Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study

Pedro Castelo-Branco, Sanaa Choufani, Stephen Mack, Denis Gallagher, Cindy Zhang, Tatiana Lipman, Nataliya Zhukova, Erin WALKER, Diana Martin, Diana Merino, Jonathan Wasserman, Cynthia Elizabeth, Noa Alon, Li Zhang, Volker Hovestadt, Marcel Kool, David Jones, Gelareh Zadeh, Sidney Croul, Cynthia Hawkins & 9 others Johann Hitzler, Jing Wang, Sylvian Baruchel, Peter Dirks, David Malkin, Stefan Pfister, Michael Taylor, Rosanna Weksberg, Uri Tabori

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Abstract

Background Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. Methods For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. Findings Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0·0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0·0001). UTSS had a positive predictive value of 1·00 (95% CI 0·95–1·00) and a negative predictive value of 0·95 (0·87–0·99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31–71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86–100) for 20 with non-hypermethylated tumours (p=0·0008). 5-year progression-free survival was 86% (68–100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10–50) for those with hypermethylated tumours (p=0·0008). Interpretation Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. Funding The Canadian Institute of Health Research and the Terry Fox Foundation
Original languageEnglish
Pages (from-to)534-542
Number of pages9
JournalLancet Oncology
Volume14
Issue number6
DOIs
Publication statusPublished - 2013
Externally publishedYes

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Brain Neoplasms
Transcription Initiation Site
Methylation
Neoplasms
Pediatrics
Ependymoma
Biomarkers
Choroid Plexus Neoplasms
Survival
Brain
Papilloma
Tumor Biomarkers
Genetic Promoter Regions
Glioma
Disease-Free Survival
Canada
Germany
Disease Progression
Real-Time Polymerase Chain Reaction
Genome

Cite this

Castelo-Branco, P., Choufani, S., Mack, S., Gallagher, D., Zhang, C., Lipman, T., ... Tabori, U. (2013). Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. Lancet Oncology, 14(6), 534-542. https://doi.org/10.1016/S1470-2045(13)70110-4
Castelo-Branco, Pedro ; Choufani, Sanaa ; Mack, Stephen ; Gallagher, Denis ; Zhang, Cindy ; Lipman, Tatiana ; Zhukova, Nataliya ; WALKER, Erin ; Martin, Diana ; Merino, Diana ; Wasserman, Jonathan ; Elizabeth, Cynthia ; Alon, Noa ; Zhang, Li ; Hovestadt, Volker ; Kool, Marcel ; Jones, David ; Zadeh, Gelareh ; Croul, Sidney ; Hawkins, Cynthia ; Hitzler, Johann ; Wang, Jing ; Baruchel, Sylvian ; Dirks, Peter ; Malkin, David ; Pfister, Stefan ; Taylor, Michael ; Weksberg, Rosanna ; Tabori, Uri. / Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. In: Lancet Oncology. 2013 ; Vol. 14, No. 6. pp. 534-542.
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abstract = "Background Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. Methods For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. Findings Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99{\%}) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72{\%}) of 201 samples from malignant tumours were hypermethylated (>15{\%} methylated; p<0·0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0·0001). UTSS had a positive predictive value of 1·00 (95{\%} CI 0·95–1·00) and a negative predictive value of 0·95 (0·87–0·99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51{\%} (95{\%} CI 31–71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95{\%} (86–100) for 20 with non-hypermethylated tumours (p=0·0008). 5-year progression-free survival was 86{\%} (68–100) for the 25 patients with non-hypermethylated UTSS tumours and 30{\%} (10–50) for those with hypermethylated tumours (p=0·0008). Interpretation Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. Funding The Canadian Institute of Health Research and the Terry Fox Foundation",
keywords = "Brain Tumor",
author = "Pedro Castelo-Branco and Sanaa Choufani and Stephen Mack and Denis Gallagher and Cindy Zhang and Tatiana Lipman and Nataliya Zhukova and Erin WALKER and Diana Martin and Diana Merino and Jonathan Wasserman and Cynthia Elizabeth and Noa Alon and Li Zhang and Volker Hovestadt and Marcel Kool and David Jones and Gelareh Zadeh and Sidney Croul and Cynthia Hawkins and Johann Hitzler and Jing Wang and Sylvian Baruchel and Peter Dirks and David Malkin and Stefan Pfister and Michael Taylor and Rosanna Weksberg and Uri Tabori",
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Castelo-Branco, P, Choufani, S, Mack, S, Gallagher, D, Zhang, C, Lipman, T, Zhukova, N, WALKER, E, Martin, D, Merino, D, Wasserman, J, Elizabeth, C, Alon, N, Zhang, L, Hovestadt, V, Kool, M, Jones, D, Zadeh, G, Croul, S, Hawkins, C, Hitzler, J, Wang, J, Baruchel, S, Dirks, P, Malkin, D, Pfister, S, Taylor, M, Weksberg, R & Tabori, U 2013, 'Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study', Lancet Oncology, vol. 14, no. 6, pp. 534-542. https://doi.org/10.1016/S1470-2045(13)70110-4

Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. / Castelo-Branco, Pedro; Choufani, Sanaa; Mack, Stephen; Gallagher, Denis; Zhang, Cindy; Lipman, Tatiana; Zhukova, Nataliya; WALKER, Erin; Martin, Diana; Merino, Diana; Wasserman, Jonathan; Elizabeth, Cynthia; Alon, Noa; Zhang, Li; Hovestadt, Volker; Kool, Marcel; Jones, David; Zadeh, Gelareh; Croul, Sidney; Hawkins, Cynthia; Hitzler, Johann; Wang, Jing; Baruchel, Sylvian; Dirks, Peter; Malkin, David; Pfister, Stefan; Taylor, Michael; Weksberg, Rosanna; Tabori, Uri.

In: Lancet Oncology, Vol. 14, No. 6, 2013, p. 534-542.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study

AU - Castelo-Branco, Pedro

AU - Choufani, Sanaa

AU - Mack, Stephen

AU - Gallagher, Denis

AU - Zhang, Cindy

AU - Lipman, Tatiana

AU - Zhukova, Nataliya

AU - WALKER, Erin

AU - Martin, Diana

AU - Merino, Diana

AU - Wasserman, Jonathan

AU - Elizabeth, Cynthia

AU - Alon, Noa

AU - Zhang, Li

AU - Hovestadt, Volker

AU - Kool, Marcel

AU - Jones, David

AU - Zadeh, Gelareh

AU - Croul, Sidney

AU - Hawkins, Cynthia

AU - Hitzler, Johann

AU - Wang, Jing

AU - Baruchel, Sylvian

AU - Dirks, Peter

AU - Malkin, David

AU - Pfister, Stefan

AU - Taylor, Michael

AU - Weksberg, Rosanna

AU - Tabori, Uri

PY - 2013

Y1 - 2013

N2 - Background Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. Methods For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. Findings Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0·0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0·0001). UTSS had a positive predictive value of 1·00 (95% CI 0·95–1·00) and a negative predictive value of 0·95 (0·87–0·99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31–71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86–100) for 20 with non-hypermethylated tumours (p=0·0008). 5-year progression-free survival was 86% (68–100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10–50) for those with hypermethylated tumours (p=0·0008). Interpretation Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. Funding The Canadian Institute of Health Research and the Terry Fox Foundation

AB - Background Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. Methods For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. Findings Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0·0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0·0001). UTSS had a positive predictive value of 1·00 (95% CI 0·95–1·00) and a negative predictive value of 0·95 (0·87–0·99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31–71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86–100) for 20 with non-hypermethylated tumours (p=0·0008). 5-year progression-free survival was 86% (68–100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10–50) for those with hypermethylated tumours (p=0·0008). Interpretation Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. Funding The Canadian Institute of Health Research and the Terry Fox Foundation

KW - Brain Tumor

U2 - 10.1016/S1470-2045(13)70110-4

DO - 10.1016/S1470-2045(13)70110-4

M3 - Article

VL - 14

SP - 534

EP - 542

JO - Lancet Oncology

JF - Lancet Oncology

SN - 1470-2045

IS - 6

ER -