2. 854 Acta Neuropathol (2014) 128:853–862
1 3
attenuated the poor outcome conferred by TP53 mutations
in specific pediatric brain tumors. Due to very poor prog-
nosis, one year overall survival was quantified in malig-
nant gliomas, while in children with choroid plexus car-
cinoma, five year overall survival was investigated. For
children with TP53 mutant malignant gliomas, one year
overall survival was 63 ± 12 and 23 ± 10 % for ALT pos-
itive and negative tumors, respectively (p = 0.03), while
for children with TP53 mutant choroid plexus carcino-
mas, 5 years overall survival was 67 ± 19 and 27 ± 13 %
for ALT positive and negative tumors, respectively
(p = 0.07). These observations suggest that the presence
of ALT is limited to a specific group of childhood brain
cancers which harbor somatic TP53 mutations and may
influence the outcome of these patients. Analysis of ALT
may contribute to risk stratification and targeted therapies
to improve outcome for these children.
Abbreviations
ALT Alternative lengthening of telomeres
qPCR Real-time polymerase chain reaction
CCA C-circle assay
TP53 Tumor protein 53
ATRX Alpha thalassemia/mental retardation syndrome
X-linked
PBT Pediatric brain tumor
BTRC Brain tumor research center
TRF Terminal restriction fragment
FISH Fluorescence in situ hybridization
FFPE Formalin-fixed paraffin extracted
CPC Choroid plexus carcinoma
SHGG Supratentorial high grade glioma
DIPG Diffuse intrinsic pontine glioma
PNET Primitive neuroectodermal tumors
LGG Low grade glioma
LFS Li-Fraumeni syndrome
IHC Immunohistochemistry
GBM Glioblastoma
Introduction
Pediatric brain tumors (PBTs), comprising multiple separate
pathological entities, are the most common group of solid
cancers in children. Recent discoveries using next-genera-
tion genomic platforms have uncovered substantial molecu-
lar heterogeneity even amongst PBTs with the same histo-
logical classification [26]. Examples include subgroups of
medulloblastoma [12, 28], primitive neuroectodermal tumor
[12], glioma [26], atypical teratoid rhabdoid tumor [7] and
ependymoma [22, 31, 37]. Although the identification of
subtype-specific genetic alterations may lead to the devel-
opment of patient specific targeted therapies, these may be
effective in a minority of children, even within so-called
histologically equivalent tumors. Identification of common
molecular features which are shared by different types of
PBTs will add a new dimension to PBT stratification and
will enable us to refine prognosis and develop therapies
applicable to a larger group of these children.
Telomere maintenance is required for tumor self-renewal
and is activated ubiquitously in most malignant cancers
[16]. Telomeres are structural elements at the ends of chro-
mosomes that consist of hexameric 5′-TTAGGG-3′ repeats,
and contain no gene-coding information [36]. They play a
critical role in preventing loss of genetic information which
occurs due to lagging-strand shortening during DNA repli-
cation [42]. Cells which are unable to maintain their telom-
eres undergo senescence and apoptosis. Therefore, in order
for cancer cells to maintain replicative ability, a telomere
maintenance pathway must be activated [16].
Telomeres are maintained in normal cells by the addition
of telomere hexameric repeats by the ribonucleoprotein
enzyme telomerase. Indeed, telomerase is activated in the
majority of tumors (85–90 %) [10]. Recently, activating
mutations in the TERT promoter were uncovered in mela-
noma [20] and other tumors, including several brain tumors
[27]. Although generally rare, the role and distribution of
TERT mutations in pediatric brain tumor subtypes is being
explored [27, 40].
Tumors which do not activate telomerase utilize the less
well-defined alternative lengthening of telomeres (ALT)
phenotype which maintains telomeres in a telomerase-inde-
pendent manner, presumably by telomere-specific homolo-
gous recombination [6, 10, 35]. Although ALT occurs in a
minority of tumors (4 %) [18], its incidence is enriched in
adult tumors of neuroectodermal and mesenchymal origin,
including brain tumors and soft-tissue sarcoma [10]. While
telomerase activity and its clinical utility has been explored
in several types of PBTs [13, 48, 50], the prevalence and
clinical impact of ALT in PBTs is still unknown.
The association between ALT and TP53 mutations
has previously been reported in adult gliomas [21, 29].
Next generation sequencing efforts have revealed a strong
J. S. Wunder
Department of Surgery, Mount Sinai Hospital, Toronto,
ON, Canada
N. Jabado
Department of Oncology, Montreal Children’s Hospital Research
Institute, Montreal, QC, Canada
N. Jabado
Department of Pediatrics, Montreal Children’s Hospital Research
Institute, Montreal, QC, Canada
D. L. Johnston
Division of Hematology/Oncology, Department of Pediatrics,
The Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada
3. 855Acta Neuropathol (2014) 128:853–862
1 3
association of ATRX mutations in several adult tumors
which exhibit ALT. ATRX is a protein involved in loading
the histone 3 variant H3.3 (the product of the H3f3a gene)
at the telomeres, favoring formation of heterochromatin
[30, 44]. As ALT occurs by homologous recombination,
loss of a heterochromatic state at the telomeres is permis-
sive for this recombination to occur [15]. Recently, muta-
tions in ATRX and H3f3a have been reported in pediatric
glioma [17, 44]; however, their association and possible
implication on the ALT phenotype remain controversial.
In order to shed light on some of these issues, we
screened a large cohort of various pediatric brain tumors
for evidence of ALT, using the C-circle assay (CCA), a
high throughput and novel tool for ALT detection. C-circles
are extrachromosomal circular telomeric repeats which
have been tightly associated with ALT, while being virtu-
ally absent in cells which do not utilize ALT [19].
We observe that ALT is found only in a subset of malig-
nant PBTs, is strongly associated with TP53 mutations, and
may impact survival of patients with TP53 mutant tumors.
Materials and methods
Patients and tumor samples
We collected tumor samples representing the major
pediatric brain cancers from the Brain Tumor Research
Center (BTRC) tissue bank at the Hospital for Sick Chil-
dren in Toronto. Childhood brain tumors were defined as
all cancers diagnosed before age 18 years. The study was
approved by the hospital’s Research Ethics Board, and
written consent was obtained for the collection and analy-
sis of each sample. For tumor types where ALT was sig-
nificantly enriched, additional demographic, treatment and
outcome data was collected.
Determination of ALT
We used three methods to determine ALT status: terminal
restriction fragment (TRF) [2], fluorescence in situ hybridi-
zation (FISH) [18] and C-circle assay (CCA) [19]. CCA
was performed using a dot blot assay and qPCR. Results
of ALT detection by CCA were validated using TRF and
FISH. Further details on each assay are available in the
Supplementary methods. After validation, all samples
(n = 517) were screened for ALT using the C-circle assay.
Detection of molecular alterations in TP53, H3f3a
and ATRX
Mutations in TP53, H3f3a and ATRX were available from
our previously reported diffuse intrinsic pontine glioma
(DIPG) genome sequencing studies [8]. For the remain-
ing tumor types, TP53 mutations were detected by Sanger
sequencing of exons 2 through 11, and up to 50 bases into
the intronic regions, as previously described [45]. In a sub-
set of tumors, where DNA was not available, the presence
of ATRX and p53 alterations were assayed by immunohis-
tochemistry (see Supplementary methods).
Statistical considerations
We compared CCA to FISH and TRF as well as the fre-
quency of genetic alterations in TP53, ATRX, and H3f3a
using the Fisher’s exact and two-tailed T test. Survival anal-
ysis was done using Kaplan–Meier estimates and log-rank
test by SPSS v20. p values 0.05 were considered statisti-
cally significant.
Results
C-circles are a robust marker for ALT detection in pediatric
brain tumors
In order to establish the accuracy of the CCA to detect ALT
in pediatric brain tumors, we compared it with two other
established ALT detection methods: FISH [18] (Fig. 1a),
and TRF [4] (Fig. 1b). C-circle detection was performed by
dot blot assay (Fig. 1c) and qPCR.
There was a high agreement in the detection of ALT in
both TRF assay and the CCA (n = 67; p = 8.62 × 10−13
).
All 16 CCA positive samples were TRF positive, and 48 of
51 CCA negative samples (94.1 %) were also TRF negative
(Fig. 1d). We also found high agreement between CCA and
FISH analysis (n = 19, p = 1.98 × 10−5
, Fig. 1d).
These results indicate that the CCA is a robust tool for
ALT detection in PBTs. Since the CCA can be performed
using very low concentrations of DNA (~32 ng per sample)
and can provide accurate results even in samples whose
DNA is extracted from formalin-fixed paraffin-embedded
(FFPE) tissues, we analyzed all of our PBT cohort using
this assay.
ALT is detected in a subset of malignant pediatric brain
tumors
In order to interrogate the prevalence of ALT in PBTs,
we performed the CCA on a cohort of 517 childhood
brain tumor samples (Table 1). ALT was prevalent in four
malignant tumor types of different histological classifica-
tion: choroid plexus carcinoma (CPC) (22.6 %), supraten-
torial high grade glioma (SHGG) (22 %), diffuse intrinsic
pontine glioma (DIPG) (18.8 %) and primitive neuroec-
todermal tumors (PNET) (11.6 %). In contrast, ALT was
4. 856 Acta Neuropathol (2014) 128:853–862
1 3
rarely observed in medulloblastoma (3/137—2.19 %), all
of which were of the sonic hedgehog subgroup, and was
absent in other malignant PBTs such as atypical teratoid
rhabdoid tumors and ependymoma.
Moreover, ALT was virtually absent in low grade
tumors. Of 60 pediatric low grade gliomas (LGG) only
one tumor exhibited ALT. This tumor was an atypical
LGG from an older teenager (age 17 years), exhibiting an
IDH1 mutation and a phenotype which may more closely
resemble that of an adult LGG. In our cohort, we found that
ALT was strongly associated with high grade glial histol-
ogy (n = 158; p = 1.124 × 10−3
, Supplementary Table 2).
Similarly, in contrast to CPC, none of the 24 choroid plexus
papillomas exhibited the ALT phenotype.
Since adult LGG frequently transform to high grade gli-
oma [23] and exhibit a high incidence of ALT [18], we per-
formed ALT analysis on a cohort of pediatric LGG which
had transformed to high grade histology (n = 12). None of
the LGGs from the transformation cohort had ALT.
We were able to perform TERT promoter analysis on
151 tumors. As expected, TERT promoter mutations and
ALT were mutually exclusive except for one medulloblas-
toma sample (Supplementary Table 1). This tumor had a
generally bland genome and lacked TP53 mutation.
ALT is associated with TP53 mutations in pediatric brain
tumors
The PBT types which exhibited a high frequency of ALT
were also previously reported to harbor somatic TP53
mutations [41, 43, 47]. These brain tumors are a part
of a group of brain tumors commonly observed in chil-
dren with Li-Fraumeni syndrome (LFS) [46], a cancer
Fig. 1 Comparison of differ-
ent methods to detect ALT in
cancer. (a) ALT negativity (left
panel), as detected by FISH,
is characterized by small and
homogeneous telomere foci
while ALT positivity (right
panel) is characterized by the
presence of heterogeneous
fluorescence foci (displayed
by arrows) with larger foci.
(b) A telomere restriction frag-
ments (TRF) blot. Red-boxed
samples are ALT positive. (c)
C-circle dot blot. Rows marked
“ϕ29” were treated with ϕ29
polymerase to amplify c-circles.
Prominent dots in these rows
represent the presence of
c-circles. (d) Summary of the
agreement between assays
Table 1 Incidence of ALT in pediatric brain tumors
Sample type Incidence of ALT
Choroid plexus carcinoma 7/31 (22.6 %)
Supratentorial high grade glioma 11/50 (22 %)
Grade IV glioblastoma multiforme 3/25 (12 %)
Grade III anaplastic glioma 8/25 (32.0 %)
Diffuse intrinsic pontine glioma 9/48 (18.8 %)
Primitive neuroectodermal tumor 5/43 (11.6 %)
Medulloblastoma 3/137 (2.19 %)
Low grade glioma 1/60 (1.67 %)
Pilocytic astrocytoma 1/35 (2.86 %)
Ganglioglioma 0/8 (0 %)
Pleomorphic xanthoastrocytoma 0/8 (0 %)
Other 0/9 (0 %)
Ependymomas 0/95 (0 %)
Atypical teratoid rhabdoid tumor 0/29 (0 %)
Choroid plexus papilloma 0/24 (0 %)
Total 36/517 (6.96 %)
5. 857Acta Neuropathol (2014) 128:853–862
1 3
predisposition syndrome characterized by very high inci-
dence of sarcomas, tumors of the adrenal cortex and cer-
tain types brain tumors diagnosed at a young age [33,
46]. We therefore performed the CCA on a cohort of two
non-brain cancers commonly observed in children with
LFS—osteosarcoma and adrenocortical carcinoma [33,
46]. Interestingly, both osteosarcoma and adrenocortical
tumors harbored a very high incidence of ALT (53 and
36.4 %, respectively, Fig. 2).
One of the criteria for diagnosis of LFS is the pres-
ence of germline TP53 mutations. We found no associa-
tion between ALT and germline TP53 mutation (n = 14;
p = 1.000) (Fig. 3a).
To clarify the association of TP53 mutations and ALT
in PBTs we analyzed 245 samples where data for both
somatic TP53 mutations and ALT was available. ALT was
highly associated with somatic TP53 mutations across the
entire spectrum of PBTs in our study—77 % of ALT tumors
also had TP53 mutations (p = 7.32 × 10−8
) (Fig. 3b).
A detailed examination of TP53 alterations in the cohort
of malignant PBTs enriched for ALT revealed that 100 %
of ALT CPCs harboured a functionally deleterious TP53
mutation, while in malignant gliomas 14/20 ALT tumors
(70 %) had a functional deleterious TP53 mutation. Of the
remaining six TP53 WT gliomas, three stained positive for
p53 by immunohistochemistry (IHC) and a fourth tumor
had a heterozygous TP53 deletion. Thus, 26 of 28 (92.9 %)
ALT malignant PBTs had a compromised p53 pathway
(Supplementary Table 3).
Association of ALT with other mutations in PBTs
Since H3f3a and ATRX mutations were previously reported
to segregate together with TP53 in pediatric glioblastoma
(GBM) [44] we tested their association with ALT. H3f3a
K27M mutations are common (64 %) in DIPG (n = 48)
but no statistical association between these mutations and
ALT was observed (p = 0.1310) (Fig. 3c) even when TP53
mutations were considered (p = 0.4325; n = 31) (Supple-
mentary Table 4).
In contrast, a significant association between ATRX
alterations and ALT was observed in a cohort of 40
pediatric malignant gliomas, where data was available
(p = 5.23 × 10−4
, Fig. 3d).
ALT is associated with improved survival in children
with TP53 mutant brain tumors
TP53 alterations have been previously shown to con-
fer worse outcome in pediatric high grade gliomas [11]
and CPCs [47]. Indeed, in our cohort of DIPG (n = 40),
Fig. 2 Prevalence of ALT in different pediatric brain tumors and Li-
Fraumeni associated cancers. ALT is prevalent in brain and non-brain
cancers common in patients with Li-Fraumeni syndrome
Fig. 3 Association of ALT with mutations in putative ALT-associated
genes—incidence of ALT was stratified by mutation/expression status
for a germline TP53, b somatic TP53, c H3f3a, d ATRX. P-values are
determined by Fisher’s exact test
6. 858 Acta Neuropathol (2014) 128:853–862
1 3
1-year overall survival was 66 ± 12 and 36 ± 9 % for
TP53 wildtype and mutant tumors, respectively (p = 0.06,
Fig. 4a).
Strikingly, the presence of ALT attenuated the delete-
rious effect of TP53 mutations. Analysis of the 24 TP53
mutant DIPGs revealed that one year overall survival was
63 ± 12 % for ALT positive tumors and 23 ± 10 % for
ALT negative tumors (p = 0.03, Fig. 4b). Survival analy-
sis of all children with high grade glial tumors harboring
TP53 mutations (n = 36) revealed one-year overall survival
of 80 ± 10 % for ALT positive tumors and 36 ± 10 % for
ALT negative tumors (p = 0.03, Fig. 4c). Similarly, in a
small cohort of TP53 mutant CPC where data was avail-
able, (n = 16), five year overall survival was 67 ± 19 %
for ALT positive tumors and 27 ± 13 % for ALT negative
tumors (p = 0.07, Fig. 4d).
Discussion
To our knowledge, this is the first study to screen for ALT
in a large cohort of all major histological types of pediat-
ric brain tumors. We report a strong association of the ALT
phenotype with TP53 mutations, and provide additional
insight into the pathogenesis and clinical implications of
telomere maintenance in childhood cancers.
The high agreement between the CCA and two other
well established methods (FISH and TRF) used to detect
ALT is of great technical importance, since TRF requires
2 µg of high quality DNA, generally unavailable in the rou-
tinely obtained diagnostic biopsies of these cancers. More-
over, FISH is a highly skill-dependent assay whose results
are difficult to interpret in a large number of tissues. On
the other hand, CCA requires only 32 ng of DNA and is
Fig. 4 Kaplan–Meier estimates of overall survival for study patients
by TP53 and ALT status. a DIPG patients (n = 40) stratified by TP53
mutation status. b TP53 mutant DIPG (n = 24) stratified by ALT sta-
tus. c All TP53 mutant malignant glioma (DIPG + HGG, n = 36)
stratified by ALT status. d TP53 mutant CPCs (n = 16) stratified by
ALT status
7. 859Acta Neuropathol (2014) 128:853–862
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reliable even with poor quality DNA extracted from paraf-
fin embedded tissues. Therefore, the CCA is an easy and
reliable clinical tool that could be readily integrated into
clinical diagnostic laboratories in the near future.
It is important to note that most cancers maintain their
telomeres through activation of telomerase and not ALT.
This hallmark of cancer [16] can be activated by specific
mutations in the TERT promoter, however this is uncom-
mon in childhood cancer [27, 49]. In our cohort of tumors,
TERT promoter mutations were rare and there was almost
mutual exclusivity between ALT and this alteration. These
findings are consistent with those in adult gliomas [25], but
will require a larger cohort for further validation.
We observed ALT in only 1 of 84 (1.19 %) low grade
pediatric tumors. This is in agreement with the fact that
most pediatric LGG lack any form of telomere mainte-
nance and are characterized by spontaneous growth arrest
[48]. In contrast, ALT is commonly observed in adult low
grade gliomas [18, 21] which tend to progress to high grade
tumors. The observation that ALT is absent in childhood
LGGs which transform further highlights the biological dif-
ferences between adult and pediatric gliomas.
Our observations expand current knowledge of the asso-
ciation between ALT and p53 in three important ways.
First, almost all ALT tumors had some form of p53 dys-
function which included, but was not limited to, TP53
mutations. Dysfunction in p53 permits cells to evade senes-
cence and apoptosis, which, under normal circumstances,
are the consequences of critical loss of telomere length
[10]. It is therefore reasonable to propose that only cells
which have dysfunctional p53 will be able to survive crisis
and that these dysfunctional telomeres are the basis of the
ALT phenotype.
Second, activation of ALT is thought to result from the
inability of some clones to activate telomerase, defining it as
a late event in carcinogenesis [10]. In gliomas and CPC, the
lack of association between ALT and germline TP53 muta-
tions, an early event in carcinogenesis, further support this
concept. Importantly, the late onset of ALT appears to con-
trast with other p53 dysfunction-linked tumor alterations,
such as chromothripsis. Chromothripsis has been reported
in the context of germline TP53 mutations and is thought to
represent an early event in tumorigenesis [32, 39].
Third, TP53 mutations are associated with a more
aggressive cancer phenotype and worse clinical outcome in
several PBT subtypes [38, 40, 47]. In agreement with the
two concepts presented above, we propose that ALT is a
late event by which a clone that is unable to activate tel-
omerase utilizes ALT as a “last resort”. This may lead to
a cancer which is always on the verge of crisis and may
be less efficient at self-renewal. Indeed, 71 % of TP53
mutant PBTs did not exhibit ALT, probably using the more
efficient telomerase for their telomere maintenance and
self-renewal. Nevertheless, due to the permissive nature of
TP53 mutations, a subset of TP53 mutant tumors will acti-
vate ALT leading to decreased proliferation efficiency [35]
and a less aggressive cancer phenotype.
ATRX mutation and lack of expression are heavily asso-
ciated with ALT in adult gliomas, especially in low grade
[24] and secondary high grade gliomas [21]. TP53 muta-
tions are almost always present suggesting requirement of
a permissive background as mentioned above. It is clear
from our study and others [1], that in childhood gliomas,
ATRX mutations are neither necessary, nor sufficient to
cause ALT. Our findings suggest that tumors with TP53
dysfunction which gain ATRX mutations will enable ALT
but tumors with ATRX mutations may already have telom-
erase based telomere maintenance and need not develop
ALT. Furthermore, several other genetic alterations enable
ALT without ATRX mutations. Importantly, most of these
still require TP53 pathway alterations to enable this dys-
functional telomere maintenance state.
We observed ALT only in SHH medulloblastoma. Inter-
estingly, this subtype is also enriched for TERT promoter
mutations [40]. SHH pathway genes activate specific tran-
scription factors which bind to the TERT promoter [34]
and potentially enhance TERT expression when mutations
occur. In parallel, SHH mutations are enriched in SHH
medulloblastoma [51] potentially facilitating ALT and
other chromosomal alterations such as chromothripsis [39].
It is important to note that most medulloblastomas do not
harbor either of these two alterations suggesting a different
mechanism of telomerase activation [9].
Although the data reported herein should be interpreted
with caution due to the retrospective nature of the study,
stratification of TP53 mutant gliomas and CPC into two
separate groups by ALT status revealed longer survival for
patients with TP53 mutant tumors exhibiting ALT. These
observations may explain some of the discrepancies in
reports of the role of TP53 mutations in these cancers [3,
38]. Similarly, ALT itself occurring in the context of wild-
type TP53 may not affect outcome of children with brain
tumors [14]. Importantly, similar results were found in
a cohort of adult gliomas [11], which suggests a general
association between ALT and improved prognosis in TP53
mutant tumors, at least in a context of brain tumors.
In summary, our study catalogues the relative incidence
of ALT in various different types of pediatric brain tumors.
These results would explain why some types of PBTs such
as atypical teratoid rhabdoid tumors, medulloblastomas
[13] and ependymomas [5] exhibit high levels of telomer-
ase activation while others such as gliomas [14] and cho-
roid plexus tumors reveal a more heterogeneous pattern.
The addition of ALT as a component of telomere mainte-
nance can also inform the potential use of telomerase as a
prognostic marker and therapeutic target in specific types
8. 860 Acta Neuropathol (2014) 128:853–862
1 3
of PBTs. Further validation on a larger prospective cohort
will determine the effect of ALT on the outcome of TP53
mutant PBTs and would allow for stratification and man-
agement decisions for these children.
Acknowledgments We thank Jeremy D. Henson for his continued
support and technical assistance during the early stages in develop-
ment of the CCA and FISH assays in our laboratory. JM received
an Ontario Graduate Scholarship for 2012–2013. This work was
supported by the Canadian Institute of Health Research Grant
MOP#86558.
Conflict of interest The authors declare no conflict of interest.
References
1. Abedalthagafi M, Phillips JJ, Kim GE, Mueller S, Haas-Kogen
DA, Marshall R, Croul SE, Santi MR, Cheng J, Zhou S, Sullivan
LM, Martinez-Lage M, Judkins AR, Perry A (2013) The alterna-
tive lengthening of telomere phenotype is significantly associated
with loss of ATRX expression in high-grade pediatric and adult
astrocytomas: a multi-institutional study of 214 astrocytomas.
Mod Pathol 26:1425–1432
2. Allshire RC, Dempster M, Hastie ND (1989) Human telomeres
contain at least three types of G-rich repeat distributed non-ran-
domly. Nucleic Acids Res 17:4611–4627
3. Antonelli M, Buttarelli FR, Arcella A, Nobusawa S, Donofrio V,
Oghaki H, Giangaspero F (2010) Prognostic significance of histo-
logical grading, p53 status, YKL-40 expression, and IDH1 muta-
tions in pediatric high-grade gliomas. J Neurooncol 99:209–215
4. Aubert G, Hills M, Lansdorp PM (2012) Telomere length meas-
urement—caveats and a critical assessment of the available
technologies and tools. Mutat Res Fundam Mol Mech Mutagen
730:59–67
5. Barszscyk, MS. (2013) Telomerase as a prognostic marker and
therapeutic target in paediatric ependymoma. Laboratory Medi-
cine and Pathobiology. University of Toronto, City, p 53
6. Bechter OE, Zou Y, Shay JW, Wright WE (2003) Homologous
recombination in human telomerase-positive and ALT cells
occurs with the same frequency. EMBO Rep 4:1138–1143
7. Birks DK, Donson AM, Patel PR, Dunham C, Muscat A, Algar
EM, Ashley DM, Kleinschmidt-DeMasters BK, Vibhakar R, Han-
dler MH, Foreman NK (2011) High expression of BMP pathway
genes distinguishes a subset of atypical teratoid/rhabdoid tumors
associated with shorter survival. Neuro Oncol 13:1296–1307
8. Buczkowicz P, Hoeman C, Rakopoulos P, Pajovic S, Letourneau
L, Dzamba M, Morrison A, Lewis P, Bouffet E, Bartels U, Zuc-
caro J, Agnihotri S, Ryall S, Barszczyk, Chornenkyy Y, Bourgey
M, Bourque G, Monpetit A, Cordero F, Castelo-Branco P, Man-
gerel J, Tabori U, Ho PKC, Huang A, Taylor KR, Mackay A,
Bendel AE, Nazarian J, Fangusaro JR, Karajannis, et al. (2014)
Comprehensive genomic analysis of diffuse intrinsic pontine
gliomas unravels three molecular subgroups and a novel cancer
driver ACVR1. Nat Genet
9. Castelo-Branco P, Choufani S, Mack S, Gallagher D, Zhang C,
Lipman T, Zhukova N, Walker EJ, Martin D, Merino D, Wasser-
man JD, Elizabeth C, Alon N, Zhang L, Hovestadt V, Kool M,
Jones DTW, Zadeh G, Croul S, Hawkins C, Hitzler J, Wang JCY,
Baruchel S, Pfister S, Taylor MD, Weksberg R, Tabori U (2013)
Methylation of the TERT promoter and risk stratifi cation of
childhood brain tumours: an integrative genomic and molecular
study. Lancet Oncol 14:534–542
10. Cesare AJ, Reddel RR (2010) Alternative lengthening of tel-
omeres: models, mechanisms and implications. Nat Rev Genet
11:319–330
11. Chen Y-J, Hakin-Smith V, Teo M, Xinarionas GE, Jellinek DA,
Carroll T, McDowell D, MacFarlane MR, Boet R, Baguley
BC, Braithwaite AW, Reddel RR, Royds JA (2006) Association
of mutant TP53 with alternative lengthening of telomeres and
favorable prognosis in glioma. Cancer Res 66:6473–6476
12. De Braganca KC, Packer RJ (2013) Treatment options for medul-
loblastoma and CNS primitive neuroectodermal tumor (PNET).
Curr Treat Options Neurol 15:593–606
13. Didiano D, Shalaby T, Lang D, Grotzer MA (2004) Telomere
maintenance in childhood primitive neuroectodermal brain
tumors. Neuro Oncol 6:1–8
14. Dorris K, Sobo M, Onar-Thomas A, Panditharatna E, Stevenson
CB, Gardner SL, DeWire MD, Pierson CR, Olshefski R, Rempel
SA, Goldman S, Miles L, Fouladi M, Drissi R (2014) Prognos-
tic significance of telomere maintenance mechanisms in pediatric
high-grade gliomas. J Neurooncol 117:67–76
15. Episkopou H, Draskovic I, Van Beneden A, Tilman G, Mattiussi
M, Gobin M, Arnoult N, Londono-Vallejo A, Decottignies A
(2014) Alternative lengthening of telomeres is characterized by
reduced compaction of telomeric chromatin. Nucleic Acids Res
42:4391–4405
16. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next
generation. Cell 144:646–674
17. Heaphy CM, de Wilde RF, Jiao Y, Klein AP, Edil BH, Shi C,
Bettegowda C, Rodriguez FJ, Eberhart CG, Hebbar S, Offerhaus
GJ, McLendon R, Rasheed BA, He Y, Yan H, Bigner DD, Oba-
Shinjo SM, Marie SKN, Riggins GJ, Kinzler KW, Vogelstein
B, Hruban RH, Maitra A, Papadopoulos N, Meeker AK (2011)
Altered telomeres in tumors with ATRX and DAXX mutations.
Science 333:425–428
18. Heaphy CM, Subhawong AP, Hong S-M, Goggins MG, Mont-
gomery EA, Grabielson E, Netto GJ, Epstein JI, Lotan TL,
Westra WH, Shih I-M, Iacobuzio-Donahue CA, Maltra A, Li
QK, Eberhart CG, Taube JM, Rakheja D, Kurman RJ, Wu TC,
Roden RB, Argani P, De Marzo AM, Terracciano L, Torbenson
M, Meeker AK (2011) Prevalence of alternative lengthening of
telomeres telomere maintenance mechanism in human cancer
subtypes. Am J Pathol 179:1608–1615
19. Henson JD, Cao Y, Huschtscha LI, Chang AC, Au AYM, Pickett
HA, Reddel RR (2009) DNA C-circles are specific and quantifi-
able markers of alternative-lengthening-of-telomeres activity. Nat
Biotechnol 27:1181–1186
20. Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A,
Kadel S, Moll I, Nagore E, Hemminki K, Schadendorf D, Kumar
R (2013) TERT promoter mutations in familial and sporadic mel-
anoma. Science 339:959–961
21. Jiao Y, Killela PJ, Reitman ZJ, Rasheed BA, Heaphy CM, de
Wilde RF, Rodriguez FJ, Rosemberg S, Oba-Shinjo SM, Marie
SZM, Bettegowda C, Agrawal N, Lipp E, Pirozzi CJ, Lopez GY,
He Y, Friedman HS, Friedman AH, Riggins GJ, Holdhoff M,
Burger P, McLendon RE, Bigner DD, Vogelstein B, Meeker AK,
Kinzler KW, Papadopoulos N, Diaz LA Jr, Yan H (2012) Fre-
quent ATRX, CIC, FUBP1 and IDH1 mutations refine the clas-
sification of malignant gliomas. Oncotarget 3:710–722
22. Johnson RA, Wright KD, Poppleton H, Mohankumar KM, Fin-
kelstein D, Pounds SB, Rand V, Leary SES, White E, Eden C,
Hogg T, Northcott P, Mack S, Neale G, Wang Y-D, Coyle B,
Atkinson J, DeWire M, Kranenburg TA, Gillespie Y, Allen JC,
Merchant T, Boop FA, Sanford RA, Gajjar A, Ellison DW, Taylor
MD, Grundy RG, Gilberston RJ (2010) Cross-species genomics
matches driver mutations and cell compartments to model epend-
ymoma. Nature 466:632–636
9. 861Acta Neuropathol (2014) 128:853–862
1 3
23. Jones C, Perryman L, Hargrave D (2012) Paediatric and adult
malignant glioma: close relatives or distant cousins? Nat Rev
Clin Oncol 9:400–413
24. Kannan K, Inagaki A, Silber J, Gorovets D, Zhang J, Kastenhu-
ber ER, Heguy A, Petrini JH, Chan TA, Huse JT (2012) Whole
exome sequencing identifies ATRX mutation as a key molecular
determinant in lower-grade glioma. Oncotarget 3:1194–1203
25. Killela PJ, Reitman ZJ, Jiao Y, Bettegowda C, Agrawal N, Diaz
Jr. LA, Friedman AH, Friedman H, Gallia GL, Giovanella B,
Grollman AP, He T-C, He Y, Hruban RH, Jallo GI, Mandahl N,
Meeker AK, Mertens F, Netto GJ, Rasheed BA, Riggins GJ,
Rosenquist TA, Schiffman M, Shih Ie-Ming, Theodorescu D,
Torbenson MS, Velculescub VE, Wang T-L, Wentzensen N, Wood
LD, et al. (2013) TERT promoter mutations occur frequently in
gliomas and a subset of tumors derived from cells with low rates
of self-renewal. PNAS 110:6021–6026
26. Kim J-H, Huse JT, Huang Y, Lyden D, Greenfield JP (2013)
Molecular diagnostics in paediatric glial tumours. Lancet Oncol
14:19
27. Koelsche C, Sahm F, Capper D, Reuss D, Sturm D, Jones DTW,
Kool M, Northcott PA, Wiestler B, Bohmer K, Meyer J, Marwin
C, Hartmann C, Mittelbronn M, Platten M, Brokinkel B, Seiz M,
Herold-Mende C, Unterberg A, Schittenhelm J, Weller M, Pfis-
ter S, Wick W, Korshunov A, von Deimling A (2013) Distribution
of TERT promoter mutations in pediatric and adult tumors of the
nervous system. Acta Neuropathol 126:907–914
28. Kool M, Koster J, Bunt J, Hasselt NE, Lakeman A, van Sluis
P, Troost D, Schouten-van Meeteren N, Caron HB, Cloos J,
Mrsic A, Ylstra B, Grajkowska W, Hartmann W, Pietsch T,
Ellison D, Clifford SC, Versteeg R (2008) Integrated genomics
identifies five medullo-blastoma subtypes with distinct genetic
profiles, pathway signatures and clinicopathological features.
PLoS one. 3
29. Liu X-Y, Gerges N, Korhunov A, Sabha N, Khuong-Quang D-A,
Fontebasso AM, Fleming A, Hadjadj D, Schwartzentruber J,
Majewski J, Dong Z, Siegel P, Albrecht S, Croul S, Jones DTW,
Kool M, Tonjes M, Reifenberger G, Faury D, Zadeh G, Pfister S,
Jabado N (2012) Frequent ATRX mutations and loss of expres-
sion in adult diffuse astrocytic tumors carrying IDH1/IDH2 and
TP53 mutations. Acta Neuropathol 124:615–625
30. Lovejoy CA, Li W, Reisenweber S, Thongthip S, Bruno J, de
Lange T, De S, Petrini JHJ, Sung PA, Jasin M, Rosenbluh J,
Zwang Y, Weir BA, Hatton C, Invanova E, Macconalli L, Hanna
M, Hahn WC, Lue NF, Reddel RR, Jiao Y, Kinzler K, Vogelstein
B, Papdopoulos N, Meeker A (2012) Loss of ATRX, genome
instability, and an altered DNA damage response are Hallmarks
of the alternative lengthening of telomeres pathway. PloS Genet.
8
31. Mack SC, Witt H, Piro RM, Gu L, Zuyderduyn S, Stutz AM,
Wang X, Gallo M, Garzia L, Zayne K, Zhang X, Ramaswamy
V, Jager N, Jones DTW, Sill M, Pugh TJ, Ryzhova M, Wani KM,
Shih DJH, Head R, Remke M, Bailey SD, Zichner T, Faria CC,
Barszczyk M, Stark S, Seker-Cin H, Hutter S, Johann P, Bender
S et al (2014) Epigenomic alterations define lethal CIMP-positive
ependymomas of infancy. Nature 506:445–450
32. Malhotra A, Lindber M, Faust GG, Leibowitz ML, Clark RA,
Layer RM, Quinlan AR, Hall IM (2013) Breakpoint profiling of
64 cancer genomes reveals numerous complex rearrangements
spawned by homology-independent mechanisms. Genome Res
23:762–776
33. Malkin D (2011) Li-Fraumeni Syndrome. Genes Cancer
2:475–484
34. Mazumdar T, Sandhu R, Qadan M, DeVecchio J, Magloire V,
Agyeman A, Li B, Houghton JA (2013) Hedgehog signaling
regulates telomerase reverse transcriptase in human cancer cells.
PLoS One. 8
35. Nabetani A, Ishikawa F (2011) Alternative lengthening of tel-
omeres pathway: recombination-mediated telomere maintenance
mechanism in human cells. J Biol Chem 149:5–14
36. Oganasian L, Karlseder J (2009) Telomeric armor: the layers of
end protection. J Cell Sci 122:4013–4025
37. Parker M, Mohankumar KM, Punchihewa C, Weinlich R, Dalton
JD, Li Y, Lee R, Tatevossian RG, Phoenix TM, Thiruvenkatam
R, White E, Tang B, Orisme W, Gupta K, Rusch M, Chen X, Li
Y, Nagahawhatte P, Hedlund E, Finkelstein D, Wu G, Shurtleff S,
Easton J, Boggs K, Yergeau D, Vadodaria B, Mulder HL, Becks-
fort J, Gupta P, Huether R et al (2014) C11orf95-RELA fusions
drive oncogenic NF-κB signalling in ependymoma. Nature
506:451–455
38. Pollack IF, Finkelstein SD, Woods J, Burnham J, Holmes EJ,
Hamilton RL, Yates AJ, Boyett JM, Finlay JL, Sposto R (2002)
Expression of p53 and prognosis in children with malignant glio-
mas. N Engl J Med 346:420–427
39. Rausch T, Jones DTW, Zapatka M, Stutz AM, Zichner T, Weis-
chenfeldt J, Jager N, Remke M, Shih D, Northcott PA, Plaff E,
Tica J, Wang Q, Massimi L, Witt H, Bender S, Pleier S, Cin H,
Hawkins C, Beck C, von Deimling A, Hans V, Brors B, Eils R,
Scheurlen W, Blake J, Benes V, Kulozik AE, Witt O, Martin D et
al (2012) Genome sequencing of pediatric medulloblastoma links
catastrophic DNA rearrangements with TP53 mutations. Cell
148:59–71
40. Remke M, Ramaswamy V, Peackock J, Shih DJH, Koelsche C,
Northcott PA, Hill N, Cavalli FMG, Kool M, Wang X, Mack
SC, Barszczyk M, Morissi AS, Wu X, Agnihotri S, Luu B, Jones
DTW, Garzia L, Dubuc AM, Zhukova N, Vanner R, Kros JM,
French PJ, Van Meir EG, Vibhakar R, Zitterbart K, Chan JA,
Bognar L, Kekner A, Lach B et al (2013) TERT promoter muta-
tions are highly recurrent in SHH subgroup medulloblastoma.
Acta Neuropathol 126:917–929
41. Rood BR, MacDonald TJ (2005) Pediatric high-grade glioma:
molecular genetic clues for innovative therapeutic approaches. J
Neurooncol 75:267–272
42. Rubtsova MP, Vasilkova DP, Malyavko AN, Naraikina YV,
Zvereva MI, Dontsova OA (2012) Telomere lengthening and
other functions of telomerase. Acta Naturae 2:44–61
43. Schroeder KM, Hoeman CM, Becher OJ (2014) Children are
not just little adults: recent advances in understanding of diffuse
intrinsic pontine glioma biology. Pediatr Res 75:205–209
44. Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff
E, Jacob K, Sturm D, Fontebasso AM, Quang DA, Tonjes M,
Hovestadt V, Albercht S, Kool M, Nantel A, Konermann C,
Lindroth A, Jager N, Rausch T, Ryzhova M, Korbel JO, Hiels-
cher T, Hauser P, Garami M, Klekner A, Bognar L, Ebinger M,
Schuhmann MU, Scheurlen W, Pekrun A, Fuhwald MC et al
(2012) Driver mutations in histone H3.3 and chromatin remod-
elling genes in paediatric glioblastoma. Nature 482:226–231.
doi:10.1038/nature10833
45. Shlien A, Tabori U, Marshall CR, Pienkowska M, Feuk L, Novo-
kmet A, Nanda S, Druker H, Scherer SW, Malkin D (2008)
Excessive genomic DNA copy number variation in the Li-
Fraumeni cancer predisposition syndrome. Proc Natl Acad Sci
105:11264–11269
46. Sorrell AD, Espenschied CR, Culver JO, Weitzel JN (2013) TP53
testing and Li-Fraumeni Syndrome: current status of clinical
applications and future directions. Mol Diag Ther 17:31–47
47. Tabori U, Shlien A, Baskin B, Levitt S, Ray P, Alon N, Hawkins
C, Bouffet E, Pienkowska M, Lafay-Cousin L, Gozali A, Zhu-
kova N, Shane L, Gonzalez I, Finlay J, Malkin D (2010) TP53
alterations determine clinical subgroups and survival of patients
with choroid plexus tumors. J Clin Oncol 28:1995–2001
48. Tabori U, Vukovic B, Zielenska M, Hawkins C, Braude I,
Rutka J, Bouffet E, Squire J, Malkin D (2006) The role of
10. 862 Acta Neuropathol (2014) 128:853–862
1 3
telomere-maintenance in the spontaneous growth arrest of paedi-
atric low-grade gliomas. Neoplasia 8:136–142
49. Vinagre J, Pinto V, Celestino R, Reis M, Populo H, Boaventura
P, Melo M, Catarino T, Lima J, Lopes JM, Maximo V, Sobrinho-
Simoes M, Soares P (2014) Telomerase promoter mutations
in cancer: an emerging molecular biomarker? Virchows Arch
465:119–133
50. Wong VCH, Ma J, Hawkins CE (2009) Telomerase inhibition
induces acute ATM-dependent growth arrest in human astrocyto-
mas. Cancer Lett 274:151–159
51. Zhukova N, Ramaswamy V, Remke M, Pfaff E, Shih DJH, Mar-
tin DC, Castelo-Branco P, Baskin B, Ray PN, Bouffet E, von
Bueren AO, Jones DTW, Northcott PA, Kool M, Sturm D, Pugh
TJ, Pomeroy SL, Cho Y-J, Pietsch T, Gessi M, Rutkowski S, Bog-
nar L, Klekner A, Cho B-K, Kim S-K, Wang K-C, Eberhart CG,
Fevre-Montange M, Fouladi M, French PJ et al (2013) Subgroup-
specific prognostic implications of TP53 mutation in medullo-
blastoma. J Clin Oncol 31:2927–2934
![854 Acta Neuropathol (2014) 128:853–862
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attenuated the poor outcome conferred by TP53 mutations
in specific pediatric brain tumors. Due to very poor prog-
nosis, one year overall survival was quantified in malig-
nant gliomas, while in children with choroid plexus car-
cinoma, five year overall survival was investigated. For
children with TP53 mutant malignant gliomas, one year
overall survival was 63 ± 12 and 23 ± 10 % for ALT pos-
itive and negative tumors, respectively (p = 0.03), while
for children with TP53 mutant choroid plexus carcino-
mas, 5 years overall survival was 67 ± 19 and 27 ± 13 %
for ALT positive and negative tumors, respectively
(p = 0.07). These observations suggest that the presence
of ALT is limited to a specific group of childhood brain
cancers which harbor somatic TP53 mutations and may
influence the outcome of these patients. Analysis of ALT
may contribute to risk stratification and targeted therapies
to improve outcome for these children.
Abbreviations
ALT Alternative lengthening of telomeres
qPCR Real-time polymerase chain reaction
CCA C-circle assay
TP53 Tumor protein 53
ATRX Alpha thalassemia/mental retardation syndrome
X-linked
PBT Pediatric brain tumor
BTRC Brain tumor research center
TRF Terminal restriction fragment
FISH Fluorescence in situ hybridization
FFPE Formalin-fixed paraffin extracted
CPC Choroid plexus carcinoma
SHGG Supratentorial high grade glioma
DIPG Diffuse intrinsic pontine glioma
PNET Primitive neuroectodermal tumors
LGG Low grade glioma
LFS Li-Fraumeni syndrome
IHC Immunohistochemistry
GBM Glioblastoma
Introduction
Pediatric brain tumors (PBTs), comprising multiple separate
pathological entities, are the most common group of solid
cancers in children. Recent discoveries using next-genera-
tion genomic platforms have uncovered substantial molecu-
lar heterogeneity even amongst PBTs with the same histo-
logical classification [26]. Examples include subgroups of
medulloblastoma [12, 28], primitive neuroectodermal tumor
[12], glioma [26], atypical teratoid rhabdoid tumor [7] and
ependymoma [22, 31, 37]. Although the identification of
subtype-specific genetic alterations may lead to the devel-
opment of patient specific targeted therapies, these may be
effective in a minority of children, even within so-called
histologically equivalent tumors. Identification of common
molecular features which are shared by different types of
PBTs will add a new dimension to PBT stratification and
will enable us to refine prognosis and develop therapies
applicable to a larger group of these children.
Telomere maintenance is required for tumor self-renewal
and is activated ubiquitously in most malignant cancers
[16]. Telomeres are structural elements at the ends of chro-
mosomes that consist of hexameric 5′-TTAGGG-3′ repeats,
and contain no gene-coding information [36]. They play a
critical role in preventing loss of genetic information which
occurs due to lagging-strand shortening during DNA repli-
cation [42]. Cells which are unable to maintain their telom-
eres undergo senescence and apoptosis. Therefore, in order
for cancer cells to maintain replicative ability, a telomere
maintenance pathway must be activated [16].
Telomeres are maintained in normal cells by the addition
of telomere hexameric repeats by the ribonucleoprotein
enzyme telomerase. Indeed, telomerase is activated in the
majority of tumors (85–90 %) [10]. Recently, activating
mutations in the TERT promoter were uncovered in mela-
noma [20] and other tumors, including several brain tumors
[27]. Although generally rare, the role and distribution of
TERT mutations in pediatric brain tumor subtypes is being
explored [27, 40].
Tumors which do not activate telomerase utilize the less
well-defined alternative lengthening of telomeres (ALT)
phenotype which maintains telomeres in a telomerase-inde-
pendent manner, presumably by telomere-specific homolo-
gous recombination [6, 10, 35]. Although ALT occurs in a
minority of tumors (4 %) [18], its incidence is enriched in
adult tumors of neuroectodermal and mesenchymal origin,
including brain tumors and soft-tissue sarcoma [10]. While
telomerase activity and its clinical utility has been explored
in several types of PBTs [13, 48, 50], the prevalence and
clinical impact of ALT in PBTs is still unknown.
The association between ALT and TP53 mutations
has previously been reported in adult gliomas [21, 29].
Next generation sequencing efforts have revealed a strong
J. S. Wunder
Department of Surgery, Mount Sinai Hospital, Toronto,
ON, Canada
N. Jabado
Department of Oncology, Montreal Children’s Hospital Research
Institute, Montreal, QC, Canada
N. Jabado
Department of Pediatrics, Montreal Children’s Hospital Research
Institute, Montreal, QC, Canada
D. L. Johnston
Division of Hematology/Oncology, Department of Pediatrics,
The Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)