RESUMEN
Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. 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 elusive. 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 type. 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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Genoma Humano/genética , Neuroblastoma/genética , Neuroblastoma/patología , Recombinación Genética/genética , Telomerasa/genética , Telomerasa/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Cromatina/genética , Cromatina/metabolismo , Cromosomas Humanos Par 5/genética , ADN Helicasas/genética , Metilación de ADN , Elementos de Facilitación Genéticos/genética , Activación Enzimática/genética , Amplificación de Genes/genética , Silenciador del Gen , Humanos , Lactante , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/clasificación , Neuroblastoma/enzimología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Pronóstico , ARN Mensajero/análisis , ARN Mensajero/genética , Riesgo , Translocación Genética/genética , Regulación hacia Arriba/genética , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , ARN Largo no Codificante/genética , Factor de Transcripción SOX9/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , Neuroblastoma/genética , Neuroblastoma/patología , Ubiquitina Tiolesterasa/genéticaRESUMEN
BACKGROUND: Current risk stratification systems for neuroblastoma patients consider clinical, histopathological, and genetic variables, and additional prognostic markers have been proposed in recent years. We here sought to select highly informative covariates in a multistep strategy based on consecutive Cox regression models, resulting in a risk score that integrates hazard ratios of prognostic variables. METHODS: A cohort of 695 neuroblastoma patients was divided into a discovery set (n=75) for multigene predictor generation, a training set (n=411) for risk score development, and a validation set (n=209). Relevant prognostic variables were identified by stepwise multivariable L1-penalized least absolute shrinkage and selection operator (LASSO) Cox regression, followed by backward selection in multivariable Cox regression, and then integrated into a novel risk score. RESULTS: The variables stage, age, MYCN status, and two multigene predictors, NB-th24 and NB-th44, were selected as independent prognostic markers by LASSO Cox regression analysis. Following backward selection, only the multigene predictors were retained in the final model. Integration of these classifiers in a risk scoring system distinguished three patient subgroups that differed substantially in their outcome. The scoring system discriminated patients with diverging outcome in the validation cohort (5-year event-free survival, 84.9±3.4 vs 63.6±14.5 vs 31.0±5.4; P<.001), and its prognostic value was validated by multivariable analysis. CONCLUSION: We here propose a translational strategy for developing risk assessment systems based on hazard ratios of relevant prognostic variables. Our final neuroblastoma risk score comprised two multigene predictors only, supporting the notion that molecular properties of the tumor cells strongly impact clinical courses of neuroblastoma patients.
Asunto(s)
Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/mortalidad , Factores de Edad , Biomarcadores de Tumor , Niño , Preescolar , Biología Computacional/métodos , Femenino , Amplificación de Genes , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de RiesgoRESUMEN
Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.
Asunto(s)
Neuronas Adrenérgicas/patología , Neuroblastoma/patología , Factor de Transcripción AP-2/metabolismo , Adolescente , Neuronas Adrenérgicas/metabolismo , Adulto , Azacitidina/análogos & derivados , Azacitidina/farmacología , Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Niño , Preescolar , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Decitabina , Dopamina beta-Hidroxilasa/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Recién Nacido , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Pronóstico , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción AP-2/genética , Tretinoina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Mutación , Recurrencia Local de Neoplasia/genética , Neuroblastoma/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , ADN Helicasas/genética , Exoma/genética , Perfilación de la Expresión Génica/métodos , Frecuencia de los Genes , Factores de Intercambio de Guanina Nucleótido/genética , Vía de Señalización Hippo , Humanos , Hibridación Fluorescente in Situ , Proteínas del Tejido Nervioso/genética , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Análisis de Secuencia de ADN/métodos , Transducción de Señal/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
PURPOSE: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression-based classification and established prognostic markers. EXPERIMENTAL DESIGN: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan-Meier estimates and Cox regression analyses. RESULTS: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients [5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001] and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 [EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20-8.02; OS: HR, 25.54; 95% CI, 8.40-77.66; both P < 0.001]. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation. CONCLUSIONS: Combination of gene expression-based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial.