1 3

Acta Neuropathol (2014) 128:853–862DOI 10.1007/s00401-014-1348-1

ORIGINAL PAPER

Alternative lengthening of telomeres is enriched in, and impacts survival of TP53 mutant pediatric malignant brain tumors

Joshua Mangerel · Aryeh Price · Pedro Castelo-Branco · Jack Brzezinski · Pawel Buczkowicz · Patricia Rakopoulos · Diana Merino · Berivan Baskin · Jonathan Wasserman · Matthew Mistry · Mark Barszczyk · Daniel Picard · Stephen Mack · Marc Remke · Hava Starkman · Cynthia Elizabeth · Cindy Zhang · Noa Alon · Jodi Lees · Irene L. Andrulis · Jay S. Wunder · Nada Jabado · Donna L. Johnston · James T. Rutka · Peter B. Dirks · Eric Bouffet · Michael D. Taylor · Annie Huang · David Malkin · Cynthia Hawkins · Uri Tabori

Received: 17 July 2014 / Revised: 23 September 2014 / Accepted: 26 September 2014 / Published online: 15 October 2014 © Springer-Verlag Berlin Heidelberg 2014

histological phenotypes and with clinical outcome. ALT was detected almost exclusively in malignant tumors (p = 0.001). ALT was highly enriched in primitive neu-roectodermal tumors (12 %), choroid plexus carcinomas (23 %) and high-grade gliomas (22 %). Furthermore, in contrast to adult gliomas, pediatric low grade gliomas which progressed to high-grade tumors did not exhibit the ALT phenotype. Somatic but not germline TP53 muta-tions were highly associated with ALT (p = 1.01 × 10−8). Of the other alterations examined, only ATRX point mutations and reduced expression were associated with the ALT phenotype (p = 0.0005). Interestingly, ALT

Abstract Although telomeres are maintained in most cancers by telomerase activation, a subset of tumors uti-lize alternative lengthening of telomeres (ALT) to sus-tain self-renewal capacity. In order to study the preva-lence and significance of ALT in childhood brain tumors we screened 517 pediatric brain tumors using the novel C-circle assay. We examined the association of ALT with alterations in genes found to segregate with specific

Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1348-1) contains supplementary material, which is available to authorized users.

J. Mangerel · A. Price · P. Castelo-Branco · P. Buczkowicz · P. Rakopoulos · M. Mistry · M. Barszczyk · D. Picard · S. Mack · M. Remke · H. Starkman · C. Elizabeth · C. Zhang · N. Alon · J. Lees · J. T. Rutka · P. B. Dirks · E. Bouffet · M. D. Taylor · A. Huang · C. Hawkins · U. Tabori The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada

J. Mangerel · D. Merino · M. Mistry Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, ON, Canada

P. Castelo-Branco Regenerative Medicine Program, Department of Medicine and Biomedical Sciences, University of Algarve, 8005-139 Faro, Portugal

P. Castelo-Branco Centre for Molecular and Structural Biomedicine, CBME/IBB, University of Algarve, 8005-139 Faro, Portugal

J. Brzezinski · J. S. Wunder · J. T. Rutka · P. B. Dirks · E. Bouffet · M. D. Taylor · A. Huang · D. Malkin · C. Hawkins · U. Tabori (*) Division of Hematology/Oncology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, ON M5G 1X8, Canadae-mail: uri.tabori@sickkids.ca

P. Buczkowicz · P. Rakopoulos · M. Barszczyk · I. L. Andrulis Laboratory Medical Pathology, University of Toronto, Toronto, ON, Canada

D. Merino · D. Malkin Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada

B. Baskin Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

J. Wasserman Department of Endocrinology, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

J. Wasserman Department of Pediatrics, University of Toronto, Toronto, ON, Canada

M. Remke Division of Neurosurgery, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada

I. L. Andrulis · J. S. Wunder The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

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.

AbbreviationsALT Alternative lengthening of telomeresqPCR Real-time polymerase chain reactionCCA C-circle assayTP53 Tumor protein 53ATRX Alpha thalassemia/mental retardation syndrome

X-linkedPBT Pediatric brain tumorBTRC Brain tumor research centerTRF Terminal restriction fragmentFISH Fluorescence in situ hybridizationFFPE Formalin-fixed paraffin extractedCPC Choroid plexus carcinomaSHGG Supratentorial high grade gliomaDIPG Diffuse intrinsic pontine gliomaPNET Primitive neuroectodermal tumorsLGG Low grade gliomaLFS Li-Fraumeni syndromeIHC ImmunohistochemistryGBM 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

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

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 %)

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

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

859Acta Neuropathol (2014) 128:853–862

1 3

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

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

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

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