RESUMO
BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.
Assuntos
Neuroblastoma , Receptores Proteína Tirosina Quinases , Criança , Humanos , Quinase do Linfoma Anaplásico/genética , Receptores Proteína Tirosina Quinases/genética , Recidiva Local de Neoplasia/genética , Neuroblastoma/genética , Neuroblastoma/patologia , GenômicaRESUMO
Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.
Assuntos
Amplificação de Genes/genética , MicroRNAs/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Deleção Cromossômica , Feminino , Deleção de Genes , Genes Neoplásicos/genética , Humanos , Camundongos , MicroRNAs/metabolismo , Modelos Genéticos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Recombinação Genética/genética , Telomerase/genética , Telomerase/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos Par 5/genética , DNA Helicases/genética , Metilação de DNA , Elementos Facilitadores Genéticos/genética , Ativação Enzimática/genética , Amplificação de Genes/genética , Inativação Gênica , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/classificação , Neuroblastoma/enzimologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Prognóstico , RNA Mensageiro/análise , RNA Mensageiro/genética , Risco , Translocação Genética/genética , Regulação para Cima/genética , Proteína Nuclear Ligada ao XRESUMO
Opsoclonus myoclonus syndrome (OMS), often called "dancing eyed syndrome," is a rare neurological condition associated with neuroblastoma in the majority of all childhood cases. Genomic copy number profiles have shown to be of prognostic significance for neuroblastoma patients. The aim of this retrospective multicenter study was to analyze the genomic copy number profiles of tumors from children with neuroblastoma presenting with OMS at diagnosis. In 44 cases of neuroblastoma associated with OMS, overall genomic profiling by either array-comparative genomic hybridization or single nucleotide polymorphism array proved successful in 91% of the cases, distinguishing tumors harboring segmental chromosome alterations from those with numerical chromosome alterations only. A total of 23/44 (52%) tumors showed an segmental chromosome alterations genomic profile, 16/44 (36%) an numerical chromosome alterations genomic profile, and 1 case displayed an atypical profile (12q amplicon). No recurrently small interstitial copy number alterations were identified. With no tumor relapse nor disease-related deaths, the overall genomic profile was not of prognostic impact with regard to the oncological outcome in this series of patients. Thus, the observation of an excellent oncological outcome, even for those with an unfavorable genomic profile of neuroblastoma, supports the hypothesis that an immune response might be involved in tumor control in these patients with OMS.
Assuntos
Neuroblastoma/complicações , Neuroblastoma/genética , Síndrome de Opsoclonia-Mioclonia/genética , Criança , Pré-Escolar , Feminino , Dosagem de Genes , Humanos , Lactente , Masculino , Estudos RetrospectivosRESUMO
The TP53 tumor suppressor pathway is abrogated by TP53 mutations in the majority of human cancers. Increased levels of wild-type TP53 in aggressive neuroblastomas appear paradox but are tolerated by tumor cells due to co-activation of the TP53 ubiquitin ligase, MDM2. The role of the MDM2 antagonist, p14(ARF), in controlling the TP53-MDM2 balance in neuroblastoma is unresolved. In the present study, we show that conditional p14(ARF) expression substantially suppresses viability, clonogenicity and anchorage-independent growth in p14(ARF)-deficient or MYCN-amplified neuroblastoma cell lines. Furthermore, ectopic 14(ARF) expression induced accumulation of cells in the G1 phase and apoptosis, which was paralleled by accumulation of TP53 and its targets. Comparative genomic hybridization analysis of 193 primary neuroblastomas detected one homozygous deletion of CDKN2A (encoding both p14(ARF) and p16(INK4A)) and heterozygous loss of CDKN2A in 22% of tumors. Co-expression analysis of p14(ARF) and its transactivator, E2F1, in a set of 68 primary tumors revealed only a weak correlation, suggesting that further regulatory mechanisms govern p14(ARF) expression in neuroblastomas. Intriguingly, analyses utilizing chromatin immunoprecipitation revealed different histone mark-defined epigenetic activity states of p14(ARF) in neuroblastoma cell lines that correlated with endogenous p14(ARF) expression but not with episomal p14(ARF) promoter reporter activity, indicating that the native chromatin context serves to epigenetically repress p14(ARF) in neuroblastoma cells. Collectively, the data pinpoint p14(ARF) as a critical factor for efficient TP53 response in neuroblastoma cells and assign p14(ARF) as a neuroblastoma suppressor candidate that is impaired by genomic loss and epigenetic repression.
Assuntos
Apoptose , Repressão Epigenética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Histonas/genética , Neuroblastoma/patologia , Proteína Supressora de Tumor p14ARF/genética , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Deleção de Genes , Expressão Gênica , Histonas/metabolismo , Humanos , Perda de Heterozigosidade , Masculino , Neuroblastoma/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The prognostic relevance of chromosome 17 gain in neuroblastoma is still discussed. This investigation specifies the frequency, type, size, and transcriptional relevance in a large patient cohort. Primary tumor material of 202 patients was analyzed using high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH) and correlated with clinical and survival data. A subset (n = 145) was correlated for differentially expressed genes (DEG) by microarray analysis. Chromosome 17 aCGH analysis showed numerical gain in 94/202 patients (47%), partial gain in 93/202 patients (46%), and no gain in 15/202 patients (7%). The frequency of partial gain was higher in stage 4 neuroblastoma (stage 1 15%; stage 2 12%; stage 3 16%; stage 4S 7%; and stage 4 50%). Overall survival (OS) was superior in patients with numerical gain compared with patients with partial gain or no gain (5-y-OS: 0.95 ± 0.02 vs. 0.63 ± 0.05 vs. 0.60 ± 0.13; P < 0.001). Gene expression analysis demonstrated 95/130 DEGs between tumors with numerical or partial chromosome/no gain. Only one DEG (CCKBR) was detected comparing tumors with partial gain and those with no gain. In patients with partial gain, the distribution of breakpoints did not correlate with stage and 11q status, but with MYCN amplification and 1p status. The "best" breakpoints in cases with partial 17q gain were at 42.5 Mb for event-free and 26.6 Mb for OS. Numerical gain of chromosome 17 is associated with a better prognosis than partial and no gain. The group of tumors with partial gain was similar to the group without gain with respect to stage distribution, outcome, and gene expression profile.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Neuroblastoma/diagnóstico , Hibridização Genômica Comparativa , Feminino , Humanos , Lactente , Masculino , Neuroblastoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , TranscriptomaRESUMO
MOTIVATION: Recurrent DNA breakpoints in cancer genomes indicate the presence of critical functional elements for tumor development. Identifying them can help determine new therapeutic targets. High-dimensional DNA microarray experiments like arrayCGH afford the identification of DNA copy number breakpoints with high precision, offering a solid basis for computational estimation of recurrent breakpoint locations. RESULTS: We introduce a method for identification of recurrent breakpoints (consensus breakpoints) from copy number aberration datasets. The method is based on weighted kernel counting of breakpoints around genomic locations. Counts larger than expected by chance are considered significant. We show that the consensus breakpoints facilitate consensus segmentation of the samples. We apply our method to three arrayCGH datasets and show that by using consensus segmentation we achieve significant dimension reduction, which is useful for the task of prediction of tumor phenotype based on copy number data. We use our approach for classification of neuroblastoma tumors from different age groups and confirm the recent recommendation for the choice of age cut-off for differential treatment of 18 months. We also investigate the (epi)genetic properties at consensus breakpoint locations for seven datasets and show enrichment in overlap with important functional genomic regions. AVAILABILITY: Implementation in R of our approach can be found at http://www.mpi-inf.mpg.de/ â¼laura/FeatureGrouping.html. CONTACT: laura@mpi-inf.mpg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Pontos de Quebra do Cromossomo , Variações do Número de Cópias de DNA , Neoplasias/genética , Genoma Humano , Genômica/métodos , Humanos , Neuroblastoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , SoftwareRESUMO
BACKGROUND: Segmental genomic copy number alterations, such as loss of 11q or 3p and gain of 17q, are well established markers of poor outcome in neuroblastoma, and have been suggested to comprise tumor suppressor genes or oncogenes, respectively. The gene forkhead box P1 (FOXP1) maps to chromosome 3p14.1, a tumor suppressor locus deleted in many human cancers including neuroblastoma. FoxP1 belongs to a family of winged-helix transcription factors that are involved in processes of cellular proliferation, differentiation and neoplastic transformation. METHODS: Microarray expression profiles of 476 neuroblastoma specimens were generated and genes differentially expressed between favorable and unfavorable neuroblastoma were identified. FOXP1 expression was correlated to clinical markers and patient outcome. To determine whether hypermethylation is involved in silencing of FOXP1, methylation analysis of the 5' region of FOXP1 in 47 neuroblastomas was performed. Furthermore, FOXP1 was re-expressed in three neuroblastoma cell lines to study the effect of FOXP1 on growth characteristics of neuroblastoma cells. RESULTS: Low expression of FOXP1 is associated with markers of unfavorable prognosis like stage 4, age >18 months and MYCN amplification and unfavorable gene expression-based classification (P < 0.001 each). Moreover, FOXP1 expression predicts patient outcome accurately and independently from well-established prognostic markers. Array-based CGH analysis of 159 neuroblastomas revealed that heterozygous loss of the FOXP1 locus was a rare event (n = 4), but if present, was associated with low FOXP1 expression. By contrast, DNA methylation analysis in 47 neuroblastomas indicated that hypermethylation is not regularly involved in FOXP1 gene silencing. Re-expression of FoxP1 significantly impaired cell proliferation, viability and colony formation in soft agar. Furthermore, induction of FOXP1 expression led to cell cycle arrest and apoptotic cell death of neuroblastoma cells. CONCLUSIONS: Our results suggest that down-regulation of FOXP1 expression is a common event in high-risk neuroblastoma pathogenesis and may contribute to tumor progression and unfavorable patient outcome.
Assuntos
Transformação Celular Neoplásica/genética , Fatores de Transcrição Forkhead/genética , Neuroblastoma/genética , Proteínas Repressoras/genética , Apoptose/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Pré-Escolar , Análise por Conglomerados , Hibridização Genômica Comparativa , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , Lactente , Estadiamento de Neoplasias , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Fenótipo , Prognóstico , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
About 50% of children with neuroblastoma (NB) show a metastatic disease and have a poor prognosis. However, disease progression is greatly variable and depends on patients' age and MYCN oncogene amplification. To investigate the role of patients' age in tumor aggressiveness, we performed array-CGH and gene expression profiles of three groups (G) of metastatic NBs: G1, stage 4S patients and MYCN single copy (MYCN-) tumors; G2, stage 4 patients, ≤ 18 months of age, MYCN- tumors and favorable outcome and G3, Stage 4 patients, ≥ 19 months with unfavorable outcome. G1 was characterized by numerical aberrations prevalently; on the contrary, all G3 tumors had structural rearrangements, whereas G2 showed an intermediate pattern. The average of numerical alterations decreased significantly from G1 to G2 to G3 (p < 0.01). Contrarily, the number of structural aberrations increased from G1 to G2 to G3 (p < 2.35 E-05). Noteworthy, G3/MYCN- NBs were characterized by several complex intrachromosome rearrangements. Expression analysis of the three groups showed significant differences in genes of Rho and Ras signaling pathways, development and adhesion, cell cycle regulation and telomerase activity. Accumulation of structural alterations increased with patients' age and was associated with a more aggressive disease. Abnormal expression of genes involved in cell cycle and telomerase in G3 may be responsible for the genomic instability in this cohort of patients. The higher DNA instability observed in G3/MYCN- NBs than in MYCN-amplified G3 may also explain why patients ≥ 19 months have a poor outcome independently by MYCN status.
Assuntos
Ciclo Celular/genética , Aberrações Cromossômicas , Neuroblastoma/genética , Neuroblastoma/patologia , Telomerase/genética , Fatores Etários , Variações do Número de Cópias de DNA , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Metástase NeoplásicaRESUMO
For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our "neuroblastoma hybrid capture sequencing panel" (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.
RESUMO
PURPOSE: MYCN amplification is an important therapy-stratifying marker in neuroblastoma. Fluorescence in situ hybridization with signal detection on the single-cell level allows a critical judgement of MYCN intratumoral heterogeneity. EXPERIMENTAL DESIGN: The MYCN status was investigated by fluorescence in situ hybridization at diagnosis and relapse. Heterogeneity was defined as the simultaneous presence of amplified cells (>/=5 cells per slide) and nonamplified cells within one tumor or sequential change of the amplification status during the course of the disease. Likewise, heterogeneity can be detected between primary tumor and metastasis. RESULTS: From 1,341 patients analyzed, 1,071 showed no amplification, 250 showed homogeneous amplification, and 20 patients showed MYCN heterogeneity. Of the patients with heterogeneity, 12 of 20 had clusters of MYCN amplifications, 3 of 20 had amplified single cells, 3 of 20 showed MYCN amplifications in the bone marrow but not in the primary tumor, and 2 of 20 acquired MYCN amplification during the course of the disease. All stage 4 patients were treated according to high-risk protocols; 7 of 8 later progressed. Four patients with localized disease were treated according to high-risk protocol because of MYCN-amplified clusters; 1 of 4 later progressed. One patient treated with mild chemotherapy experienced progression. Seven patients with localized/4S disease underwent no chemotherapy: 4 of 5 patients with MYCN heterogeneity at diagnosis remained disease-free, and 1 of 5 experienced local progression. Two patients had normal MYCN status at diagnosis but acquired MYCN amplification during the course of the disease. CONCLUSION: MYCN heterogeneity is rare. Our results suggest that small amounts of MYCN-amplified cells are not correlated to adverse outcomes. More patients with heterogeneity are warranted to clarify the role of MYCN heterogeneity for risk classification.
Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Oncogenes , Amplificação de Genes , Humanos , Proteína Proto-Oncogênica N-Myc , Estadiamento de Neoplasias , Neuroblastoma/patologiaRESUMO
Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.
Assuntos
Carcinogênese/patologia , DNA Circular/genética , Herança Extracromossômica/genética , Rearranjo Gênico , Genoma Humano , Neuroblastoma/patologia , Oncogenes/genética , Recombinação Genética , Humanos , Neuroblastoma/genética , Células Tumorais CultivadasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
For many decades, neuroblastoma has remained a challenging disease for both clinicians and researchers. Now, techniques that efficiently specify both comprehensive genetic and gene-expression alterations of neuroblastoma tumors have provided molecular markers that indicate tumor behavior and patient outcome with very high accuracy. Once the anticipated value of these markers has been confirmed in ongoing studies, patients may profit from more accurate risk assessment by integrating these markers into clinical routine. Moreover, disclosing further tumor-initiating events, such as the recently revealed oncogenic mutations of ALK, will further promote the elucidation of the genetic etiology of the disease. Together with recent information on altered signaling pathways in aggressively growing tumors, this knowledge will help to establish therapeutic strategies specifically targeting molecular key factors of neuroblastoma tumor progression.
Assuntos
Neuroblastoma/classificação , Neuroblastoma/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients.
Assuntos
Neuroblastoma/classificação , Neuroblastoma/mortalidade , Homeostase do Telômero/genética , Criança , Pré-Escolar , Intervalo Livre de Doença , Exoma/genética , Genoma Humano , Humanos , Redes e Vias Metabólicas/genética , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Prognóstico , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
Background: Neuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations. Methods: In an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome. Results: In this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval [CI] = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001). Conclusions: Using a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 6 , Amplificação de Genes , Genômica , Neuroblastoma/genética , Neuroblastoma/mortalidade , Biomarcadores Tumorais , Pré-Escolar , Variações do Número de Cópias de DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Genômica/métodos , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc/genética , Estadiamento de Neoplasias , Neuroblastoma/patologia , Neuroblastoma/terapia , PrognósticoRESUMO
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.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Recidiva Local de Neoplasia/genética , Neuroblastoma/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , DNA Helicases/genética , Exoma/genética , Perfilação da Expressão Gênica/métodos , Frequência do Gene , Fatores de Troca do Nucleotídeo Guanina/genética , Via de Sinalização Hippo , Humanos , Hibridização in Situ Fluorescente , Proteínas do Tecido Nervoso/genética , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Análise de Sequência de DNA/métodos , Transdução de Sinais/genética , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Neuroblastoma/genética , Neuroblastoma/mortalidade , Análise por Conglomerados , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Neuroblastoma/diagnóstico , Neuroblastoma/terapia , Prognóstico , Análise de Regressão , Reprodutibilidade dos Testes , Medição de Risco , Fatores de RiscoRESUMO
We aimed to identify novel molecular prognostic markers to better predict relapse risk estimate for children with high-risk (HR) metastatic neuroblastoma (NB). We performed genome- and/or transcriptome-wide analyses of 129 stage 4 HR NBs. Children older than 1 year of age were categorized as "short survivors" (dead of disease within 5 years from diagnosis) and "long survivors" (alive with an overall survival time ≥ 5 years). We reported that patients with less than three segmental copy number aberrations in their tumor represent a molecularly defined subgroup with a high survival probability within the current HR group of patients. The complex genomic pattern is a prognostic marker independent of NB-associated chromosomal aberrations, i.e., MYCN amplification, 1p and 11q losses, and 17q gain. Integrative analysis of genomic and expression signatures demonstrated that fatal outcome is mainly associated with loss of cell cycle control and deregulation of Rho guanosine triphosphates (GTPases) functioning in neuritogenesis. Tumors with MYCN amplification show a lower chromosome instability compared to MYCN single-copy NBs (P = .0008), dominated by 17q gain and 1p loss. Moreover, our results suggest that the MYCN amplification mainly drives disruption of neuronal differentiation and reduction of cell adhesion process involved in tumor invasion and metastasis. Further validation studies are warranted to establish this as a risk stratification for patients.
Assuntos
Instabilidade Genômica , Neuroblastoma/genética , Neuroblastoma/mortalidade , Criança , Pré-Escolar , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Amplificação de Genes , Perfilação da Expressão Gênica , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc , Metástase Neoplásica , Estadiamento de Neoplasias , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , PrognósticoRESUMO
PURPOSE: Genomic alterations of the anaplastic lymphoma kinase (ALK) gene have been postulated to contribute to neuroblastoma pathogenesis. This study aimed to determine the interrelation of ALK mutations, ALK expression levels, and clinical phenotype in primary neuroblastoma. EXPERIMENTAL DESIGN: The genomic ALK status and global gene expression patterns were examined in 263 primary neuroblastomas. Allele-specific ALK expression was determined by cDNA cloning and sequencing. Associations of genomic ALK alterations and ALK expression levels with clinical phenotypes and transcriptomic profiles were compared. RESULTS: Nonsynonymous point mutations of ALK were detected in 21 of 263 neuroblastomas (8%). Tumors with ALK mutations exhibited about 2-fold elevated median ALK mRNA levels in comparison with tumors with wild-type (WT) ALK. Unexpectedly, the WT allele was preferentially expressed in 12 of 21 mutated tumors. Whereas survival of patients with ALK mutated tumors was significantly worse as compared with the entire cohort of WT ALK patients, it was similarly poor in patients with WT ALK tumors in which ALK expression was as high as in ALK mutated neuroblastomas. Global gene expression patterns of tumors with ALK mutations or with high-level WT ALK expression were highly similar, and suggested that ALK may be involved in cellular proliferation in primary neuroblastoma. CONCLUSIONS: Primary neuroblastomas with mutated ALK exhibit high ALK expression levels and strongly resemble neuroblastomas with elevated WT ALK expression levels in both their clinical and molecular phenotypes. These data suggest that high levels of mutated and WT ALK mediate similar molecular functions that may contribute to a malignant phenotype in primary neuroblastoma.