RESEARCH ARTICLE Open Access
Clinical associa ion analysis o
ependymomas and pilocy ic as ocy omas
e eals ele a ed FGFR3 and FGFR1
exp ession in agg essi e ependymomas
Bi gi a Leh inen
1†
, Annina Rai a
2,3†
, Juha Kesseli
1
, Ma i Annala
1
, K is iina No d o s
2,4
, Olli Yli-Ha ja
5
, Wei Zhang
5,6
,
Tapio Visako pi
1,2
, Ma i Nyk e
1,7
, Hannu Haapasalo
2,3*
and Ki si J. G anbe g
1,5,7*
Abs ac
Backg ound: Fib oblas g ow h ac o ecep o s (FGFRs) a e well-known p o o-oncogenes in se e al human
malignancies and a e cu en ly he apeu ically a ge ed in clinical ials. Among glioma sub ypes, ac i a ing FGFR1
al e a ions ha e been obse ed in a subpopula ion o pilocy ic as ocy omas while FGFR3 usions occu in IDH
wild- ype di use gliomas, esul ing in high FGFR3 p o ein exp ession. The pu pose o his s udy was o associa e
FGFR1 and FGFR3 p o ein le els wi h clinical ea u es and gene ic al e a ions in ependymoma and pilocy ic as ocy oma.
Me hods: FGFR1 and FGFR3 exp ession le els we e de ec ed in ependymoma and pilocy ic as ocy oma issues using
immunohis ochemis y. Selec ed cases we e u he analyzed using a ge ed sequencing.
Resul s: Exp ession o bo h FGFR1 and FGFR3 a ied wi hin all umo ypes. In ependymomas, inc eased FGFR3 o
FGFR1 exp ession was associa ed wi h high umo g ade, ce eb al loca ion, young pa ien age, and poo p ognosis.
Mode a e- o-s ong exp ession o FGFR1 and/o FGFR3 was obse ed in 76% o ce eb al ependymomas. Cases wi h
mode a e- o-s ong exp ession o bo h p o eins had poo clinical p ognosis. In pilocy ic as ocy omas,
mode a e- o-s ong FGFR3 exp ession was de ec ed p edominan ly in non-pedia ic pa ien s. Ta ge ed
sequencing o 12 umo s ound no p o ein-al e ing mu a ions o usions in FGFR1 o FGFR3.
Conclusions: Ele a ed FGFR3 and FGFR1 p o ein exp ession is common in agg essi e ependymomas bu
likely no d i en by gene ic al e a ions. Fu he s udies a e wa an ed o e alua e whe he ependymoma
pa ien s wi h high FGFR3 and/o FGFR1 exp ession could bene i om ea men wi h FGFR inhibi o based
he apeu ic app oaches cu en ly unde e alua ion in clinical ials.
Keywo ds: Tissue mic oa ay, Deep-sequencing, FGFR inhibi ion, Immunohis ochemis y s aining
Backg ound
Fib oblas g ow h ac o ecep o s (FGFRs) a e a amily
o ecep o y osine kinases ha a e ac i a ed in a a ie y
o cance s and ha e well-es ablished oncogenic p ope ies
[1, 2]. Since he disco e y o ecu en FGFR gene usions
in glioblas oma [3, 4], FGFR inhibi o based ea men
egimens ha e been iewed as a p omising he apeu ic op-
ion o b ain umo s wi h FGFR al e a ions. The mecha-
nisms o FGFR ac i a ion in b ain umo s a y by umo
ype, bu include oncogenic FGFR3 and FGFR1 usions,
FGFR1 ea angemen s, and FGFR1 mu a ions [2–8].
Mo eo e , gene usions appea o be he sole ecu en
oncogenic FGFR3 al e a ion in b ain umo s. Al hough
FGFR3 is commonly used o a ans o ming acidic coiled-
coil-con aining p o ein 3 (TACC3) gene, o he usion
pa ne s exis . Fo example, ecu en FGFR3–BAIAP2L1
usions ha e been de ec ed in bladde cance [9]. Se e al
FGFR inhibi o s a e cu en ly unde p e-clinical and
* Co espondence: hannu.haa[email p o ec ed];[email p o ec ed]
†
Equal con ibu o s
2
Fimlab Labo a o ies L d., Tampe e Uni e si y Hospi al, Bioka u 4, 33520
Tampe e, Finland
1
BioMediTech Ins i u e and Facul y o Medicine and Li e Sciences, Bioka u 8,
33520 Tampe e, Finland
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Leh inen e al. BMC Cance (2017) 17:310
DOI 10.1186/s12885-017-3274-9
clinical e alua ion, and ecen epo s ha e shown good
ea men esponses in FGFR3 usion posi i e cells and
umo s [8, 10, 11]. While mos o he FGFR inhibi o
s udies, o da e, ha e been pe o med in cases in ol ing
ca cinomas, esponses o FGFR inhibi o s ha e also been
epo ed in cases wi h glioblas oma [8, 12].
Ependymomas and pilocy ic as ocy omas a e nondi -
use gliomas, in which neoplas ic cells do no subs an-
ially in il a e in o su ounding no mal issue. They
ep esen di e en g ades, ypes o g ow h and clinical
cou ses. Nondi use g ow h pa e n acili a es e icien
su gical emo al o he umo , which pa ly explains he
be e p ognosis in hese pa ien s ela i e o hose wi h
di use gliomas. Howe e , umo ecu s in some o he
pa ien s, and o e all su i al a es a e wo se wi h mo e
agg essi e ependymomas [13].
Ependymomas a e he hi d mos common b ain
umo in child en, ep esen ing 8–10% o pedia ic in a-
c anial umo s and app oxima ely 4% o all adul b ain
umo s [13]. Ependymomas a e ound in all loca ions o
he cen al ne ous sys em, and may be in ac anial
(in a en o ial o sup a en o ial) o spinal. In a en o ial
pos e io ossa ependymomas can be u he subclassi-
ied in o pos e io ossa g oup A (PFA) and g oup B
(PFB) umo s [14]. Adul ependymomas a e ypically
g ade I myxopapilla y ependymomas localized in he
spinal co d, while pedia ic ependymomas a e ypically
in ac anial g ade II–III umo s [13, 15]. Al hough epen-
dymomas in young child en a e ypically associa ed wi h
poo p ognosis [15, 16], adul sup a en o ial ependymo-
mas a e also associa ed wi h lowe su i al a es [13].
Apa om copy numbe al e a ions [13], signi ican
gene ic and epigene ic d i e s o ependymoma de elop-
men ha e been ecen ly epo ed. C11o 95–RELA u-
sions ha e been obse ed o occu in wo- hi ds o
pedia ic cases o sup a en o ial ependymomas and a e
belie ed o be oncogenic due o inc eased NF-kB signaling
[17]. Fu he mo e, a sub ype o ce ebella ependymomas
ha is associa ed wi h young pa ien age and poo p og-
nosis is cha ac e ized by a CpG island me hyla o pheno-
ype (CIMP) and Polycomb ep essi e complex 2 d i en
ime hyla ion o H3K27. These umo s a e esponsi e o
pha macological he apies a ge ing epigene ic egula o s
[18]. The au ho s also highligh ed he low a e o ecu -
en mu a ions and copy numbe al e a ions in ce ebella
ependymomas. Fu he mo e, FGFR al e a ions ha e no
been epo ed in high- h oughpu sequencing s udies wi h
he excep ion o FGFR1 missense mu a ion N544 K [17]
localized o he y osine kinase domain o FGFR1.
Pilocy ic as ocy oma (PA), he mos common b ain
neoplasm in he pedia ic popula ion, is classi ied as
WHO g ade I [19, 20]. They a ise mos commonly in he
ce ebellum, b ains em and he op ic ne e. Familial PAs
a e cha ac e ized by inac i a ion o he neu o ib oma osis
1(NF1) umo supp esso gene, while ac i a ing BRAF
usions and mu a ions a e ypical o spo adic PAs [19].
BRAF al e a ions subsequen ly lead o ac i a ion o he
MEK-ERK pa hway [19], which is also an impo an
downs eam signalling pa hway o FGFR-induced sig-
naling [19, 21]. Addi ionally, FGFR1-TACC1 usion has
been epo ed in a BRAF wild- ype pilocy ic as ocy-
oma o he diencephalon and se e al s udies ha e
epo ed oncogenic s uc u al FGFR1 a ian s wi h du-
plica ion o he y osine kinase domain [6, 7]. Fu he -
mo e, app oxima ely 5% o PAs ha bo an FGFR1
mu a ion a ge ing codons Asn546 o Lys656 in he
kinase domain [7]. The Lys656 mu a ion has been
associa ed wi h dec eased pa ien su i al [22]. Mos
FGFR1-mu an umo s s udied ha e been ex a-
ce ebella , loca ed mos ly in midline loca ions, and mu-
ually exclusi e wi h BRAF, NF1, and o he ecu en
MAPK pa hway al e a ions [7, 22]. Al hough hese
s udies did no epo mu a ions o s uc u al a ian s
in FGFR3, hey emphasized he u ili y o FGFR1 as a
ma ke o PA sub yping.
In di use gliomas, FGFR3 p o ein le el is an in o m-
a i e ma ke o usion s a us [34]. Mos umo s in a co-
ho o 791 cases did no ha e any de ec able FGFR3
p o ein exp ession, and all he usion-posi i e cases we e
s ongly s ained (s aining sensi i i y 100% and speci ici y
88% in he a ge ed sequencing coho ). In non-di use
gliomas, FGFR1 al e a ions a e commonly p esen in a
subg oup o pilocy ic as ocy omas ha lack o he yp-
ical MAPK pa hway al e a ions [6, 7], bu FGFR1 and
FGFR3 exp ession le els ha e no been sys ema ically
e alua ed. Fu he mo e, FGFR usions o inc eased FGFR
p o ein exp ession le els ha e no , o da e, been e-
po ed o occu in ependymomas. In he p esen s udy,
we sough o in es iga e he clinical signi icance o
FGFR3 and FGFR1 exp ession in wo di e en nondi -
use gliomas: ependymomas and pilocy ic as ocy omas.
We used immunohis ochemis y o de ec FGFR1 and
FGFR3 p o ein le els in ependymomas and pilocy ic
as ocy omas, and e alua ed he ela ionship be ween
p o ein exp ession le els, clinical ea u es and selec ed
gene ic al e a ions.
Me hods
Pa ien samples
This s udy was app o ed by he E hical Commi ee o
Tampe eUni e si yHospi aland heNa ionalAu-
ho i y o Medico-legal A ai s in Finland. The s udy
coho included 108 ependymal umo s om 88
pa ien s, 97 pilocy ic as ocy omas om 97 pa ien s
(Table 1).
Ependymoma pa ien s unde wen neu osu gical ope -
a ion wi h he in en ion o g oss adical umo esec ion
be ween 1984 and 2009 a Tampe e Uni e si y Hospi al,
Leh inen e al. BMC Cance (2017) 17:310 Page 2 o 12
be ween 1979 and 1998 a Kuopio Uni e si y Hospi al,
and be ween 1986 and 1999 a Tu ku Uni e si y Hospi al,
Finland. The clinical da a de ail abou adicali y o umo
esec ion is impe ec , bu adical esec ion has always
been pe o med when possible o he pa ien . G ade I
umo s included 17 myxopapilla y ependymomas and
1 subependymoma. G ade II umo s included 68 epen-
dymomas, while G ade III umo s included 22 anaplas-
ic ependymomas, as classi ied acco ding o WHO
c i e ia [23].
Pilocy ic as ocy oma pa ien s unde wen umo su ge y
a he Tampe e Uni e si y Hospi al be ween 1985 and
1999, a he Kuopio Uni e si y Hospi al be ween 1980 and
1992, a he Tu ku Uni e si y Hospi al be ween 1981 and
1992, and a he Helsinki Uni e si y Hospi al be ween
1986 and 1993.
Tissue his opa hology and mic oa ays
Tumo samples we e ixed in o maldehyde (bu e ed
wi h 4% phospha e) and embedded in pa a in. The
samples we e p ocessed in o pa a in blocks and sec ions
we e s ained wi h hema oxylin and eosin (H&E). His o-
pa hological yping and g ading, e alua ion, and iden i i-
ca ion o his ologically ep esen a i e umo egions on
each slide we e pe o med by an expe ienced neu o-
pa hologis . Tissue mic oa ay (TMA) blocks we e
cons uc ed using ep esen a i e sample egions and a
cus om-buil ins umen (Beeche Ins umen s, Sil e
Sp ing, MD, USA). The diame e o he issue co e
on he mic oa ay block was 0.6 o 1 mm, depending
on he TMA ype. Fi e-mic ome e - hick sec ions we e
cu om ep esen a i e a ay pa a in blocks.
Immunohis ochemis y
Pa a in was emo ed wi h hexane. A e ehyd a ion in
e hanol, he p e-p ocessing s age was pe o med using
Ta ge Re ie al Solu ion ci a e bu e (Dako). The sam-
ples we e s ained using abbi monoclonal FGFR1 an i-
body (#9740, Cell Signaling Technology, 1:100 dilu ion)
and mouse monoclonal FGFR3 an ibody (sc-13,121, San a
C uz Bio echnology, 1:600 dilu ion). ‘En ision + Sys em-
ho se adish pe oxidase and diaminobenzidine (DAB)’ki
(Dako) was used o FGFR3. The nuclei we e s ained
wi h hema oxylin. A mouse monoclonal an ibody MIB-
1 (Ki-67 an igen, dilu ion 1:40, Immuno ech, S.A.
Ma seille, F ance) was used o analyze cell p oli e a ion.
The issue sec ions we e coun e s ained wi h me hyl
g een. The pe cen age o issue MIB-1-posi i e nuclei
was quan i a i ely e alua ed using a compu e -assis ed
image analysis sys em (CAS-200 TM So wa e, Bec on
Dickinson & Co., USA) and ImmunoRa io analysis.
Only neoplas ic cells we e included in he analysis
(nec o ic and hemo hagic a eas we e omi ed).
The in ensi y o FGFR3 and FGFR1 immunoposi-
i i y was sco ed by wo obse e s (HH and KG) on
a scale om 0 o 3 as ollows: 0 (no s aining), 1
(weak immunos aining), 2 (mode a e immunos aining), o
3 (s ong immunos aining).
S a is ical analysis
All da a we e analyzed using R packages o IBM SPSS
s a is ics 21.0 so wa e (SPSS Inc., Chicago, IL, USA) o
Windows. Tes s o pai wise associa ion be ween
disc e e a iables we e pe o med using Fishe ’s exac
es o coun da a. Fo ables la ge han 2 × 2, he
p- alues o Fishe ’s exac es s we e calcula ed using
Mon e Ca lo simula ion wi h 2.5*10^7 eplica es.
p- alues we e no co ec ed o mul iple es ing. Log-
Table 1 Pa ien demog aphics and clinical cha ac e is ics wi hin
ependymoma and pilocy ic as ocy oma umo pa ien coho s
Ependymomas Pilocy ic
as ocy omas
Pa ien s 88 80
Male 48 42
Female 40 38
Age (yea s)
Median (Mean ± SD) 37 (35 ± 21) 9 (14 ± 14)
Minimum 1 0
Maximum 73 58
Follow-up o p ima y umo pa ien s
Su i o s in he end o he
ollow-up
60 69
Follow-up ime o su i o s
(m) (median (mean ± SD))
125 (135 ± 82) 70 (111 ± 89)
5-yea esidi e- ee su i al (%) 71 82
5-yea su i al (%) 82 93
Tumo s 108 80
P ima y 74 73
Second 14 5
Thi d 14 1
Fou h-six h 6 1
His ological g ade
I1880
II 68 0
III 22 0
Topog aphy
Sup a en o ial 35 3
In a en o ial 28 69
Spinal 43 2
C anial ne e 0 6
Pa ien age and ollow-up in o ma ion we e calcula ed using p ima y cases.
Follow-up imes a e shown in mon hs (m)
SD s anda d de ia ion
Leh inen e al. BMC Cance (2017) 17:310 Page 3 o 12
ank es was used o he analysis o p ognos ic ac o s.
In cox eg ession analysis, cox model was buil using a
s epwise o wa d likehood- a io es ing.
Ta ge ed sequencing
All he issue samples we e o malin ixed and pa a in
embedded (FFPE). A u XTRAC FFPE DNA ki
(Co a is) o AllP ep DNA/RNA Mini Ki (Qiagen) was
used o DNA isola ion. We used 1 μg o ex ac ed
DNA o a ge ed sequencing using he Su eselec XT
Ta ge en ichmen sys em oge he wi h cus om-
designed RNA p obes (Addi ional ile 1: Table S1).
The sequencing lib a y was p epa ed acco ding o he
ki ins uc ions (200 ng o DNA samples) wi h a
sho e DNA-shea ing p o ocol (220 s) and sequenced
wi h MiSeq (Illumina). Tumo s Epe002 and Epe003
we e de i ed om he i s and he hi d umo su ge y
(a e second ecu ence) o one pa ien . In addi ion, he u-
mo s Epe004 (1s umo su ge y) and Epe005 (2nd umo
su ge y) we e de i ed om a sepa a e ependymoma
pa ien .
The esul ing da a we e aligned agains he GRCh37
human e e ence genome using Bow ie 2.2.4 [24]. Mu a-
ions we e iden i ied in umo samples by sea ching o
si es wi h an al e na e allele ac ion o a leas 10%, and
a leas 5 eads wi h he mu a ion. Addi ionally, he al-
lele ac ion was equi ed o be 20 imes highe han he
backg ound e o a e (i.e., he a e age allele ac ion
ac oss con ol blood samples om heal hy pa ien s).
P o ein-le el consequences o a ian s we e p edic ed
using ANNOVAR so wa e ool [25]. Mu a ions wi h a
known o suspec ed pa hological unc ion we e iden i-
ied manually. To disco e ch omosomal ea angemen s
o usion de ec ion, unaligned eads om each sample
we e spli in o wo 30 bp ancho s (one om bo h ends)
ha we e aligned o he hg38 genome using Bow ie-
1.1.2. Disco dan ancho pai s we e g ouped by posi ion,
and g oups wi h 8 o mo e suppo ing eads we e
lagged as ea angemen candida es and manually cu-
a ed using IGV and BLAT.
Log a ios o amplicon ead coun s we e used o
DNA copy numbe calling. Di e ences in a e age co e -
age be ween samples we e co ec ed on he basis o con-
ol amplicons in ch omosomes 5, 8, 11, and 18 (14–21
amplicons pe ch omosome), posi ioned in egions wi h
he lowes a e o epo ed copy numbe al e a ions.
Blood-de i ed DNA om heal hy indi iduals was used as
a nega i e con ol o he copy numbe analysis.
Resul s
We used an an ibody ha a ge s amino acids 25–124 in
he FGFR3 N- e minus o pe o m immunohis ochemi-
cal (IHC) s aining on 188 cases including ependymomas
o pilocy ic as ocy omas (Table 1). FGFR3 s aining was
localized o he cy oplasm and plasma memb ane
(Fig. 1). S aining was ypically he e ogeneous in all
umo ypes s udied. Nega i ely s ained blood essels
p o ided an in e nal con ol o an ibody speci ici y.
No mal b ain issue was immunonega i e, wi h he
excep ion o he ce ebella and ce eb al molecula
laye s, whe e weak- o-mode a e s aining was obse ed
(Addi ional ile 1: Figu e S1a).
In ependymomas, FGFR3 s aining is associa ed wi h
disease agg essi eness
Immunohis ochemis y was used o in es iga e FGFR3
exp ession le els in 108 ependymal umo samples ap-
plied o TMAs. The TMA coho (Table 1), ep esen ing
di e en g ades o ependymomas and disease sub ypes,
has been pa ly epo ed p e iously [26]. FGFR3 immu-
no eac i i y was de ec ed in 27 (37%) o he cases; 11
(15%) showed weak immunos aining, 11 (15%) showed
mode a e immunos aining and 5 (7%) we e s ongly
immunoposi i e. Inc eased s aining was also obse ed in
pseudo ose e s uc u es (Addi ional ile 1: Figu e S1b).
Recu en umo s showed ypically simila s aining le els
as he p ima y umo . Wi h espec o he associa ion
analysis (Addi ional ile 1: Figu e S2), FGFR3 s aining
was signi ican ly associa ed wi h a highe umo g ade
(p< 0.01, Fishe ’s exac es , Fig. 1b, Table 2). None o
he g ade I cases showed de ec able FGFR3 exp ession.
Mode a e- o-s ong FGFR3 immunos aining was p e-
dominan ly de ec ed in ce eb al umo s as compa ed o
o he loca ions (p< 0.001, Fishe ’s exac es , Fig. 1c,
Table 2). Ele a ed FGFR3 immunoposi i i y in high-
g ade ce eb al umo s sugges s ha FGFR3 immuno-
s aining may be ypical o pedia ic ependymomas.
Indeed, pa ien s wi h age < 20 yea s a umo onse had
a highe equency o FGFR3 immunoposi i e s aining
(p< 0.05, Fishe ’s exac es , Fig. 1d). Cases wi h
mode a e- o-s ong FGFR3 immunos aining end o
show a high p oli e a ion a e (Fig. 1e), al hough his
associa ion was no s a is ically signi ican (p= 0.07,
Fishe ’s exac es ). Impo an ly, mode a e- o-s ong
FGFR3 immunos aining was signi ican ly associa ed wi h
sho e o e all pa ien su i al (p< 0.05, log- ank es ,
Fig. 1 ) and sho e ime o umo ecu ence (p< 0.01,
log- ank es , Fig. 1g). The associa ion wi h disease- ee
su i al emained signi ican a e adjus men o umo
loca ion, g ade, and p oli e a ion (p= 0.003, RR = 1.82,
95% CI 1.23–2.68 o FGFR3, o he a iables no signi ican
in he inal equa ion, N= 77, s epwise Cox eg ession), bu
only umo loca ion (p= 0.022, RR = 2.47, 95% CI 1.42–
5.34, N= 77, s epwise Cox eg ession) was a signi ican
p ognos ic p edic o o disease-speci ic su i al in mul i-
ac o ial analysis. I is ele an o no e he pa ien numbe s
(N= 77) a e a he low o mul i ac o ial analysis using ou
di e en a iables. S ill, he ob ained esul s sugges ha
Leh inen e al. BMC Cance (2017) 17:310 Page 4 o 12
a)
b) c)
d) e)
) g)
Fig. 1 Mode a e- o-s ong FGFR3 immunos aining was p edic i e o poo pa ien su i al in ependymomas. aRep esen a i e s aining images.
bDis ibu ion o FGFR3 immunos aining in g ade I–III ependymomas. FGFR3 immunos aining was posi i ely associa ed wi h umo g ade (p< 0.01, Fishe ’s
exac es ). cMode a e- o-s ong FGFR3 immunos aining was associa ed wi h ce eb al umo loca ion (p< 0.0001, Fishe ’s exac es ). To al numbe o
umo s o each loca ion is ma ked in o he igu e. dMode a e- o-s ong FGFR3 exp ession was mo e common in younge pa ien s (p < 0.05, Fishe ’sexac
es ). Only newly-diagnosed cases we e included in he analysis and hese we e di ided in o hose wi h nega i e- o-weak s. mode a e- o-s ong FGFR3
immunos aining. eCases wi h mode a e- o-s ong FGFR3 exp ession ended o ha e highe p oli e a ion index (p= 0.07, Fishe ’sexac es ).Sampleswe e
di ided based on FGFR3 s aining and p oli e a ion a e (1: low, 2: in e media e, and 3: high p oli e a ion index). -gMode a e- o-s ong FGFR3
immunos aining was associa ed wi h wo se g) disease-speci ic su i al (N= 73, p< 0.05, log- ank es ) and g) ecu ence- ee su i al (N= 70,
p< 0.01, log- ank es ). Only newly-diagnosed cases we e included in o he analysis
Leh inen e al. BMC Cance (2017) 17:310 Page 5 o 12

FGFR3 immunoposi i i y is associa ed wi h mo e agg essi e
ependymomas.
As pedia ic and adul ependymomas di e in many
espec s and he age associa ion migh in luence he ob-
se ed associa ions, we analyzed he pedia ic and adul
sample coho s independen ly. Pa ien s ha we e a leas
16 yea s old we e conside ed as adul s acco ding o gen-
e al p ac ice in Finnish pedia ic clinics. The e we e 35
pedia ic and 73 adul samples in ou coho . Mode a e-
o-s ong FGFR3 s aining was sligh ly mo e common in
pedia ic han adul samples (34.3% s 13.7%, p= 0.055,
Fishe ’s exac es , Table 2). In pedia ic pa ien s, mode -
a e FGFR3 immunos aining was obse ed in ce ebella
(31%, n= 16) and ce eb al (29%, n= 14) umo s and
s ong FGFR3 s aining only in ce eb al umo s (21%,
n= 14), whe eas all he spinal cases (n= 5) we e nega-
i e o FGFR3 (p= 0.065, Fishe ’s exac es ). FGFR3
s aining was no associa ed wi h umo g ade o p oli e -
a ion index in pedia ic ependymomas. In adul s, FGFR3
associa ions we e la gely e y simila as in he whole
sample coho : s onge FGFR3 s aining was associa ed
wi h umo g ade (p< 0.01, n= 73, Fishe ’s exac es ),
umo loca ion (p< 0.001, n= 71, Fishe ’s exac es )
and he e was a close- o-signi ican associa ion wi h p o-
li e a ion index (p= 0.095, n= 66, Fishe ’s exac es ).
P ognos ic associa ions we e mos ly nonsigni ican in
sepa a e su i al analyses in pedia ic (n= 14) and adul
(n= 30) sample coho s, bu his was likely due o low
sample coun in he analysis, as he end emain he simi-
la . O no e, when FGFR3 s aining was di ided in o ou
g oups, i was associa ed wi h wo se disease-speci ic
(p< 0.01, log- ank es ) and disease- ee (p<0.001,log-
ank es ) su i al in pedia ic pa ien s.
FGFR1 s aining is associa ed wi h highe umo g ade and
ce eb al loca ion
The in e p e a ion o he FGFR1 immunos aining da a
was no as s aigh o wa d as FGFR3 s aining, pa ly be-
cause mac ophages, neu ons, and nec o ic a eas showed
immunoposi i e s aining. The e o e, FGFR1 immunohis-
ochemical sco ing was based on he p esence o
FGFR1-posi i e malignan cell clus e s o la ge umo
a eas (i.e. di use s aining), and sco ing o indi idual cells
was omi ed in he analysis. Spo adic mode a e- o-
s ong FGFR1 immunoposi i i y was also de ec ed and
cha ac e ized by high ou lie exp ession in indi idual
malignan cells. These obse a ions suppo hose om
p e ious epo s [27]. FGFR1 s aining was de ec ed in
he cy oplasm and memb ane compa men s, while
g anula s aining was also obse ed in a subpopula ion
o posi i ely-s ained samples. In e es ingly, mode a e- o-
s ong FGFR1 immunos aining was also obse ed in
ependymal ose es (Addi ional ile 1: Figu e S3).
Di use FGFR1 immuno eac i i y was de ec ed in 42
(58%) o ependymal umo s. Twen y- ou cases (33%)
showed weak immunos aining, 15 (21%) cases showed
mode a e immuno eac i i y, and 3 (4%) cases showed
s ong immunoposi i i y (Fig. 2a). Consis en wi h
FGFR3 exp ession, FGFR1 immunos aining was signi i-
can ly associa ed wi h a highe umo g ade (p< 0.05,
Fishe ’s exac es , Fig. 2b, Table 2) and ce eb al loca ion
(p< 0.01, Fishe ’s exac es , Fig. 2c, Table 2). Di use
FGFR1 s aining was no signi ican ly associa ed wi h
o e all o ecu ence- ee su i al bu cases wi h high
FGFR1 exp ession had a endency owa d dec eased su -
i al a es in his coho (Addi ional ile 1: Figu e S4).
When ependymomas we e di ided in o pedia ic (n=34)
and adul (n= 72) pa ien s, no associa ions we e obse ed
o FGFR1 in he pedia ic coho . Howe e , FGFR1
s aining was simila ly associa ed wi h umo loca ion
(p< 0.001, n= 70, Fishe ’sexac es )andhighe
umo g ade (p<0.01,n= 72, Fishe ’s exac es ) in
he adul coho as in he whole sample coho . Fu -
he mo e, a weak associa ion was obse ed be ween
s onge FGFR1 s aining and highe umo p oli e a ion
index (p=0.061,n=68,Fishe ’s exac es ) among adul
pa ien s.
FGFR1 and/o FGFR3 le els a e ele a ed in majo i y o he
ce eb al ependymomas
Among ependymomas, ma ked (mode a e- o-s ong)
immunos aining o FGFR1, FGFR3, o bo h p o eins oc-
cu ed mo e equen ly in ce eb al han in non-ce eb al
umo s (76, 32, and 19% in ce eb al, ce ebella , and spinal
umo s, espec i ely, p< 0.001, Fishe ’s exac es , Fig. 2d).
Table 2 Samples numbe s in FGFR1 low, FGFR1 high, FGFR3
low, and FGFR3 high g oups in espec o umo loca ion,
umo g ade and pa ien age
FGFR1 low FGFR1 high FGFR3 low FGFR3 high
Tumo loca ion
Spinal 37 6 41 2
Ce ebella 21 4 23 5
Ce eb al 16 20 20 15
p- alue 0.0001 0.0002
Tumo g ade
I 16 2 18 0
II 50 15 54 14
III 10 13 14 8
p- alue 0.002 0.013
Pa ien age
<162212 2312
> =16 50 18 61 10
p- alue 0.15 0.055
p- alues ha e been calcula ed using Fishe ’s exac es . High: Mode a e- o-s ong
immunos aining, Low: Nega i e- o-low immunos aining
Leh inen e al. BMC Cance (2017) 17:310 Page 6 o 12
0 20406080100
G ade 1
G ade 2
G ade 3
% o cases
n=23
n=65
n=12
Spinal Ce ebella Ce eb al
0
20
40
60
80
100
nega i e
weak
mode a e
s ong
%o cases
n=43 n=25 n=36
n=42 n=25 n=34
1.0
0.8
0.6
0.4
0.2
0.0
0 100 200 300 400
p<0.05
1.0
0.8
0.6
0.4
0.2
0.0
0 100 200 300 400
O e all su i al Recu ence- ee su i al
FGFR1+FGFR3 low (N = 43)
FGFR1 high (N = 11)
FGFR3 high (N = 8)
FGFR1+FGFR3 high (N = 7)
a)
b) c
)
d)
e) )
p < 0.05
Spinal Ce ebella Ce eb al
0
20
40
60
80
100
%o cases
nega i e
weak mode a e s ong
FGFR1+FGFR3 high
FGFR3 high
FGFR1 high
FGFR1+FGFR3 low
FGFR1
FGFR1+FGFR3
low (N = 41)
FGFR1 high
(N=11)
FGFR3 high
(N=7)
FGFR1+FGFR3
high (N = 7)
Fig. 2 Mode a e- o-s ong FGFR1 and/o FGFR3 exp ession is cha ac e is ic o agg essi e ependymomas. aRep esen a i e images o FGFR1
s aining in ependymomas. bThe dis ibu ion o FGFR1 immunos aining in g ade I-III ependymomas. FGFR1 s aining was associa ed wi h highe
umo g ade (p< 0.05, Fishe ’s exac es ). cMode a e- o-s ong FGFR1 immunos aining was associa ed wi h ce eb al umo loca ion (p< 0.01,
Fishe ’s exac es ). To al numbe o umo s o each loca ion is ma ked in o he igu e. dMode a e- o-s ong immunos aining o FGFR1 and/o
FGFR3 was de ec ed in a majo i y o ce eb al ependymoma samples (p< 0.0001, Fishe ’s exac es ). e- ). Mode a e- o-s ong immunos aining o
bo h FGFR3 and FGFR1 was associa ed wi h e) poo disease-speci ic su i al (N= 69, p< 0.05, log- ank es ) and wo se ecu ence- ee su i al
(N= 66, p< 0.05, log- ank es ). Newly diagnosed cases we e di ided in o ou ca ego ies based on he exp ession o bo h FGFR1 and FGFR3.
High: Mode a e- o-s ong immunos aining, Low: Nega i e- o-low immunos aining
Leh inen e al. BMC Cance (2017) 17:310 Page 7 o 12
Inc eased FGFR1 and/o FGFR3 exp ession was he e o e
a common cha ac e is ic o ce eb al umo s. S ikingly,
umo issues exp essing ma ked (mode a e- o-s ong)
le els o bo h FGFR1 and FGFR3 we e associa ed wi h sig-
ni ican ly wo se pa ien su i al han issues ob ained
om o he cases, in e ms o bo h o e all mo ali y
(p< 0.05, log- ank es , Fig. 2e) and ecu ence- ee su -
i al (p< 0.05, log- ank es , Fig. 2 ). Fu he mo e, he
combined a iable o FGFR1 and FGFR3 (bo h a e
nega i e- o weak, ei he s aining is mode a e- o-s ong o
bo h a e mode a e- o-s ong) was he only signi ican p e-
dic o o he disease-speci ic su i al (p= 0.014, RR = 1.91,
95% CI 1.14–3.20, N= 77, s epwise Cox eg ession) and
disease- ee su i al (p= 0.007, RR = 1.75, 95% CI 1.17–
2.62, N= 77, s epwise Cox eg ession), when i was com-
bined oge he wi h umo loca ion, g ade, and p oli e a-
ion index as explana o y ac o s in he mul i ac o ial
analysis. I is good o emembe ha he pa ien numbe s
(N= 77) a e a he low o mul i ac o ial analysis using
ou di e en a iables when in e p e ing hese esul s.
S ill, he ob ained esul s suppo he agg essi e na u e o
umo s wi h mode a e- o-s ong s aining o bo h FGFR1
and FGFR3. Ou esul s a e also conco dan wi h p e ious
no ions (e.g. [28]) ha sup a en o ial and in a en o ial
ependymomas a e la gely di e en and appea o ep e-
sen dis inc umo en i ies.
FGFR3 s aining is associa ed wi h inc eased pa ien age in
pilocy ic as ocy oma
In he pilocy ic as ocy oma coho , 60 (82%) samples
we e nega i e o FGFR3 exp ession, while only 21 cases
(22%) ailed o show any FGFR1 exp ession (Fig. 3c-d).
Among samples wi h FGFR3 immuno eac i i y, 7 sam-
ples (9%) showed weak immunos aining, 5 samples (6%)
showed mode a e immunos aining, and 2 samples (3%)
we e s ongly immunoposi i e. Immunoposi i e FGFR3
s aining was de ec ed in bo h mic ocys ic and pilocy ic
a eas. Among samples wi h posi i e FGFR1 s aining, 59
samples (61%) showed weak immunoposi i i y, 16 sam-
ples (16%) samples showed mode a e immunoposi i i y,
and 1 sample (1%) was s ongly immunoposi i e.
Mode a e- o-s ong FGFR1 immunos aining was de-
ec ed p edominan ly in mic ocys ic a eas. Clinical asso-
cia ion analysis (Addi ional ile 1: Figu e S5) did no
e eal any signi ican associa ions be ween FGFR1
s aining and o he clinical ac o s. In e es ingly,
mode a e- o-s ong FGFR3 p o ein le els we e associ-
a ed wi h inc eased pa ien age (≥16 yea s, p< 0.01,
Fishe ’s exac es , Fig. 3e). All bu one o he six p i-
ma y cases showing mode a e- o-s ong FGFR3 im-
munos aining we e om pa ien s who we e a leas
15 yea s old. FGFR3 immunos aining was no associ-
a ed wi h umo loca ion o aneuploidy.
Absence o FGFR1 o FGFR3 usions in a ge ed
sequencing coho
Ten umo s showing mode a e- o-s ong FGFR1 o FGFR3
immunos aining we e selec ed o a ge ed sequencing
analysis. All analyzed ependymomas we e sup a en o ial.
In addi ion o FGFR3 and FGFR1, he sequencing panel in-
co po a ed genes wi h epo ed al e a ions in gliomas, in-
cluding IDH1, IDH2,TP53,ATRX,CIC,CDKN2A, RB1,
RELA, and BRAF (Addi ional ile 1: Table S1). We did no
de ec FGFR coding mu a ions o usions in any o he
samples (Fig. 4, Addi ional ile 2: Table S2, Addi ional
ile 1: Figu e S6). FGFR3 usions we e de ec ed wi h
high sensi i i y om la ge di use glioma coho using
he same sequencing panel and me hodology [34], sug-
ges ing ha he lack o de ec able FGFR usions was
no due o me hodological limi a ions. The umo s se-
lec ed o analysis con ained many known al e a ions, in-
cluding a C11o 95-RELA usion and CDKN2A al e a ions
in ependymoma umo s (Epe001, Epe002 and Epe003).
RELA usions and loss o CDKN2A ha e been ou inely
obse ed in agg essi e ependymomas [17, 29, 30]. A TERT
p omo e mu a ion was obse ed in umo s Epe004 and
Epe005 ob ained om he same ependymoma pa ien . In
addi ion, one pilocy ic as ocy oma umo ha bo ed he
KIAA1549-BRAF usion, which is he mos equen
MAPK pa hway al e a ion in his umo ype [7]. I is in-
e es ing ha majo i y o sequenced PA samples did no
ca y any BRAF o FGFR1 al e a ions, bu limi ed sample
size does no allow ull gene aliza ion o his esul . A o al
o 4 cases in ou coho did no ca y any al e a ions in
a ge ed genes. This may be due, in pa , o he ac ha
all genomic egions we e no co e ed du ing a ge ed se-
quencing. In addi ion, pilocy ic as ocy omas a e known
o ha bo e y ew al e a ions [7].
Discussion
Ou esul s demons a e ha mode a e- o-s ong FGFR3
and/o FGFR1 immunos aining was de ec able in mos
o he sup a en o ial ependymomas. In ependymoma,
mode a e- o-s ong FGFR3 s aining was associa ed wi h
umo loca ion, highe p oli e a ion index, and highe
g ade. Simila associa ions we e ob ained when only
adul pa ien s we e included in o he analysis. Mode a e-
o-s ong FGFR3 s aining was mo e equen ly obse ed
among pedia ic pa ien s han among adul s, bu only
he associa ion be ween FGFR3 and umo loca ion
emained signi ican in he pedia ic coho . This migh
be pa ly due o a small numbe o pedia ic cases
(n= 35) and sho age o g ade I umo s (n= 1) among
child en. In any case, he da a sugges ha clinical asso-
cia ions o FGFR3 we e no solely due o age- ela ed
di e ences. The si ua ion was simila o FGFR1:
mode a e- o-s ong s aining was associa ed wi h umo
loca ion and highe g ade in bo h he whole and he
Leh inen e al. BMC Cance (2017) 17:310 Page 8 o 12
adul coho , despi e he lack o clinical associa ions in
he pedia ic coho .
Tumo s wi h high exp ession o bo h FGFR3 and
FGFR1 we e associa ed wi h poo clinical p ognosis in
ependymoma, sugges ing ha agg essi e sup a en o ial
ependymomas may bene i om ea men egimens
based on FGFR inhibi ion. Addi ional wo k is equi ed
o elucida e he signi icance o high FGFR1 and/o
FGFR3 exp ession as independen p ognos ic ac o s o
ea men esponse. The absence o FGFR al e a ions in
hese umo s does no ule ou he possibili y o ea -
men esponse. In head and neck squamous cell cance s
and a ious lung cance s, FGFR1 exp ession has, in ac ,
been shown o p edic ea men esponses be e han
0 20406080100
FGFR1
FGFR3
nega i e
weak mode a e s ong
% o cases
0 20 40 60 80 100
nega i e weak mode a e s ong
% o cases
16 yea s
<16 yea s
FGFR3
FGFR1 FGFR3
Posi i e
Nega i e Posi i e
Nega i e
a)
b) c)
N=22
N=54
n=80
n=97
Fig. 3 FGFR3 and FGFR1 s aining in pilocy ic as ocy oma. aRep esen a i e immunohis ochemical images in pilocy ic as ocy oma. bDis ibu ion
o immunohis ochemis y sco es. The majo i y o samples we e nega i e o FGFR3. cNea ly all o he pilocy ic as ocy oma samples showing
mode a e- o-s ong FGFR3 immunos aining we e ob ained om non-pedia ic pa ien s (p< 0.01, Fishe ’s exac es ). Only newly-diagnosed umo s
we e included in o his analysis
Fig. 4 Summa y o gene ic al e a ions in he cases ha we e analyzed using a ge ed sequencing. No coding mu a ions o gene usions we e
de ec ed in FGFR3 o FGFR1. FGFR1 and FGFR3 immunohis ochemical s aining sco es a e shown abo e he igu e. I s ained whole-moun issue
slides we e a ailable, hey we e used o sco ing. Pilocy ic: pilocy ic as ocy oma
Leh inen e al. BMC Cance (2017) 17:310 Page 9 o 12