Acta Neuropathol (2014) 128:853–862 DOI 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
Abstract Although telomeres are maintained in most cancers by telomerase activation, a subset of tumors utilize alternative lengthening of telomeres (ALT) to sustain self-renewal capacity. In order to study the prevalence 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.
histological phenotypes and with clinical outcome. ALT was detected almost exclusively in malignant tumors (p = 0.001). ALT was highly enriched in primitive neuroectodermal 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 mutations 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
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
P. Buczkowicz · P. Rakopoulos · M. Barszczyk · I. L. Andrulis Laboratory Medical Pathology, University of Toronto, Toronto, ON, Canada
J. Mangerel · D. Merino · M. Mistry Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, ON, Canada
B. Baskin Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
P. Castelo-Branco Regenerative Medicine Program, Department of Medicine and Biomedical Sciences, University of Algarve, 8005-139 Faro, Portugal
J. Wasserman Department of Endocrinology, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
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, Canada e-mail: [email protected]
D. Merino · D. Malkin Department of Medical Biophysics, 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
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attenuated the poor outcome conferred by TP53 mutations in specific pediatric brain tumors. Due to very poor prognosis, one year overall survival was quantified in malignant gliomas, while in children with choroid plexus carcinoma, 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 positive and negative tumors, respectively (p = 0.03), while for children with TP53 mutant choroid plexus carcinomas, 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
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
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Acta Neuropathol (2014) 128:853–862
Introduction Pediatric brain tumors (PBTs), comprising multiple separate pathological entities, are the most common group of solid cancers in children. Recent discoveries using next-generation genomic platforms have uncovered substantial molecular heterogeneity even amongst PBTs with the same histological 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 development 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 chromosomes 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 replication [42]. Cells which are unable to maintain their telomeres 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 melanoma [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-independent manner, presumably by telomere-specific homologous recombination [6, 10, 35]. Although ALT occurs in a minority of tumors (
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
Abstract Although telomeres are maintained in most cancers by telomerase activation, a subset of tumors utilize alternative lengthening of telomeres (ALT) to sustain self-renewal capacity. In order to study the prevalence 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.
histological phenotypes and with clinical outcome. ALT was detected almost exclusively in malignant tumors (p = 0.001). ALT was highly enriched in primitive neuroectodermal 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 mutations 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
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
P. Buczkowicz · P. Rakopoulos · M. Barszczyk · I. L. Andrulis Laboratory Medical Pathology, University of Toronto, Toronto, ON, Canada
J. Mangerel · D. Merino · M. Mistry Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, ON, Canada
B. Baskin Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
P. Castelo-Branco Regenerative Medicine Program, Department of Medicine and Biomedical Sciences, University of Algarve, 8005-139 Faro, Portugal
J. Wasserman Department of Endocrinology, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
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, Canada e-mail: [email protected]
D. Merino · D. Malkin Department of Medical Biophysics, 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
13
854
attenuated the poor outcome conferred by TP53 mutations in specific pediatric brain tumors. Due to very poor prognosis, one year overall survival was quantified in malignant gliomas, while in children with choroid plexus carcinoma, 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 positive and negative tumors, respectively (p = 0.03), while for children with TP53 mutant choroid plexus carcinomas, 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
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
13
Acta Neuropathol (2014) 128:853–862
Introduction Pediatric brain tumors (PBTs), comprising multiple separate pathological entities, are the most common group of solid cancers in children. Recent discoveries using next-generation genomic platforms have uncovered substantial molecular heterogeneity even amongst PBTs with the same histological 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 development 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 chromosomes 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 replication [42]. Cells which are unable to maintain their telomeres 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 melanoma [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-independent manner, presumably by telomere-specific homologous recombination [6, 10, 35]. Although ALT occurs in a minority of tumors (
