Nature GeNetics VOLUME 45 | NUMBER 8 | AUGUST 2013 927
L E T T E R S
Pilocytic astrocytoma, the most common childhood brain
tumor
1
, is typically associated with mitogen-activated protein
kinase (MAPK) pathway alterations
2
. Surgically inaccessible
midline tumors are therapeutically challenging, showing
sustained tendency for progression
3
and often becoming a
chronic disease with substantial morbidities
4
. Here we describe
whole-genome sequencing of 96 pilocytic astrocytomas,
with matched RNA sequencing (n = 73), conducted by the
International Cancer Genome Consortium (ICGC) PedBrain
Tumor Project. We identified recurrent activating mutations
in FGFR1 and PTPN11 and new NTRK2 fusion genes in
non-cerebellar tumors. New BRAF-activating changes were
also observed. MAPK pathway alterations affected all tumors
analyzed, with no other significant mutations identified,
indicating that pilocytic astrocytoma is predominantly a
single-pathway disease. Notably, we identified the same FGFR1
mutations in a subset of H3F3A-mutated pediatric glioblastoma
with additional alterations in the NF1 gene
5
. Our findings thus
identify new potential therapeutic targets in distinct subsets of
pilocytic astrocytoma and childhood glioblastoma.
Pilocytic astrocytoma is the most common central nervous sys-
tem (CNS) neoplasm in childhood, accounting for ~20% of all
pediatric brain tumors. Tumor locations in our cohort reflect the
fact that pilocytic astrocytomas occur throughout the CNS, with
about half arising outside the cerebellum (Supplementary Fig. 1).
Extracerebellar tumors are often surgically inaccessible, leading to
chronic disease with multiple recurrences, visual and neurological
impairment and/or side-effects of therapy
1,4
. Genetic alterations
within the MAPK signaling pathway are a hallmark of this tumor,
with KIAA1549-BRAF fusion being the most frequent event
6–8
.
A smaller number of tumors harbor BRAF or KRAS point mutations,
alternative BRAF-RAF1 fusions or germline NF1 mutations
2
. Pilocytic
astrocytoma has therefore been hypothesized to represent a single-
pathway disease
2
. Previously, however, no MAPK pathway changes
were identifiable in 15–20% of tumors (mostly non-cerebellar)
2
.
To investigate the full range of genetic alterations in pilocytic
astrocytoma, we performed whole-genome sequencing of tumor and
blood DNA from 96 affected individuals (Supplementary Table 1).
Corresponding RNA sequencing data and data from mate-pair
sequencing with larger inserts (for enhanced detection of structural
rearrangements) were generated for 73 and 68 samples, respectively.
The average somatic mutation rate was extremely low (<0.1 mutation
per megabase), with a mean of 1.6 nonsynonymous single-nucleotide
variants (SNVs) per tumor (range of 0–9; Supplementary Table 1),
similar to the rate described in NF1-associated pilocytic astrocytomas
9
.
The somatic mutation rate in our series was markedly lower than
those recently reported for the malignant pediatric brain tumor
Recurrent somatic alterations of FGFR1 and NTRK2 in
pilocytic astrocytoma
David T W Jones
1,39
, Barbara Hutter
2,39
, Natalie Jäger
2,39
, Andrey Korshunov
3,4
, Marcel Kool
1
,
Hans-Jörg Warnatz
5
, Thomas Zichner
6
, Sally R Lambert
7
, Marina Ryzhova
8
, Dong Anh Khuong Quang
9
,
Adam M Fontebasso
9
, Adrian M Stütz
6
, Sonja Hutter
1
, Marc Zuckermann
10
, Dominik Sturm
1
, Jan Gronych
10
,
Bärbel Lasitschka
11
, Sabine Schmidt
11
, Huriye Şeker-Cin
1
, Hendrik Witt
1,12
, Marc Sultan
5
, Meryem Ralser
5
,
Paul A Northcott
1
, Volker Hovestadt
10
, Sebastian Bender
1
, Elke Pfaff
1
, Sebastian Stark
1
, Damien Faury
9
,
Jeremy Schwartzentruber
13
, Jacek Majewski
13
, Ursula D Weber
10
, Marc Zapatka
10
, Benjamin Raeder
6
,
Matthias Schlesner
2
, Catherine L Worth
5
, Cynthia C Bartholomae
14
, Christof von Kalle
14,15
, Charles D Imbusch
2
,
Sylwester Radomski
2,16,17
, Chris Lawerenz
2
, Peter van Sluis
18
, Jan Koster
18
, Richard Volckmann
18
,
Rogier Versteeg
18
, Hans Lehrach
5
, Camelia Monoranu
19
, Beate Winkler
20
, Andreas Unterberg
21
,
Christel Herold-Mende
21
, Till Milde
12,22
, Andreas E Kulozik
12
, Martin Ebinger
23
, Martin U Schuhmann
24
,
Yoon-Jae Cho
25
, Scott L Pomeroy
26,27
, Andreas von Deimling
3,4
, Olaf Witt
12,22
, Michael D Taylor
28,29
,
Stephan Wolf
11
, Matthias A Karajannis
30
, Charles G Eberhart
31
, Wolfram Scheurlen
32
, Martin Hasselblatt
33
,
Keith L Ligon
26,34,35
, Mark W Kieran
26,36
, Jan O Korbel
6
, Marie-Laure Yaspo
5
, Benedikt Brors
2
, Jörg Felsberg
37
,
Guido Reifenberger
37
, V Peter Collins
7
, Nada Jabado
9,38
, Roland Eils
2,15–17,40
, Peter Lichter
10,15,40
&
Stefan M Pfister
1,12,40
, for the International Cancer Genome Consortium PedBrain Tumor Project
A full list of author affiliations appears at the end of the paper.
Received 26 March; accepted 3 June; published online 30 June 2013; doi:10.1038/ng.2682
npg
© 2013 Nature America, Inc. All rights reserved.
928 VOLUME 45 | NUMBER 8 | AUGUST 2013 Nature GeNetics
L E T T E R S
medulloblastoma
10–12
and for several other pediatric solid tumors
13
.
The average number of small insertion-deletion alterations (indels)
affecting coding sequences was <1 per case. All coding somatic SNVs
and indels are listed in Supplementary Table 2.
In line with other tumor types
10,14,15
, pilocytic astrocytomas had
genome-wide mutation rates that positively correlated with the age
of the affected individual (r = 0.42; P = 2.3 × 10
−5
, Pearson’s product-
moment correlation; Supplementary Fig. 2a). The observed muta-
tions were predominantly cytosine-to-thymine transitions at CpG
sites (likely arising from deamination of methylated cytosines),
suggesting that the age-dependent increase in mutation frequency
may largely be due to background processes occurring in progeni-
tor cells before tumorigenesis, as recently reported in leukemia
15
(Supplementary Fig. 2b).
Most of the known events activating the MAPK pathway were
also found in our series, including KIAA1549-BRAF fusion variants
(70 cases), a FAM131B-BRAF fusion
16
, 4 BRAF
V600E
mutations and
1 BRAF
ins599T
alteration (Supplementary Table 1). Three tumors were
associated with neurofibromatosis type 1. This prevalence is lower
than would be expected for prospective cohorts (5–10%), as material
for biological studies from these typically optic pathway–associated
tumors is limited. NF1 has been reported to follow a classical tumor
suppressor model in pilocytic astrocytoma, with a somatic second hit
in addition to a germline alteration
9
. This model also held true in our
series (Supplementary Table 1).
Analysis of copy number and structural alterations using DNA
and RNA sequencing identified four new BRAF fusions (Fig. 1 and
Supplementary Fig. 3). As expected, all variants resulted in loss of the
N-terminal regulatory region of BRAF. An RNF130-BRAF fusion derived
from a reciprocal t(5;7)(q35;q34) translocation was seen in two cases
(Fig. 1a), with single examples identified of CLCN6-BRAF, MKRN1-
BRAF and GNAI1-BRAF fusions (Supplementary Fig. 3a–c). Thus,
non–KIAA1549-BRAF fusions comprise a notable minority of activating
events, with BRAF seeming to be a promiscuous fusion partner.
Another new BRAF alteration was identified in ICGC_PA65, result-
ing in a three-amino-acid insertion (p.Arg506_insValLeuArg, insVLR)
in the interdomain cleft of BRAF—a structural region linked to
BRAF activity
17
and homodimerization
18
. Protein modeling pre-
dicted that the insertion of these residues stabilizes a dimeric form
of BRAF (known to be active independent of RAS stimulation
19
)
(Fig. 1b). Homodimerization was confirmed by immunoprecipi-
tation, and the BRAF
insVLR
mutant increased extracellular signal–
regulated kinase (ERK) phosphorylation as effectively as the
BRAF
V600E
mutant (Fig. 1c,d).
New alterations in KRAS were also observed. ICGC_PA117
and ICGC_PA142 both showed two distinct mutations (encoding
p.[Glu63Lys]+[Arg73Met] and p.[Leu19Phe]+[Gln22Lys], respec-
tively). DNA and RNA sequencing data confirmed that both altera-
tions affected the same allele (Supplementary Fig. 4). Although there
are reports of double KRAS mutations in entities such as colon can-
cer
20
, these typically involve at least one hotspot residue (codon 12,
13 or 61) and often represent heterogeneous tumor subclones rather
than two hits in one allele (although this has also been described; for
example, see ref. 21). The alterations identified in our tumors did
not encompass classical mutational hotspots, suggesting that further
characterization of downstream effects is warranted.
All but one of the cerebellar tumors in our series harbored a BRAF
fusion, with this one exception having a KRAS alteration. Nine of
48 (19%) of the non-cerebellar tumors, however, lacked the above
alterations. Further assessment of structural rearrangements iden-
tified two new gene fusions in a total of three samples, involving
the region encoding the kinase domain of NTRK2 (also known as
TrkB)—an oncogene implicated in the tumorigenesis of neuroblas-
toma, among other cancers
22,23
. The related NTRK1 and NTRK3
genes have previously been shown to be activated by fusion events
(for example, TPM3-NTRK1 in papillary thyroid cancer
24
and
ETV6-NTRK3 in multiple tumors
25
). The QKI-NTRK2 and NACC2-
NTRK2 fusions identified here were verified by PCR (Fig. 2 and
Supplementary Fig. 3d). Both 5′ partners contained regions encoding
dimerization domains and are therefore predicted to induce ligand-
independent dimerization. Notably, N-terminal TrkB truncation has
recently been shown to induce transformation of neural crest cells
26
.
Figure 1 New BRAF alterations in pilocytic
astrocytoma. (a) Schematic of the RNF130-
BRAF fusion gene in ICGC_PA112 resulting
from a translocation between chromosomes
5 and 7. A similar fusion was observed in
ICGC_PA96. The cDNA sequence at the fusion
breakpoint (dashed line) and resulting exon
and protein structures are shown. A reciprocal
fusion between RUFY1 (encoding RUN and
FYVE domain–containing 1) and TMEM178B
(encoding transmembrane protein 178B)
on the derivative chromosome 5 in ICGC_PA112
was also found to be expressed in RNA
sequencing analysis (data not shown).
RPM, reads per million; KD, kinase domain.
(b) Computational modeling of two BRAF
monomers (light and dark gray) with a ValLeuArg
insertion (blue and magenta) between Arg506
and Lys507 (green), as identified in ICGC_PA65
(p.Arg506insValLeuArg, insVLR). Protein
Data Bank (PDB) structure 4E26 was used as
a template. Val600, a mutational hotspot, is shown in yellow. A new dimer interface is formed between the protomers, with hydrogen bonds formed
between the new arginine side chains (dashed lines) and a hydrophobic interaction between the leucine side chains (magenta). (c) Protein blot analysis
of NIH3T3 cells transfected with empty vector (EV) or with vector expressing wild-type (WT) BRAF, BRAF
V600E
or BRAF
insVLR
. The newly identified
BRAF
insVLR
mutant results in greater phosphorylation of ERK1 and ERK2 (pERK1/2), with phosphorylation at a similar level to that seen with the
known oncogenic BRAF
V600E
form. (d) Pulldown assay with immunoprecipitation (IP) of HA-tagged BRAF
insVLR
, showing that this new mutant forms
homodimers with coexpressed AU1-tagged BRAF
insVLR
mutant but does not seem to form a strong heterodimer with wild-type BRAF.
a b
c d
RNF130-BRAF fusion
Derivative
chr. 5
Derivative
chr. 7
t(5;7)
2.9
1.8
0
0
BRAF RNF130
TMEM178BRUFY1
RNF130 exons 1–3 BRAF exons 9–18
...ATGCACGCGACAGGAACCAG GACTTGATTAGAGACCAAGG...
N- -C
RPM
RNF130 expression
RPM
BRAF expression
KD
(ICGC_PA112)
BRAF
insVLR
-HA + EV
BRAF
insVLR
-Flag + EV
BRAF
insVLR
-HA + WT BRAF–Flag
BRAF
insVLR
-HA + BRAF
insVLR
-Flag
EV
WT BRAF–Flag
BRAF
V600E
-Flag
BRAF
insVLR
-Flag
BRAF-Flag
ERK1/2
pERK1/2
Flag-BRAF
IP: HA
Input
HA-BRAF
Flag-BRAF
HA-BRAF
npg
© 2013 Nature America, Inc. All rights reserved.
Nature GeNetics VOLUME 45 | NUMBER 8 | AUGUST 2013 929
L E T T E R S
The downstream effects of TrkB activation are mediated, at least in
part, via MAPK pathway activation
27
.
A second new recurrent alteration, namely, mutation of two
hotspots (codons for Asn546 and Lys656) within the kinase domain
of FGFR1, was seen in five tumors (Fig. 3a and Supplementary
Table 3). FGFR1 is more commonly activated through amplification
in tumors such as breast
28
and lung
29,30
cancer. Occasional FGFR1
mutations have been observed in adult glioblastoma (GBM)
31,32
, a
highly malignant astrocytoma, as have FGFR1-TACC1 or FGFR3-
TACC3 fusion genes
33
. Mutations in homologous codons in FGFR2
and FGFR3 are commonly found in other tumor types, particularly
bladder, skin and endometrial cancers (see the Catalogue of Somatic
Mutations in Cancer (COSMIC) database
34
). Both mutations result
in midbrain hyperproliferation in developing chick embryos
35
. The
p.Asn546Lys variant alters FGFR1 autophosphorylation, resulting
in higher kinase activity and transforming potential
36
, whereas the
p.Lys656Glu variant is also transforming in vitro
37
. Notably, the latter
study suggested that fibroblast growth factor 2 (FGF2, also known as
bFGF) ligand was necessary in addition to FGFR1 mutation to main-
tain neurosphere formation in vitro. Gene expression array data of 118
pilocytic astrocytomas, including 66 from the present series, showed
significantly increased FGF2 expression in pilocytic astrocytomas
compared with 158 other astrocytic tumors
38,39
or normal tissues
40
.
This increase was not restricted to only FGFR1-mutant or wild-type
tumors, suggesting that ligand-mediated pathway activation may
have a general role in tumorigenesis (Fig. 3b). Immunohistochemical
detection of phosphorylated FGFR1 showed strong, diffuse positivity
in all seven pilocytic astrocytomas harboring an FGFR1 mutation
for which material was available. No positivity was observed in 11
tumors with wild-type FGFR1. All samples showed strong staining for
phosphorylated ERK (Supplementary Fig. 5). Notably, ICGC_PA89
harbored an alternative alteration in FGFR1 consisting of a ~4.5-kb
internal tandem duplication (ITD) of the portion of the gene encod-
ing the kinase domain, reminiscent of the activating internal tan-
dem duplications of the FLT3 kinase observed in acute myeloid
leukemia
41
(Fig. 3c).
Further recurrent mutations were found in the phosphatase gene
PTPN11 (also called SHP-2) encoding a RAS-MAPK–related adap-
tor protein (Fig. 3d). Both encoded alterations (p.Glu69Lys and
p.Glu76Ala) were previously reported in juvenile monomyelocytic
leukemia, which is frequently associated with SHP-2 activa-
tion
42,43
. Notably, both alterations were found in FGFR1-mutant
tumors (ICGC_PA84 and ICGC_PA166), suggesting a cooperative
role of these factors in tumorigenesis (Supplementary Table 3).
Overexpression of mutant SHP-2 alone did not elevate the levels of
phosphorylated ERK in vitro, whereas the two FGFR1 mutants, either
alone or in combination with mutant SHP-2, upregulated the levels
of phosphorylated ERK (Supplementary Fig. 6). This finding sup-
ports the hypothesis that PTPN11 mutation alone is insufficient for
pilocytic astrocytoma development but may have a modifying role in
FGFR1-mutant tumors. Of note, PTPN11 expression was higher in
pilocytic astrocytomas compared with other astrocytomas or normal
tissues (Fig. 3e), suggesting that this phosphatase has a broader role
in the biology of this entity. An additional cohort of 45 non-cerebellar
pilocytic astrocytomas, negative for KIAA1549-BRAF fusion, was
screened for FGFR1 (exons 12 and 14) and PTPN11 (exon 3) muta-
tions. Nine cases harbored FGFR1 mutations encoding a p.Asn546 or
p.Lys656 alteration, and one additionally carried a PTPN11 mutation
encoding a p.Glu69Lys change (Supplementary Table 3), confirming
our whole-genome sequencing findings. Germline PTPN11 muta-
tions are one of the causes of the hereditary developmental disorders
Noonan syndrome
44
and multiple lentigines syndrome (also known as
LEOPARD syndrome)
45
. A few case reports have described pilocytic
astrocytomas occurring in individuals with these syndromes
46–49
.
Thus, together with NF1, there are three known ‘RASopathies’, char-
acterized by germline MAPK pathway mutations
50
, linked with pilo-
cytic astrocytoma tumorigenesis. In our germline sequencing data,
however, NF1 was the only RASopathy-related gene disrupted at a
higher frequency than in the 1000 Genomes Project (see URLs).
Notably, all of the pilocytic astrocytomas in our cohort harbored
a MAPK pathway alteration. BRAF, FGFR1, KRAS and NF1 were
the only genes found to be significantly mutated using the Genome
MuSiC algorithm (see URLs; Supplementary Table 4). With the
exception of FGFR1 and PTPN11, each case typically harbored only
one pathway alteration (P < 0.0001, permutation test; Fig. 4). Together
with the finding that BRAF kinase activation alone is sufficient to
induce pilocytic astrocytomas in mice
51,52
, these data strongly support
the concept of pilocytic astrocytoma as a prototypic single-pathway
disease driven by a limited number of oncogenic hits (possibly only
one in some cases; Supplementary Fig. 7).
One of the FGFR1-mutant tumors (ICGC_PA69) also had an
H3F3A mutation encoding a p.Lys27Met alteration and somatic
mutations of NF1—both of which are more commonly encountered
in pediatric GBM
5
. Three experienced neuropathologists agreed on
pilocytic astrocytoma histology for this case, although a diagnosis of
GBM cannot be conclusively excluded, owing to limited material. By
examining previous exome sequencing data for pediatric GBM
5
, we
identified 3 of 48 samples (6%) with an FGFR1 mutation. Notably, all
three harbored the same constellation of an H3F3A p.Lys27Met altera-
tion, a somatic NF1 alteration and FGFR1 activation (Supplementary
Table 3). They were also wild type for TP53, which is mutated in
most GBMs or diffuse intrinsic pontine gliomas
5,53
with the H3F3A
p.Lys27Met alteration. One tumor reported in a targeted sequenc-
ing cohort of medulloblastoma
10
had a similar triple alteration,
QKI-NTRK2 fusion
Derivative chr. 9
Derivative chr. 6
t(6;9)
QKI exons 1–6 NTRK2 exons 16–21
...TGAACCTAGTGGTGTATTAG GCCCAGCCTCCGTTATCAGC...
RPM
QKI expression
RPM
NTRK2 expression
QKI NTRK2
(ICGC_PA159)
-C
KD
N-
QUA1
10
0
15
0
Figure 2 NTRK2 is a new gene fusion target in pilocytic astrocytoma.
Schematic of the QKI-NTRK2 fusion gene in ICGC_PA159 resulting from
a translocation between chromosomes 6 and 9. A similar fusion was
observed in ICGC_PA82. The cDNA sequence at the fusion breakpoint
(dashed line) and resulting exon and protein structures are shown.
QUA1, Qua1 dimerization domain.
npg
© 2013 Nature America, Inc. All rights reserved.
930 VOLUME 45 | NUMBER 8 | AUGUST 2013 Nature GeNetics
L E T T E R S
with an H3F3A p.Lys27Met alteration, an NF1 alteration and an
FGFR2 p.Lys659Glu alteration (homologous to FGFR1 p.Lys656Glu),
making a total of five cases with this combination. Gene expression
analysis indicated that this tumor was likely a GBM previously mis-
classified as medulloblastoma. It is not currently clear why these alter-
ations occur in concert, and additional work will be required to assess
their roles. One possibility is that NF1 mutation may mimic elevated
PTPN11 expression, as activation of SHP-2 inhibits the recruitment
of Ras GTPase–activating proteins (RasGAPs, including NF1) to the
plasma membrane
54
.
All FGFR1-mutant tumors were extracerebellar, mostly in midline
locations (Supplementary Table 3), suggesting a link between cell of
origin and/or microenvironment with FGFR1-driven tumorigenesis.
The H3F3A p.Lys27Met alteration is also associated with midline
GBM
39
. Notably, FGFR1 has a role in neural stem cell self-renewal
55
and is essential for midline glial cell development
56
. This spatial cluster-
ing may also reflect differential sensitivity of distinct neural precursors
to activating stimuli, particularly NF1 loss
57,58
. The type and timing of
second hits (H3F3A or NF1 mutation) and/or the differentiation stage
of the cell of origin may contribute to determining a fate of oncogene-
induced senescence and slow growth (pilocytic astrocytoma)
59,60
versus aggressive malignancy with poor outcome (GBM).
In summary, this study has provided new insights into the tum-
origenesis of pilocytic astrocytoma. Each tumor harbored very few
mutations, in keeping with generally benign behavior. Our findings
confirm the concept that pilocytic astrocytomas are predominantly
d
a
Ig I
Ig II
Ig III
N546K (6)
K656E, K656D,
K656N, K656M
TK1
TM
TK2
FGFR1
b
0
200
400
600
800
1,000
1,200
PA (118)
DA (20)
AA (43)
K27 GBM (13)
G34 GBM (11)
IDH1 GBM (12)
Other pediatric GBM (58)
Fetal CBM (5)
Adult CBM (4)
Normal CNS (169)
Normal non-CNS (184)
FGF2 mRNA expression
P < 0.001
P < 0.001
SH2
PTP
SH2
E69K (2)
E76A (1)
SHP-2
e
0
1,000
2,000
3,000
4,000
PTPN11 mRNA expression
PA (118)
DA (20)
AA (43)
K27 GBM (13)
G34 GBM (11)
IDH1 GBM (12)
Other pediatric GBM (58)
Fetal CBM (5)
Adult CBM (4)
Normal CNS (169)
Normal non-CNS (184)
P < 0.001
P < 0.001
c
RPM
FGFR1 expression
FGFR1-ITD
Ig I Ig II Ig III TM TK1 TK2
TK1′ TK2′
dup(8)
FGFR1
(PA89)
Derivative chr. 8
Exons 11–18
TFIPLSARSWVKYREVEMGCLSRFELHQPQLMPLSVSPER
+ Intron 10 linker
3.5
0
Figure 3 FGF pathway signaling
molecules are recurrently altered in pilocytic
astrocytoma. (a) Schematic of the domain
structure of FGFR1, indicating the position and
frequency of the hotspot alterations in pilocytic
astrocytomas sequenced in the present study (including replication cases).
Ig, immunoglobulin-like domain; TM, transmembrane domain; TK, tyrosine
kinase domain. (b) Gene expression data for FGF2 indicating significantly
elevated expression in pilocytic astrocytomas (red) compared with other
astrocytic tumors (blue), normal cerebellum (black) and other normal
tissues (green); P < 0.001, two-sided t test. The pilocytic astrocytomas with
expression data that harbor FGFR1 alterations (four mutants plus FGFR1-ITD)
are circled. Horizontal gray bars indicate mean expression values per group.
PA, pilocytic astrocytoma; DA, World Health Organization (WHO)
grade 2 diffuse astrocytoma; AA, anaplastic astrocytoma; K27, G34 and
IDH1 GBM, glioblastoma carrying a mutation affecting Lys27 or Gly34 of
H3F3A or IDH1, respectively; CBM, cerebellum. (c) Schematic of an additional
alteration in FGFR1 identified in ICGC_PA89 comprising an internal tandem
duplication of part of intron 10, exons 11–17 and part of exon 18 (boundaries highlighted by dashed lines).
The duplicated amino acids are residues 478–820 (numbered according to the α A1 isoform), with an additional 40-residue linker sequence encoded
by part of intron 10. The whole kinase domain is therefore duplicated in the resulting predicted protein (TK1′ and TK2′). (d) Schematic of the structure
of SHP-2 (PTPN11), indicating the position and frequency of alterations in pilocytic astrocytomas sequenced in the present study. SH2, src homology
2 domain; PTP, protein tyrosine phosphatase domain. (e) Gene expression data for PTPN11 indicating significantly elevated expression in pilocytic
astrocytomas compared with other groups as defined in b; P < 0.001, two-sided t test. The pilocytic astrocytomas with expression data that harbor
FGFR1 alterations (four mutants plus FGFR1-ITD) are circled.
KIAA1549-BRAF
FGFR1 mutation
Other BRAF fusion
BRAF mutation
PTPN11 mutation
KRAS mutation
NF1 mutation
NTRK2 fusion
Figure 4 Summary of MAPK pathway alterations in pilocytic astrocytoma.
An overview of MAPK pathway alterations identified in the 96 whole-genome
sequencing cases included in the present study, indicating the mutual
exclusivity of the majority of these hits (with the exception of ones
affecting FGFR1 and PTPN11); P < 0.0001, permutation test on 10,000
iterations. Each column represents one tumor sample. Red boxes indicate
that a given alteration is present in this sample. The blue box represents
FGFR1-ITD rather than a point mutation. The yellow box indicates a
BRAF p.Glu451Asp alteration in a case with a KIAA1549-BRAF fusion
(of unknown functional significance but included in the exclusivity
testing). The black/red split boxes represent one germline and one somatic
NF1 alteration per case.
npg
© 2013 Nature America, Inc. All rights reserved.
Nature GeNetics VOLUME 45 | NUMBER 8 | AUGUST 2013 931
L E T T E R S
driven by aberrant activation of the MAPK pathway. Most notably,
however, we report new recurrent mutations in NTRK2, FGFR1 and
PTPN11, which were mutually exclusive with other RAF and RAS
changes. Combined with the observation of FGF2 and PTPN11 over-
expression, these results indicate upstream contributors to MAPK
pathway activation in this entity. Emerging preclinical data suggest
that BRAF inhibitors may trigger paradoxical activation in tumors
harboring KIAA1549-BRAF fusions, that is, the majority of pilocytic
astrocytomas
61
. Single-drug or combination therapy with FGFR,
NTRK2 and/or MAPK/ERK kinase (MEK) inhibitors, several of which
are currently in preclinical and clinical trials
62–64
, may therefore rep-
resent rational treatment options. BRAF
V600E
-specific agents may also
be a logical choice for ~5% of patients. Finally, the identification of
recurrent FGFR1 mutations in a subset of pediatric GBMs provides an
opportunity for the therapeutic targeting of FGFR signaling in these
clinically challenging brain tumors.
URLs. ICGC PedBrain Tumor Project, http://www.pedbraintumor.
org/; 1000 Genomes Project, http://www.1000genomes.org/;
GenomeMuSiC, http://gmt.genome.wustl.edu/gen
ome-music/0.2/
index.html; Oncotator, http://www.broadinstitute.org/oncotator/;
R2 tool, http://r2.amc.nl.
METHODS
Methods and any associated references are available in the online
version of the paper.
Accession code. Sequencing data have been deposited at the European
Genome-phenome Archive, which is hosted by the European
Bioinformatics Institute (EBI), under accession EGAS00001000381.
Note: Supplementary information is available in the online version of the paper.
ACKNOWLEDGMENTS
For technical support and expertise, we thank B. Haase, D. Pavlinic and
B. Baying (EMBL Genomics Core Facility); M. Wahlers and R. Lück (EMBL
High-Performance Computing Facility); the DKFZ Genomics and Proteomics
Core Facility; M. Knopf (NCT Heidelberg); K. Schlangen, M. Metsger, K. Schulz,
A. Nürnberger, A. Kovacsovics and M. Linser (Max Planck Institute for Molecular
Genetics); S. Peetz-Dienhart and Y. Floer (University Hospital Münster);
D.M. Pearson (University of Cambridge); and B. Huang, G. Zipprich, M. Heinold,
R. Kabbe, A. Wittmann, L. Sieber and L. Linke (DKFZ). W. Stummer (Münster),
B. Hoffmann (Münster), B. Rama (Osnabrück), H. Ebel (Hamm), H.A. Trost
(Bayreuth) and U. Wildförster (Gelsenkirchen) provided detailed clinical
information. We also thank GATC Biotech for sequencing services. This work
was principally supported by the PedBrain Tumor Project contributing to the
International Cancer Genome Consortium, funded by German Cancer Aid
(109252) and by the German Federal Ministry of Education and Research
(BMBF, grants 01KU1201A, MedSys 0315416C and NGFN
plus
01GS0883).
Additional support came from the German Cancer Research Center–Heidelberg
Center for Personalized Oncology (DKFZ-HIPO), the Max Planck Society,
Genome Canada and the Canadian Institute for Health Research (CIHR) with
cofunding from Genome BC, Génome Quebec, CIHR-ICR (Institute for Cancer
Research) and C17 (N. Jabado), Ian’s Friend Foundation (M.A.K.), the US National
Institutes of Health (NIH; grants RO1CA105607 and P30HD018655 to S.L.P.), the
Dutch Cancer Foundations KWF (2010-4713) and KIKA (M.K.), the Brain Tumour
Charity (S.R.L. and V.P.C.) and the Pediatric Low-Grade Astrocytoma Foundation
(M.W.K. and K.L.L.).
AUTHOR CONTRIBUTIONS
D.T.W.J., S.R.L., D.A.K.Q., A.M.F., H.-J.W., A.M.S., S.H., M. Zuckermann,
J.G., S. Schmidt, H.Ş.-C., H.W., S.B., E.P., S. Stark, B.R., D.F., C.C.B., C.v.K.,
P.v.S., R. Versteeg, M. Sultan, S.W., M.H. and J.F. performed and/or coordinated
experimental work. B.H., N. Jäger, D.T.W.J., M.K., H.-J.W., T.Z., B.L., P.A.N.,
V.H., J.S., J.M., M. Zapatka, M. Schlesner, C.L.W., C.D.I., S.R., C.L., P.v.S., J.K.,
R. Volckmann and M. Ralser performed data analysis. A.K., M. Ryzhova, C.M.,
B.W., A.U., C.H.-M., T.M., A.E.K., M.E., M.U.S., Y.-J.C., S.L.P., A.v.D., O.W., M.H.,
M.A.K., C.G.E., W.S., K.L.L., M.W.K., V.P.C. and N. Jabado collected data and
provided materials from study subject. D.T.W.J., B.H., N. Jäger, D.S., N. Jabado,
R.E., P.L. and S.M.P. prepared the initial manuscript and figures. A.K., U.D.W.,
M.D.T., J.O.K., H.L., M.-L.Y., B.B., G.R., V.P.C., N. Jabado, R.E., P.L. and S.M.P.
provided project leadership. All authors contributed to the final manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
1. Central Brain Tumor Registry of the United States. Statistical Report: Primary Brain
and Central Nervous System Tumors Diagnosed in the United States, 2004–2008
(CBTRUS, Hinsdale, IL, 2012).
2. Jones, D.T.W., Gronych, J., Lichter, P., Witt, O. & Pfister, S.M. MAPK pathway
activation in pilocytic astrocytoma. Cell Mol. Life Sci. 69, 1799–1811 (2012).
3. Gnekow, A.K. et al. Long-term follow-up of the multicenter, multidisciplinary
treatment study HIT-LGG-1996 for low-grade glioma in children and adolescents
of the German Speaking Society of Pediatric Oncology and Hematology. Neuro-oncol.
14, 1265–1284 (2012).
4. Armstrong, G.T. et al. Survival and long-term health and cognitive outcomes after
low-grade glioma. Neuro-oncol. 13, 223–234 (2011).
5. Schwartzentruber, J. et al. Driver mutations in histone H3.3 and chromatin
remodelling genes in paediatric glioblastoma. Nature 482, 226–231 (2012).
6. Jones, D.T.W. et al. Tandem duplication producing a novel oncogenic BRAF fusion
gene defines the majority of pilocytic astrocytomas. Cancer Res. 68, 8673–8677
(2008).
7. Jones, D.T.W. et al. Oncogenic RAF1 rearrangement and a novel BRAF mutation
as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in
pilocytic astrocytoma. Oncogene 28, 2119–2123 (2009).
8. Pfister, S. et al. BRAF gene duplication constitutes a mechanism of MAPK pathway
activation in low-grade astrocytomas. J. Clin. Invest. 118, 1739–1749 (2008).
9. Gutmann, D.H. et al. Somatic neurofibromatosis type 1 (NF1) inactivation
characterizes NF1-associated pilocytic astrocytoma. Genome Res. 23, 431–439
(2013).
10. Jones, D.T.W. et al. Dissecting the genomic complexity underlying medulloblastoma.
Nature 488, 100–105 (2012).
11. Pugh, T.J. et al. Medulloblastoma exome sequencing uncovers subtype-specific
somatic mutations. Nature 488, 106–110 (2012).
12. Robinson, G. et al. Novel mutations target distinct subgroups of medulloblastoma.
Nature 488, 43–48 (2012).
13. Downing, J.R. et al. The Pediatric Cancer Genome Project. Nat. Genet. 44, 619–622
(2012).
14. Stephens, P.J. et al. The landscape of cancer genes and mutational processes in
breast cancer. Nature 486, 400–404 (2012).
15. Welch, J.S. et al. The origin and evolution of mutations in acute myeloid leukemia.
Cell 150, 264–278 (2012).
16. Cin, H. et al. Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion
comprises an alternative mechanism of MAPK pathway activation in pilocytic
astrocytoma. Acta Neuropathol. 121, 763–774 (2011).
17. Wan, P.T. et al. Mechanism of activation of the RAF-ERK signaling pathway by
oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).
18. Rushworth, L.K., Hindley, A.D., O’Neill, E. & Kolch, W. Regulation and role of
Raf-1/B-Raf heterodimerization. Mol. Cell Biol. 26, 2262–2272 (2006).
19. Terai, K. & Matsuda, M. The amino-terminal B-Raf–specific region mediates
calcium-dependent homo- and hetero-dimerization of Raf. EMBO J. 25,
3556–3564 (2006).
20. Macedo, M.P. et al. Multiple mutations in the Kras gene in colorectal cancer: review
of the literature with two case reports. Int. J. Colorectal Dis. 26, 1241–1248
(2011).
21. Naguib, A., Wilson, C.H., Adams, D.J. & Arends, M.J. Activation of K-RAS by co-
mutation of codons 19 and 20 is transforming. J. Mol. Signal. 6, 2 (2011).
22. Schramm, A. et al. Biological effects of TrkA and TrkB receptor signaling in
neuroblastoma. Cancer Lett. 228, 143–153 (2005).
23. Thiele, C.J., Li, Z. & McKee, A.E. On Trk—the TrkB signal transduction pathway
is an increasingly important target in cancer biology. Clin. Cancer Res. 15,
5962–5967 (2009).
24. Greco, A., Miranda, C. & Pierotti, M.A. Rearrangements of NTRK1 gene in papillary
thyroid carcinoma. Mol. Cell Endocrinol. 321, 44–49 (2010).
25. Lannon, C.L. & Sorensen, P.H. ETV6-NTRK3: a chimeric protein tyrosine kinase
with transformation activity in multiple cell lineages. Semin. Cancer Biol. 15,
215–223 (2005).
26. Dewitt, J. et al. Constitutively active TrkB confers an aggressive transformed
phenotype to a neural crest–derived cell line. Oncogene published online;
doi:10.1038/onc.2013.39 (4 March 2013).
27. Kaplan, D.R. & Miller, F.D. Neurotrophin signal transduction in the nervous system.
Curr. Opin. Neurobiol. 10, 381–391 (2000).
28. Theillet, C. et al. FGFRI and PLAT genes and DNA amplification at 8p12 in breast
and ovarian cancers. Genes Chromosom. Cancer 7, 219–226 (1993).
29. Dutt, A. et al. Inhibitor-sensitive FGFR1 amplification in human non–small cell
lung cancer. PLoS ONE 6, e20351 (2011).
npg
© 2013 Nature America, Inc. All rights reserved.