL system in pediatric OMS: relation to severity, neuroinflammation, and immunotherapy.

0.97 MB·English

by  Pranzatelli, Michael R

Pranzatellietal.JournalofNeuroinflammation2013,10:10 JOURNAL OF
http://www.jneuroinflammation.com/content/10/1/10 NEUROINFLAMMATION
RESEARCH Open Access
BAFF/APRIL system in pediatric OMS: relation to
severity, neuroinflammation, and immunotherapy
Michael R Pranzatelli1*, Elizabeth D Tate1, Nathan R McGee1, Anna L Travelstead2, Jerry A Colliver3,
Jayne M Ness4 and Richard M Ransohoff5
Abstract
Background: B-cell dysregulation has been implicated but not fully characterized inpediatric
opsoclonus-myoclonus syndrome (OMS), a neuroblastoma-associatedneuroinflammatory disorder.
Objective: Toassess the role ofB-cell activatingfactor (BAFF) and a proliferation-inducing ligand (APRIL), two
critical B cell-modulating cytokines, as potential biomarkers of disease activityand treatmentbiomarkers inOMS.
Methods: SolubleBAFF and APRIL were measuredin cerebrospinal fluid (CSF) and serum by ELISA in433 children
(296OMS, 109 controls, 28 other inflammatoryneurological disorders (OIND)). BAFF-R receptors on circulating CD19
+B cellswere measured by flow cytometry.A blinded scorer rated motor severity on theOMS Evaluation Scale.
Immunotherapieswere evaluatedcross-sectionally and longitudinally.
Results: The mean CSF BAFF concentration, which was elevated inuntreated OMS and OIND, correlated with OMS
severity category (P = 0.006), and reduction by adrenocorticotropic hormone or corticotropin(ACTH)(−61%)or
corticosteroids (−38%) was seen ateach level ofseverity.In contrast, CSF APRIL was normal in OMS and OIND and
unaffected by immunotherapy. When the entire OMS dataset was dichotomized into ‘high’versus ‘normal’CSF BAFF
concentration, the phenotype of thehighgroup included greater motor severity and number ofCSF oligoclonal
bands, and a higher concentration of inflammatory chemokines CXCL13 and CXCL10 in CSF and CXCL9 and CCL21
inserum. Serum APRIL was 6.7-fold higher inthe intravenous immunoglobulins (IVIg) group, whereas serum BAFF
was 2.6-fold higher in therituximabgroup. The frequency ofB cell BAFF-R expression was similar in untreated and
treated OMS. Longitudinal studies of CSF BAFF revealed a significant decline in ACTH-treated patients (with or
without rituximab)(P < 0.0001). Longitudinal studies ofserum APRIL showed a 2.9-fold increase after 1 to 2g/kg
IVIg monotherapy (P =0.0003).
Conclusions: Striking distinctionsin BAFF/APRIL signaling were found. OMS displayed heterogeneity in CSF BAFF
expression, which met many but not allcriteria as a potentialbiomarker of disease activity.We speculate that CSF
BAFF mayhave more utility in a biomarker panelthanas a stand-alone biomarker, and that theselective
upregulation of both serum APRIL byIVIgand BAFF by rituximab, as well as downregulationof CSF BAFF by ACTH/
steroids, may have utility as treatment biomarkers.
Keywords: BAFF-R, Cerebrospinalfluid cytokines,Childhood neuroinflammatory disorders, Corticosteroids,
Corticotropin, IVIg,Neuroblastoma, Opsoclonus-myoclonus, Paraneoplastic syndrome, Rituximab
*Correspondence:[email protected]
1DepartmentofNeurology,NationalPediatricMyoclonusCenterand
NeuroimmunologyLaboratory,andSouthernIllinoisUniversitySchoolof
Medicine,POBox19643,Springfield,IL62794-9643,USA
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Pranzatellietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Introduction or sedation/anesthesia, drug treatments outside the
Opsoclonus-myoclonus syndrome (OMS) is a neuroin- study scope, orconcurrent autoimmunedisorders.
flammatorydisorderofchildrenandadults,whichisdem- The cross-sectional part of the study involved 276 chil-
onstrably paraneoplastic in about 50% of the cases [1]. drenwithOMS,meanage(SD)3.8±3.0years(range0.89
Our research on CSF in pediatric OMS has shown that B to 17 years; boys n = 120; girls n = 156), who were en-
cellsareexpanded[2],BcellchemoattractantCXCL13[3] rolled after consent was signed by parents and assent by
and B/T cell chemoattractant CXCL10 [4] are likewise older children.OMS groupsincludeduntreated,currently
overexpressed,and 35% of the patients harbor oligoclonal treated, and previously treated patients. Videotapes were
bands.[5]Responsestocurrentimmunotherapeuticstrat- made using a standardized procedure and later scored
egies, such as intravenous immunoglobulins (IVIg), corti- blindedontheOMSEvaluationScaletoyieldatotalscore
costeroids, and anti-CD20, also are consistent with B-cell [2]. Further details of treatment and scoring are provided
involvement[2]. in the figure legends. The children underwent a morning
Two critical cytokines for B-cell activation, prolifera- lumbarpuncturefollowingourspecifiedprotocol[2].
tion, homeostasis, and survival are a proliferating- Controlscomprisedchildrenwithvariousnon-inflammatory
inducing ligand (APRIL) and B-cell activating factor neurological disorders (NIND), including ataxia, devel-
(BAFF) [6]. They are members 13 and 13B, respectively, opmentaldisorders,headache,movementdisorders,sei-
of the tumor necrosis factor ligand superfamily (TNFLS) zures, and miscellaneous disorders. Their mean age was
[7,8]. Both are expressed by astrocytes [9,10], and also 8.4±5.6years,and96wereboysand91weregirls.Chil-
dendritic cells, macrophages, and monocytes [11]. In drenwithOIND,whowereevaluatedlocallyandthrough
human cerebrospinal fluid (CSF) and serum, they are theCenterforPediatric-OnsetDemyelinatingDiseasein
found insoluble form and often studied together in neu- Alabama, served as a comparison group for specificity.
roinflammatory diseases [12-14]. The BAFF receptor Theirdiagnosesincludedacutedisseminatedencephalo-
(BAFF-R or BR3), one of three receptors to which BAFF myelitis, encephalitis, meningitis, multiple sclerosis and
binds [11], is the principal BAFF receptor [15] expressed related disorders, and miscellaneous disorders. The
bynearly allmatureB cells [10]. OINDmeanagewas7.7±5.8years(range0.1to18years;
Our preliminary work on BAFF showed an increase in boysn=17;girlsn=23).
OMS and decrease by corticotropin (ACTH) or corticos- Two longitudinal OMS studies were performed. In the
teroids[16].ThepresentreportinanincreasedOMSsam- BAFF study, 42 children from the cross-sectional study
ple size tests the hypothesis that BAFF is a biomarker of were treated using ACTH-based conventional therapy
disease activity and treatment biomarker (not diagnostic with (n = 31) or without (n = 11) rituximab. There were
biomarker). It includes additional immunotherapy cross- 16 boys and 26 girls, mean age 3.7 ± 2.8 years (range 1.8
sectional groups; analysis of the relation to OMS severity to 18 years). CSF was obtained before and at 8.0 ± 2.1
(videodocumentedandscored)andduration;comparison months of treatment. In the APRIL study, 20 new chil-
withAPRIL,inflammatorychemokines,oligoclonalbands, dren with OMS were recruited for monotherapy with
and lymphocytesubsets;longitudinal studies of BAFFand intravenous immunoglobulins (IVIg) under FDA BB-
APRIL, and comparative studies in other inflammatory IND No. 5839. There were 11 boys and nine girls, mean
neurologicaldisorders(OIND).Toidentifythe‘phenotype’ age4.6±1.4years(range1.8to6.4years).Theyreceived
of the patients with high CSF values compared to those standard monthly clinical doses of 1 or 2 g/kg, and
with normal (control-like) values, we are now able to serum was collected before and at 4.1 ± 3.9 months
dichotomize the dataset. We also measured the BAFF-R oftreatment.
receptoroncirculatingBcellsinthecurrentstudy.
Cytokine/chemokinedetection
Methods CSF and serum were collected on ice, aliquotted, and
Patientsandprocedures stored at −80°C in biorepository freezers. Samples were
This prospective, case–control, translational research thawedontheassaydayandBAFFandchemokineswere
study is part of the ‘Study of Cytokines in Children with measured using Quantikine ELISA kits per instructions
Opsoclonus-Myoclonus Syndrome’ (ClinicalTrials.gov by the manufacturer (R&D Systems, Inc., Minneapolis,
NCT 00806182) [3]. Approximately 300 children were MN, USA). The comparison chemokine panel included
recruited through the National Pediatric Myoclonus CXCL9, CXCL10, CXCL12, CXCL13, CCL17, CCL21,
Center, regardless of severity or treatment status. Inclu- and CCL22 kits from the same vendor. Human APRIL
sion criteria included clinically confirmed diagnosis of ELISA kits were purchased from eBioscience (formerly
OMS and ages 0.6 to 18 years,without restriction due to Bender MedSystems, Vienna, Austria). Assays were
gender, race, ethnicity, or socioeconomic status. Exclu- performed in duplicate on undiluted samples, each
sion criteria were contraindications to lumbar puncture 96-well plate containing both control and OMS samples.
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Samples with values above the highest standard were re- analysis utilized Pearson correlations. Although the mean
run at a 1:2 or higher dilution. Outliers were verified by ageoftheOMSgroupwassignificantlylowerthanincon-
repeat measurement.The kit userhad no patient contact. trols or the OIND group(which did not differ), no correl-
BAFF sensitivity was 0.73 ± 6.7 pg/mL in CSF, and 1.5 to ation between age of controls and CSF BAFF
11.9 pg/mL in serum; APRIL, 0.4 pg/mL. The inter-assay concentration (P = 0.72) or CSF APRIL (P = 0.19) was
coefficient of variance (CV) was 9.4% (n = 22) for CSF found,andtheagerangeofthegroupswasequivalent.
BAFF, 8.5% (n = 20) for serum BAFF, 7.8% (n = 10) for Secondary analysis was performed on the OIND group
CSFAPRIL,and4.8%(n=8)forserumAPRIL.Thecorre- compared to the other groups. Also, OMS immunother-
spondingintra-assayCVwas4.8%(n=11),6.0%(n=10), apy groups were bundled based on common treatment
7.2% (n = 6), and 6.7% (n = 6). Freezer storage time effects for graphic presentation. Lastly, the dataset was
and the concentration of CSF BAFF (P = 0.21) or APRIL dichotomized based on cytokine levels 2 SD above the
(P=0.16),orserumconcentrations,werenotcorrelated. control mean to determine if two different phenotypes
emerged, and family-wise Bonferroni corrections (α at
BAFF-Randlymphocytesubsets 0.5/n) were performed separately on six clinical variables
BAFF-R receptors were measured ex vivo by flow cyto- (P < 0.008), nine chemokines/cytokines and oligoclonal
metry [17]. Peripheral venous blood was delivered to the bands (P <0.0055), and five lymphocyte subsets
flow cytometrist within 1 h of collection. A 100 μL ali- (P < 0.01) to control for the multiple comparisons that
quot was blocked with 0.2 mg/mL normal mouse IgG were made.
for 15 min at room temperature. Directly conjugated
monoclonal antibody probes (anti-BAFF-R, anti-CD19, Results
anti-CD45),purchased from Becton Dickinson(San Jose, BAFFcross-sectionalstudy
CA, USA), were added to the remaining cell suspension. DifferencesinCSF BAFFwerehighly significantbetween
Then 2 mL FACS lysing solution was added to lyse ery- groups (Figure 1A). Mean CSF BAFF was 57% higher in
throcytes, followed by a 10-min incubation at room untreated OMS than in controls. Twenty-three percent
temperature in the dark. The assay suspension was cen- of untreated OMS had CSF BAFF concentrations >2 SD
trifuged at 4°C for 7 min at 600 × g, supernatant was above the control mean. The three ACTH groups are
removed, the pellet was washed twice with 2 mL cold shown bundled, as are the three steroid groups, for lack
FACSbuffer,andrecentrifuged.Afterdecanting,100μLof of significant difference in CSF BAFF among them. CSF
1%paraformaldehydewasadded,andafter5minatroom BAFF was 56% lower in ‘All ACTH Groups’ and 45%
temperatureinthedark,therewasanothercentrifugation, lowerin‘AllSteroid Groups.’
decanting,andresuspensionofcellsinFACSbuffer. IncomparisonsofindividualOMStreatmentgroupsver-
Lymphocyte subsets, measured by flow cytometry as sus untreated OMS (data not shown), significantly lower
described previously [2], included cells that were CD19 mean CSF BAFF concentrations (pg/mL) were found for
+CD3- (B cells), CD4+CD3+ (T helper/inducer cells), ACTH (88 ± 59, -61%, P < 0.0001), steroid (140 ± 148,
CD8+CD3+ (T cytotoxic/suppressor cells), TCRγδ+ -38%, P = 0.002), ACTH + IVIg (113 ± 92, -50%,
(gamma/deltaTcells),orCD16/56+(NKcells).Datawere P < 0.0001), steroid + IVIg (114 ± 42, -49%, P = 0.0002),
acquired ona FACSCalibur cytometer and analyzedusing ACTH + other (89 ± 76, -60%, P < 0.0001), and steroid +
Cell Quest(Becton-Dickinson,SanJose,CA,USA).Qual- other(115±52,-49%,P=0.005).Ineachofthosegroups,
ity control measures wererigorous, as reported elsewhere the mean BAFF concentration was below, though not
[2].Theflowcytometristhadnopatientcontact. significantly different than, the control mean. Of actively
treated patients, 22% had CSF BAFF concentrations
Statisticalanalysis below the lowest controls. In previously treated patients,
Because of inter-individual differences and the large range mean CSF BAFF concentration did not differ significantly
of valuesinherentinhumancytokineandchemokinedata, fromcontrols.
both means and medians were analyzed. Cross-sectional The OIND group also manifested an elevated mean
groups were compared by analysis of variance (ANOVA) CSF BAFF concentration compared to untreated OMS
withTukey post-hoc tests, and primary comparisons were (P = 0.04) and controls (P < 0.0001), though there was no
between controls and untreated OMS, and between un- significant difference in medians. In four OIND, whose
treatedandtreatedOMS.Medianswereanalyzedsecondar- diagnoses included encephalitis, neurolupus, and develop-
ily by the Kruskal-Wallis test with Dunn’s post-hoc test. mental delay, the CSF BAFF concentration was above the
Twogroupcomparisonsweremadebytwo-tailedttestsor highestcontrolinthe369to1,398pg/mLrange.TheCSF:
Mann–Whitneytests,depending onvariance,but pre-and serumBAFFratiosdidnotdifferbetweenthesegroups.
post-treatment comparisons utilized the paired t test. Per- ACTH and steroid monotherapy groups did not differ
centages were compared by Fisher’s exact test. Correlation significantly in CSF BAFF concentrations (P = 0.06,
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treatments (0.07 ± 0.05, -70%, P = 0.009), and steroid +
othertreatments(0.08±0.05,-63%,P=0.02).
The mean serum BAFF concentration did not reflect
CSFconcentrations(Figure1B).Itwassignificantlyhigher
only in rituximab-treated patients receiving combination
immunotherapy. The BAFF elevation was two-fold in the
ACTH + other group and 2.3-fold in the steroid + other
group. When the ACTH + other and steroid + other
groups were combined, the 50% treated with rituximab
had a 2.6-fold higher serum BAFF (3,186 ± 1,774 pg/mL)
thantheresttreatedwithchemotherapyorsteroidsparers
(1,236±779pg/mL)(P<0.0001).
APRILcross-sectionalstudy
The CSF concentration of APRIL did not differ signifi-
cantlybetweenthecontrolgroupandOMScross-sectional
groups (Figure 2A). Neither ACTH nor steroids lowered
the concentration of CSFAPRIL, and IVIg also had no ef-
fect.CSFAPRILwasnotelevatedintheOINDgroup.
Combined IVIg treatment groups displayed signifi-
cantly higher serum APRIL concentrations (Figure 2B);
Figure1Cross-sectionalBAFFconcentrations.(A)CSFBAFF.Box so did individual IVIg groups (see figure legend). In con-
andwhiskergraphsdepictthemeanasaplussign,themedianasa trast, serum APRIL in ACTH- or steroid-treated groups
linewithinthebox,interquartilerangesastheupperandlowerbox did not differ from untreated OMS or controls, which
lines,andtherangeasTukeyerrorbars.Primarystatistical
didnot differfromeachother. TheAPRILconcentration
comparisonswereofmeansforuntreatedOMSversuscontrols(*)
andtreatedversusuntreatedOMS(†).Statisticalsignificancelevelis inserumdidnotreflectthatinCSF.
indicatedbythenumberofsymbols:*0.05>P≥0.01,***P≤0.0001. As a result of increased serum APRIL, the CSF:serum
Thepre-bundledtreatmentgroupswereACTH(n=37),steroids(n= APRIL ratio was lower by 53% in the IVIg monotherapy
22),IVIG(n=24),ACTH+IVIg(n=39),steroids+IVIg(n=27), group, 60% in the ACTH + IVIg group, and 81% in the
ACTH+other(rituximab,chemotherapy,orsteroidsparers)(n=14),
steroid + IVIg group compared to untreated OMS. In
andsteroids+other(n=13).NoneofthethreeACTHgroups
controls, the CSF:serum APRIL ratio was 5.8-fold higher
differedfromeachotherinBAFFconcentration,sotheywere
bundledinto‘AllACTHGroups’.Steroidgroupswerebundledthe than the CSF:serum BAFF ratio (P < 0.0001); in
sameway.OINDwassignificantlydifferentthanallothergroups(‡). untreatedOMS,itwas2.8-fold higher (P < 0.0001).
ThemeanACTHdoseinthecombinedACTHgroupswas28±22
IU/m2/day(alternatedaydosesaveragedtoprovideadailydose
Relationtoclinicaldata
equivalent).Themeansteroiddosewas1.3±1.3mg/kg/day.(B)
Patients were separated according to OMS severity or
SerumBAFF.TheACTHmonotherapygroupandtheACTH+IVIg
group,whichwerenotsignificantlydifferentinBAFFconcentrations, duration. Mean CSF BAFF concentration increased sig-
werebundledintogroup2;steroidswerehandledlikewiseingroup nificantly with OMS severity category in the combined
3.The‘Rituximab+Other’groupincludedrituximab,IVIg,andeither OMS dataset (n = 271) (P = 0.006, ANOVA); so did the
steroidsorACTH. median (P = 0.02, Kruskal-Wallis test). For OMS dur-
ation category, there was no significant relation to CSF
t-test and Mann–Whitney test). When all ACTH groups BAFF (P = 0.11). The CSF:serum BAFF ratio correlated
(n = 90) were compared to all steroid groups (n = 62), with OMS total score (r = 0.38, P = 0.018), OMS dur-
there was no significant difference in CSF BAFFconcen- ation (r = −0.48, P = 0.0018), and CSF B cell frequency
trations: 91 ± 78 vs 123 ± 95 pg/mL, respectively. Only (r = 0.59, P = 0.0087). When all groups receiving either
the combined ACTH group was significantly lower than ACTH or steroids were analyzed separately from all
theIVIggroup(184 ±136pg/mL). remaining OMS groups, they manifested the lowest CSF
ThemeanCSF/serumBAFFratiowas64%higherinun- BAFF concentrations regardless of OMS severity
treated OMS than in controls (P < 0.0001). Compared to (Figure 3A) or duration category (Figure 3B). Only in
untreated OMS, there were significant ratio reductions in the remaining OMS groups did CSF BAFFconcentration
treated groups. The lowest ratios were for ACTH (0.11 ± trend withOMSseverityandOMSduration.
0.06, -55%, P = 0.0007), steroids (0.12 ± 0.05, -49%, ForserumAPRIL,therewerenosignificant differences
P=0.02),ACTH+IVIg(0.14±0.10,-38%,P=0.02),ster- between severity or duration categories when the entire
oid + IVIg (0.15 ± 0.06, -49%, P = 0.008), ACTH + other OMS dataset was used (P = 0.27). However, when the
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resulting ‘high’ BAFF group (n = 23), there was a signifi-
cantlyhigherconcentrationofCSFCXCL13andCXCL10,
CSF oligoclonal band number and frequency, and serum
CXCL9andCCL21comparedtothe‘normal’BAFFgroup
(n = 253). The clinical characteristics of this group were
greatertotalscore(motorseverity)andahigherfrequency
of untreated patients. CSF Tcell frequency was lower in
the ‘high’ BAFF group (80.3 ± 8.1%) than the ‘normal’
BAFFgroup(85.2±7.0%)(P=0.004).Therewerenosig-
nificant between-group differences after Bonferroni cor-
rectionsforCSForserumAPRIL,plasmaCXCL12,serum
CCL17, serum CCL22, or for CSF lymphocyte subset fre-
quencies (CD19+ B cells, γδ Tcells, CD4+ Tcells, CD8+
Tcells,orNKcells)(datanotshown).
Because certain treatments alter CSF BAFF concentra-
tions,theanalysiswasthenperformedonlyforuntreated
OMS, for which there were 10 ‘high’ and 34 ‘normal,’
with non-significant trends: CSF CXCL13 (P = 0.06),
serum CXCL9 (P = 0.08). When the sample was
increased by inclusion of all non-ACTH/steroid groups,
there were 10 ‘high’ and 106 ‘normal’. The statistically
Figure2Cross-sectionalAPRILconcentrations.(A)CSFAPRIL, significant results were for CSF CXCL13 (P = 0.01), CSF
expressedasng/mL(1000×pg/mL).Thepre-bundledtreatment OCB frequency (P= 0.04) and number(P= 0.03), serum
groupswereACTH(n=33),steroids(n=11),IVIg(n=14),ACTH+ CXCL9 (P = 0.02), and total score (P = 0.02); CXCL10
IVIg(n=29),steroids+IVIg(n=14).(B)SerumAPRIL,expressedas
and CCL21 were not significant. Thus, the ‘high’ group
ng/mL.ThethreeIVIggroups(IVIg,ACTH+IVIg,steroids+IVIg)did
notdiffersignificantlyfromeachotherinserumAPRIL appeared statistically underpowered when the dataset
concentration,sotheywerecombinedinto‘AllIVIggroups’. wasdelimited.
Comparedtocontrols,theserumAPRILconcentrationwashigherby
6.7-foldintheIVIgmonotherapygroup(32.6±34ng/mL),by8.2-
BAFF-R
foldintheACTH+IVIggroup(39.9±45ng/mL),andby6.9-foldin
BAFF-R wasexpressed onalmostall circulatingCD19+ B
thesteroid+othergroup(33.3±26ng/mL).IVIgwasinfused
monthly,andmostevaluationswerescheduledjustbeforethenext cells, whether from controls or OMS. Mean BAFF-R
IVIgwasdue. frequency was 94.5 ± 9.3% for untreated OMS (n = 6),
97.1±5.4%forconventionally-treatedOMS(n=9),90.1±
OMS dataset was split into no IVIg groups versus all 12.2%formultimodalimmunotherapy(n=13),and96.9±
IVIg groups, significant differences were found for IVIg 2.2% in controls, with no significant differences between
at each level of OMS severity (Figure 3C) and for two groups (P = 0.45). Median frequencies also did not differ
categories of OMS duration (Figure 3D). There was no significantly(P=0.62,Kruskal-Wallistest).
significant difference acrossseveritycategories.
The tumor OMS group (1.14 ± 95 pg/mL, n = 137) and LongitudinalCSFBAFFstudy
the non-tumor OMS group (1.11 ± 0.68 pg/mL, n = 130) The mean CSF BAFF concentration in pre-treatment
did not differ significantly in mean serum BAFFconcentra- OMSwashigherthanincontrols(P<0.0001).Itdeclined
tion (P = 0.79), or in medians (P = 0.42). Mean serum significantly in children receiving ACTH-based immuno-
APRILconcentrationinthetumorgroup(30.3±70ng/mL) therapy (Figure 4A), whether or not they also received
and non-tumor group (19.7 ± 30 ng/mL) also was not sig- rituximab (Figure 4B,C). Of the 42 patients, BAFF
nificantlydifferent(P=0.22). decreased in 36, did not change in two, and increased in
TheOMScombineddatasetwasanalyzedforvariouscor- four. The only distinguishing characteristic of patients
relations. CSF BAFF, not APRIL, correlated with CSF showing a BAFF decrease was lower CSF CXCL13 (2.6
leukocyte count (P = 0.0003, Pearson correlation), though pg/mL) than the others (4.1 pg/mL) (P = 0.02). Post-
thervaluewaslow(r=0.24).Therewerenosignificantcor- treatment CSF BAFF concentration was in the normal
relationswithCSFIgGconcentrationorthealbuminratio. range. Although there was an overall 71% reduction in
motor severity (total score) (P < 0.0001), the change in
Secondaryanalysis BAFF did not correlate with the change in total score,
The whole OMS dataset was divided into two groups as even when patients without a decrease in BAFF were
described under statistical analysis (Figure 4). In the excluded(r=0.19,P=0.29).
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A C
B D
Figure3CSFBAFFandserumAPRILconcentrationversusOMSseverityanddurationcategory.(A)DotplotcomparisonofBAFFversus
OMSseverityinallACTH/steroidgroupsversusallotherOMSgroups.AsteriskdenotesP<0.05bytwo-tailedttestswithincategories.Severity
categorieswerepre-definedbasedontotalscoreasmild(0–12),moderate(13–24),orsevere(25–36).Between-categorycomparisonsweremade
byANOVAandKruskal-Wallistests.(B)ComparisonofBAFFversusOMSdurationinallACTH/steroidgroupsversusallotherOMSgroups.OMS
durationwaspre-definedasacute(0to3months),subacute(>3monthsto1year),orchronic(>1year).(C)SerumAPRILconcentrationsversus
OMSseverityinallIVIggroupsversusallnon-IVIggroups.(D)SerumAPRILversusOMSdurationinallIVIggroupsversusallnon-IVIggroups.
LongitudinalserumAPRILstudy changevariedgreatlybetweenpatients.Insixpatients,the
The post-IVIg serum APRIL concentration (Figure 5D) post-IVIgAPRILconcentrationwas>1SDabovethecon-
wassignificantlyhigherthanthepre-treatmentconcentra- trol mean; in four, >2 SD. The increase was significant in
tion(meanby2.8-fold,medianby2.6-fold).SerumAPRIL children who received 2 g/kg (Figure 5F), but the sample
was increased in all patients, though the amount of sizewassmallinthe1g/kggroup(Figure5E).
Figure4DichotomizationofCSFBAFFconcentrationinto‘high’and‘normal’groupsforphenotyping.Percentageswerecomparedby
Fisher’sexacttestsandmeansbyttests.Thehighandnormalgroupsdidnotdiffersignificantlyingenderratio,patientage,orOMSduration
(datanotshown).
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Figure5LongitudinaleffectsofimmunotherapyonCSFBAFFandserumAPRIL.(A)CSFBAFFpre-andpost-treatmentwithACTH-based
combinationimmunotherapy.GroupmeanswithSEMandpercentreductionareshowntoeithersideofthelineplot.(B)ACTH-based
immunotherapywithoutrituximab.Comparisonsweremadebypairedttests.(C)ACTH-basedimmunotherapywithrituximab.Pre-treatment
meansinFiguresBandCdidnotdiffersignificantly;neitherdidpost-treatmentmeans.(D)Pre-andpost-IVIgtreatmentserumAPRIL.(E)Data
from1g/kgIVIgdose.(F)Datafrom2g/kgIVIgdose.Nosignificantdifferenceswerefoundbetweenpre-treatmentmeansorbetweenpost-
treatmentmeansinFiguresEandF.
Discussion reflected CSF levels in other disorders [9,13,19] or in
This study provides new insights on the BAFF/APRIL the present study.
system in pediatric OMS. Correlation of CSF BAFF with Differences in APRIL and BAFF responses also may
clinical severity and co-segregation of high CSF BAFF reflect differences in their functions [11]. Both BAFF
with CSF inflammatory chemokines and oligoclonal and APRIL bind to BCMA and TACI receptors, how-
bands suggest a potential role of CSF BAFF as one of ever, only BAFF binds to BAFF-R, and APRIL also
severalbiomarkers of disease activity inOMS.BAFFalso binds to surface proteoglycans [21]. The expression of
showed promise as a treatment biomarker in its remark- these receptors differs on pre-immune B cells and
able sensitivity to ACTH or corticosteroids in cross- antigen-experienced B cells (memory B cells and long-
sectional as well as longitudinal studies. APRIL was not lived plasma cells) [22]. BAFF and APRIL may localize
a biomarker of disease activity in OMS, but showed a to different anatomic niches, with distinctive local
striking treatment effect of IVIg. BAFF-R expression on interactions. APRIL modulates certain aspects of B cell
circulating B cells was not altered in OMS, nor is it in activation and isotype switching [22]. Also, BAFF plays
multiple sclerosis[10]ormyastheniagravis[18]. a role in T cell activation and polarization to Th1
The fact that CSF BAFF and APRIL did not trend [11,15], and APRIL suppressesTh2 cytokine production
in the same direction in OMS is not without prece- and antibody responses in vitro [23].
dent [12]. Although both are increased in neuropsychi- Extraordinary differential effects of immunotherapy
atric lupus [12,14], only CSF BAFF is increased in were found for APRIL and BAFF. To our knowledge,
untreated multiple sclerosis [19,20]. Another difference increased serum APRIL concentration as an effect of
between BAFF and APRIL signaling in neuroinflamma- IVIg therapy has not been reported previously in a
tory diseases is that both CSF and serum BAFF are neurological disorder. The only previous report we
elevated in neuro-Behcet’s disease [13], whereas only encountered was of 11 children with Kawasaki disease,
CSF BAFF is elevated in multiple sclerosis. The highest an acute vasculitis that responds to IVIg [24], in whom
CSF BAFF concentrations in multiple sclerosis were an IVIg dose of 2 g/kg raised APRIL (6.7-fold) but low-
found in patients with more than six oligoclonal bands ered BAFF (−41%) in plasma. In OMS, we found no
[21], which is similar to the higher band counts we IVIg-lowering effect on serum BAFF. Serum BAFF
found in OMS. Serum BAFF and APRIL have not increasedonly afterrituximabtherapy,whichwe showed
Pranzatellietal.JournalofNeuroinflammation2013,10:10 Page8of9
http://www.jneuroinflammation.com/content/10/1/10
to be an early response to B cell depletion in OMS [25]. (CXCL10, CXCL13), and oligoclonal bands [2-5]. Such
Only ACTH and corticosteroids lowered CSF BAFF, but molecules necessary for B-cell recruitment, activation,
they did not affect CSF APRIL. Previously, a reduced and survival may be working together to promote neu-
concentration of serum BAFF has been reported in dis- roinflammation [20]. Because of the exceptional sensi-
orders with elevated BAFF, such as in Wegener’s granu- tivity of CSF BAFF to peripherally administered ACTH
lomatosis [26]. These differences might be clinically or corticosteroid therapy, its potential utility as a re-
exploited should they be found to serve as response pre- sponse predictive biomarker or response identification
dictive biomarkers (identifying subpopulation according biomarker warrants validation. The immunomodulatory
to response potential) or response identification (relating effect of IVIg on APRIL signaling merits evaluation in a
biological and clinical responses to treatment) biomar- variety of neuroinflammatory disorders.
kers inlongitudinalstudies.
The clinicalimpactof IVIg-inducedelevation in serum Abbreviations
ACTH:Adrenocorticotropichormoneorcorticotropin;APRIL:Aproliferating-
APRIL is difficult to predict from observational data. In inducingligand;ANOVA:Analysisofvariance;BAFF:Bcellactivatingfactor;
diseases associated with increased APRIL in serum, BAFF-R:BAFFreceptor;CD:Clusterofdifferentiation;ELISA:Enzyme-linked
APRIL is thought to have a pathologic role. However, immunoassay;IVIg:Intravenousimmunoglobulins;NIND:Non-inflammatory
neurologicaldisorders;OIND:Otherinflammatoryneurologicaldisorders;
IVIg induces clinical improvement in OMS [1], and OMS:Opsoclonus-myoclonussyndrome.
APRIL is not elevated in untreated OMS, raising the
possibility that boosting serum APRIL could be thera- Competinginterests
RichardM.RansohoffisamemberoftheScientificAdvisoryBoardof
peutic in OMS. Interestingly, APRIL has been suggested
Chemocentryxandholdsstockinthecompany.Heisamemberofthe
to be involved in downregulation of serological and clin- ScientificAdvisoryBoardofVertex.Therestoftheauthorshavenothingto
ical activity in patients with systemic lupus erythemato- disclose.
sus [27]. Thisview is consistent with the proposed mode
Authors’contributions
of IVIg action, which is thought to be immunomodula-
MRPwastheprincipalinvestigator,andparticipatedinthedrafting/revising
tion of the cytokine network [28]. However, modulation ofthemanuscript,thestudyconceptanddesign,acquisitionofdata,analysis
andinterpretationofdata,andstatisticalanalysis.EDTwastheco-
ofAPRILmaynotbethemechanisminvolved.
investigator,andparticipatedinpatientrecruitmentandevaluation,study
Treatment-induced reduction of BAFF in the central
conceptanddesign,acquisitionofdata,andrevisionofthemanuscript.NRM
nervous system might decrease BAFF-dependent sur- wasthelaboratoryresearcher,andparticipatedintheacquisitionand
analysisofdata,andstatisticalanalysis.ALTwastheflowcytometrist,and
vival of plasma cells [9], which express BAFF-R [29]. In
participatedintheacquisitionandanalysisofdata,andrevisingofthe
pediatric OMS, elevated CSF BAFF (not APRIL) corre-
manuscript.JACwasthestudystatistician,andparticipatedinthereviewof
lated with CSF cerebellar autoantibodies [30], though themanuscript.JMNparticipatedinOINDpatientrecruitmentand
evaluation,andvettingofthemanuscript.RMRwastheprojectconsultant,
the effect of treatment was not studied. Increased serum
andrevisedthemanuscript.Allauthorshavereadandapprovedthefinal
BAFF after rituximab, which may be important to B-cell
versionofthemanuscript.
repopulation [31], could also increase survival of auto-
reactive circulatingB cells [29]. Acknowledgements
ThisresearchwasfundedbyfoundationgrantsfromtheThrasherResearch
BAFF adds to the evolving picture of B-cell involve- Fund(02826–2),theSpasticParalysisResearchFoundation(Illinois-Eastern
ment in OMS, which includes CSF B-cell subset expan- IowaDistrictofKiwanisInternational),TheChicagoInstituteofNeurosurgery
sion [2], positive oligoclonal bands [5], intrathecal over- andNeuroresearch,RonaldMcDonaldHouseCharitiesofCentralIllinois,and
investigator-sponsoredgrantsfromQuestcorPharmaceuticals(UnionCity,
production of CXCL13 [3], selective over-expression of
CA,USA)andGenentechInc./BiogenIDEC(SouthSanFrancisco/SanDiego,
CXCR5 receptors on CSF memory B cells [3], and clin- CA,USA).Theauthorsthankthepatients,theirfamilies,andreferring
ical response to adjunctive anti-CD20 monoclonal anti- physiciansfortheirparticipation.WeespeciallythankJamesR.andMichelle
L.ShopeIII,SallyE.Durdan,ShannonBoone,DavidandSherryRitzenthaler,
body rituximab [1]. One potential limitation of CSF
JackL.andEllynJ.Pittsley,SteveandDonnaPittsley,andCharlesandKerri
BAFF as a biomarker is its inter-individual variability. Drabekfortheirgenerousresearchdonations.
Moreover, only 23% of patients with untreated OMS had
Authordetails
CSF BAFFconcentration >2 SD above the control mean.
1DepartmentofNeurology,NationalPediatricMyoclonusCenterand
This shows significant overlap of CSF BAFF values be- NeuroimmunologyLaboratory,andSouthernIllinoisUniversitySchoolof
tween patients and controls. Our current research Medicine,POBox19643,Springfield,IL62794-9643,USA.2FlowCytometry
Center,SouthernIllinoisUniversitySchoolofMedicine,POBox19626,
explores the hypothesis that inflammatory cytokines,
Springfield,IL62794-9626,USA.3StatisticsandResearchConsulting,Southern
none of which is elevated in all patients, may have IllinoisUniversitySchoolofMedicine,POBox19623,Springfield,IL
greater predictive value in OMS as a biomarker cluster 62794-9623,USA.4DepartmentofPediatrics,UniversityofAlabamaat
BirminghamSchoolofMedicine,Birmingham,AL35233,USA.5Cleveland
than individually.
ClinicFoundation,TheLernerResearchInstitute,MellenCenterforMS
In conclusion, BAFF, not APRIL, joins a short list of TreatmentandResearch,MailCodeNC30,9500EuclidAvenue,Cleveland,
other putative CSF biomarkers of disease activity in OH44195,USA.
pediatric OMS that includes B cell frequency (total B
Received:29October2012Accepted:8January2013
cells and B-cell subsets), B-cell chemoattractants Published:16January2013
Pranzatellietal.JournalofNeuroinflammation2013,10:10 Page9of9
http://www.jneuroinflammation.com/content/10/1/10
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