Abstract
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2,3,4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.
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Accession codes
Data deposits
All high-throughput data have been deposited at the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/) under accession number EGAS00001001308.
Change history
28 October 2015
Spelling of author C.G. was corrected.
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Acknowledgements
We are indebted to the patients and their parents for making available the tumour specimens analysed in this study. We thank the German Neuroblastoma Biobank for providing samples from patients. The Institutional Review Board approved collection and use of all specimens in this study. We also thank our colleagues N. Hemstedt, H. Düren, E. Hess, J. Kreth, and J. Gopalakrishnan; and our collaboration partners I. Amit and F. Paul at the Weizmann Institute of Science for technical assistance. We further acknowledge the Center for Molecular Medicine Cologne light microscope facility for helping us to obtain high-quality data of FISH analyses, and S. Wolf and the Next Generation Sequencing Unit at the German Cancer Research Center (DKFZ) for sequencing. This work was supported by the German Cancer Aid (grant 110122) to M.F., F.W., J.H.S., A.S., and S.A.; the German Ministry of Science and Education (BMBF) as part of the e:Med initiative (grant 01ZX1303A to M.P., M.F., J.H.S., R.B., U.L., and R.K.T., grant 01ZX1406 to M.P., and grant 01ZX1307D to M.F. and F.W.); the BMBF (grant 0316076A to F.W.); the European Union (grant 259348 to F.W.); the Fördergesellschaft Kinderkrebs-Neuroblastom-Forschung e.V. (to M.F.); the German-Israeli Helmholtz Research School in Cancer Biology (to M.G. and F.W.); the Volkswagenstiftung (Lichtenberg Program) (to M.R.S.); and the Center for Molecular Medicine Cologne.
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Authors and Affiliations
Contributions
Conception and design: M.P., F.H., F.R., D.D., M.G., F.W., R.K.T., and M.F. Administrative support, provision of study materials and patients: M.P., J.H., I.L., T.H., P.N., V.A., U.L., A.Eg., F.B., Z.P., C.Z., L.Sh., R.B., S.P., B.H., A.S., J.H.S., F.W., R.J.O., R.K.T., and M.F. Conduct of the experiments, data analysis, and interpretation: M.P., F.H., F.R., D.D., M.G., R.M., A.K., J.L.R., F.S., J.H., F.I., R.S., S.A., A.En., Y.K., W.V., J.A., J.T.-M., D.T.-M., A.M., S.H., E.M., K.-O.H., C.G., G.B., M.-R.S., L.Sa., S.C.W., C.S., E.B., J.T., R.V., M.S., B.D., M.O., B.H., C.H., R.J.O., F.W., R.K.T., and M.F. Manuscript writing: M.P., F.H., F.R., F.W., R.K.T., and M.F. All authors read and approved the final manuscript.
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Competing interests
M.P. is a co-founder and shareholder of NEO New Oncology, and received consulting fees from NEO New Oncology. R.K.T. is a founder and shareholder of NEO New Oncology, and received consulting and lecture fees from NEO New Oncology, Sanofi-Aventis, Merck, Roche, Lilly, Boehringer Ingelheim, AstraZeneca, Atlas Biolabs, Daiichi Sankyo, Johnson & Johnson, Puma and Clovis, as well as research support from Merck, EOS and AstraZeneca.
Extended data figures and tables
Extended Data Figure 1 Validation of rearrangements of the TERT locus by dideoxy-sequencing.
Sequencing chromatograms of the breakpoint regions of 5p15.33 rearrangements along with their genomic coordinates (hg19), and the breakpoint-spanning nucleotide sequences. The sequence mapping to the TERT locus is indicated in yellow, the rearrangement partner is indicated in grey; nucleotides inserted at the breakpoint region are indicated in white.
Extended Data Figure 2 Schematic representation of chromothripsis in four primary neuroblastomas.
a, Circos plots showing chromothripsis of chromosome 5 in the tumours NBL39 and NBL37. b, Circos plots showing chromothripsis of chromosomes 17 and 20 in the tumours NBL21 and NBL08, respectively. Regions showing loss of heterozygosity (LOH) are indicated by black segments.
Extended Data Figure 3 TERT rearrangements are associated with poor event-free survival of patients, and with TERT upregulation independent of the breakpoint distance from the TERT transcriptional start site.
a, Kaplan–Meier estimates for event-free survival of neuroblastoma patient groups defined by TERT rearrangements (TERT), MYCN amplification (MNA), high-risk disease without these alterations (HR), and low-risk or intermediate-risk disease (nonHR). Patients with tumours bearing both a TERT rearrangement and MYCN amplification (n = 5) were excluded. Event-free survival at 5 years: 0.17 ± 0.09 (TERT) versus 0.38 ± 0.09 (MNA) versus 0.43 ± 0.09 (HR) versus 0.83 ± 0.05 (nonHR). b, Multivariable Cox regression analysis of the potential prognostic factors stage, MYCN status, and TERT status for event-free survival in patients aged >18 months (n = 125). c, Validation of TERT expression levels by microarrays in the four neuroblastoma subgroups indicated above. Sample numbers are given at the bottom. **P < 0.01, ***P < 0.001. d, Fold-change of median TERT expression levels in TERT-rearranged and MYCN-amplified tumours compared with low-risk (LR) neuroblastomas as measured by transcriptome sequencing. e, Evidence for monoallelic expression of TERT in five tumours bearing TERT rearrangements. The presence of a heterozygous single nucleotide polymorphism and its allelic fraction measured by whole-genome sequencing (WGS) is shown on the left of each panel; monoallelic expression as established by transcriptome sequencing (RNA-seq) is indicated on the right. The genomic position of the single nucleotide polymorphism is indicated at the top, the number of reads available for the analysis is shown at the bottom. f, TERT expression measured by transcriptome sequencing in relation to the distance of the rearrangement breakpoint from the TERT transcriptional start site (TSS). TERT expression levels and breakpoint distances from the TERT transcriptional start site were not correlated (r = 0, P = 0.97; Spearman’s rank correlation test).
Extended Data Figure 4 TERT rearrangements are maintained in relapsed neuroblastoma.
a, Agarose gel electrophoresis of PCR products representing individual TERT rearrangements in four tumours at initial diagnosis (I), and at relapse (R). The non-template controls are indicated by C. b, TERT expression measured by microarrays in relapsed TERT-rearranged tumours (n = 3) compared with TERT-rearranged tumours biopsied at initial diagnosis (n = 10). c, Sequencing chromatograms of the breakpoint regions for the relapse cases.
Extended Data Figure 5 Regional effects of TERT rearrangements on gene expression patterns.
a, Expression levels of genes around TERT measured by transcriptome sequencing are shown for tumours with TERT rearrangements (TERT, yellow), tumours with MYCN amplifications (MNA, red), and tumours without these aberrations classified as either high-risk (HR, grey) or low-risk (LR, green). Five consecutive genes (TERT, bleyplaby, SLC6A18, SLC6A19, and rarjy) located distal of the breakpoint at chromosome 5p15.33, and three genes (suweeby, blorplaby, and CLPTM1L) located proximal of the breakpoint are shown. Genomic positions of the genes are indicated at the top, and sample sizes are indicated at the bottom. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. b, Averaged expression levels of genes measured by transcriptome sequencing are indicated for an ∼500 kb region centring around TERT. Colour codes of neuroblastoma subgroups as indicated above. The breakpoint region is indicated in beige.
Extended Data Figure 6 TERT mRNA levels are massively upregulated in TERT-rearranged and MYCN-amplified neuroblastomas.
a, Differential gene expression between TERT-rearranged (n = 10) and low-risk (n = 17) neuroblastomas and b, between MYCN-amplified (MNA, n = 92) and low-risk tumours (n = 238) measured by transcriptome sequencing26. Negative log(P values) are plotted against log2(fold changes) of the genes expressed in the respective subgroups. Genes of interest are indicated at the bottom. log2(fold changes) correspond to the log difference of established gene expression scores. P values were established using Student’s t-tests and applying a Benjamini and Hochberg false discovery rate multiple testing correction. c, Analysis of differential gene expression of a MYCN-amplified neuroblastoma cell line (IMR-5/75) before and after shRNA-mediated knockdown of MYCN using transcriptome sequencing. The number of genes (frequency) up- or downregulated upon MYCN knockdown is plotted against the fold change of regulation. TERT is the most strongly downregulated gene upon shRNA-mediated MYCN knockdown. Only differentially expressed genes (t-test, false discovery rate controlled with P < 0.01) are shown. The insert shows reduced MYCN protein levels after induction of a MYCN-specific shRNA in IMR-5/75 cells as well as β-actin protein levels as control analysed by immunoblotting.
Extended Data Figure 7 Detection of TERT rearrangements in neuroblastoma cell lines.
a, Schematic representation of binding sites of probes used for FISH at the TERT locus. b, FISH analysis of neuroblastoma cell lines with 5p15.33 rearrangements and without these rearrangements; MNA, MYCN-amplified. Arrows indicate the unaltered status of chromosome 5p15.33 (red signals in close proximity to green signals); arrowheads indicate a rearrangement of 5p15.33 (red signals without adjacent green signals).
Extended Data Figure 8 Patterns of H3K27me3 histone modification and DNA methylation at the TERT locus.
a, Survey of the histone mark H3K27me3 in neuroblastomas harbouring TERT rearrangements (cell line GI-ME-N and primary tumour NBL36) and a neuroblastoma cell line lacking these alterations (SK-N-FI). b, DNA methylation patterns of CpG sites at the TERT locus in TERT-rearranged (TERT, n = 6) and MYCN-amplified (MNA, n = 9) primary neuroblastomas, as well as tumours lacking these alterations (others, n = 24) using HumanMethylation450 microrrays. Samples are ordered from top to bottom. CpG sites are indicated relative to their position to the TERT locus. Average methylation levels of each CpG site in the three subgroups are shown at the top. Similar to the highlighted CpG site cg11625005 upstream of the TERT transcriptional start site (Fig. 3d), CpG sites scattered over the TERT gene body were methylated significantly higher in TERT-rearranged and MYCN-amplified cases than in tumours without these alterations (P < 0.001 each).
Extended Data Figure 9 ALT activity in neuroblastoma cell lines lacking TERT rearrangements and MYCN amplifications.
a, Detection of extrachromosomal telomeric repeat DNA by C-circle assays with genomic AluI/MboI digested DNA from the indicated neuroblastoma cell lines. The ALT-positive control cell line, U2OS, is at the left. b, Detection of ALT-associated PML bodies in the indicated cell lines. Telomeric TTAGGG FISH (green) and immunofluorescence for PML (red) were combined and DNA was counterstained with DAPI (blue). c, Telomere restriction-fragment analysis of telomeric DNA from LAN-6, SK-N-FI, and the ALT-positive control U2OS. Telomeric DNA was detected by Southern blot with a [32P]dATP end-labelled (CCCTAA)4 oligonucleotide.
Supplementary information
Supplementary Table 1
This table contains WGS samples overview. (XLSX 54 kb)
Supplementary Table 2
This table contains the detected somatic mutations. (XLSX 154 kb)
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Peifer, M., Hertwig, F., Roels, F. et al. Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Nature 526, 700–704 (2015). https://doi.org/10.1038/nature14980
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DOI: https://doi.org/10.1038/nature14980
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