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1.
Br J Cancer ; 128(8): 1559-1571, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36807339

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 , Genômica
2.
Cell Biosci ; 12(1): 160, 2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36153564

RESUMO

BACKGROUND: Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. METHODS: Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). RESULTS: Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. CONCLUSIONS: We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.

3.
Cell Oncol (Dordr) ; 45(5): 991-1003, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35953764

RESUMO

BACKGROUND: The majority of high-risk neuroblastomas harbor telomerase activity, and telomerase-interacting compounds, such as 6-thio-2'-deoxyguanosine (6-thio-dG), have been found to impair the growth of telomerase-positive neuroblastoma cell lines. It has remained unclear, however, how such drugs can be combined with other compounds used in current treatment concepts for neuroblastoma patients. METHODS: Growth-inhibitory effects of varying concentrations of 6-thio-dG in combination with etoposide, doxorubicin or ceritinib were determined in eight telomerase-positive neuroblastoma cell lines with distinct genetic backgrounds. Tumor growth inhibition of subcutaneous xenografts from three different cell lines was assessed upon treatment with 6-thio-dG, the competitive telomerase inhibitor imetelstat, etoposide, or combinations of these compounds. RESULTS: Robust synergistic anti-tumor effects were observed for combinations of 6-thio-dG and etoposide or doxorubicin, but not for 6-thio-dG and ceritinib, in telomerase-positive neuroblastoma cell lines in vitro. Treatment of mouse xenografts with combinations of 6-thio-dG and etoposide significantly attenuated tumor growth and improved mouse survival over etoposide alone in two of three cell line models. Treatment of xenograft tumors by imetelstat monotherapy decreased telomerase activity by roughly 50% and significantly improved survival over control in all three models, whereas treatment with imetelstat plus etoposide led to enhanced survival over etoposide monotherapy in one model. Mechanistically, the synergistic effect was found to be due to both increased apoptosis and cell cycle arrest. CONCLUSION: Our study indicates that telomerase is an actionable target in telomerase-positive neuroblastoma, and demonstrates that combination therapies including telomerase-interacting compounds may improve the efficacy of established cytotoxic drugs. Targeting telomerase may thus represent a therapeutic option in high-risk neuroblastoma patients.


Assuntos
Neuroblastoma , Telomerase , Humanos , Camundongos , Animais , Telomerase/genética , Etoposídeo/farmacologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células , Neuroblastoma/tratamento farmacológico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico
4.
Mol Cancer ; 21(1): 126, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35689207

RESUMO

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.


Assuntos
Neuroblastoma , Medicina de Precisão , Quinase do Linfoma Anaplásico/genética , Linhagem Celular Tumoral , Criança , Humanos , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais
5.
Nat Genet ; 53(12): 1673-1685, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34782764

RESUMO

The mechanisms behind the evolution of complex genomic amplifications in cancer have remained largely unclear. Using whole-genome sequencing data of the pediatric tumor neuroblastoma, we here identified a type of amplification, termed 'seismic amplification', that is characterized by multiple rearrangements and discontinuous copy number levels. Overall, seismic amplifications occurred in 9.9% (274 of 2,756) of cases across 38 cancer types, and were associated with massively increased copy numbers and elevated oncogene expression. Reconstruction of the development of seismic amplification showed a stepwise evolution, starting with a chromothripsis event, followed by formation of circular extrachromosomal DNA that subsequently underwent repetitive rounds of circular recombination. The resulting amplicons persisted as extrachromosomal DNA circles or had reintegrated into the genome in overt tumors. Together, our data indicate that the sequential occurrence of chromothripsis and circular recombination drives oncogene amplification and overexpression in a substantial fraction of human malignancies.


Assuntos
Cromotripsia , Amplificação de Genes , Rearranjo Gênico , Neoplasias/genética , Oncogenes , Linhagem Celular Tumoral , Estudos de Coortes , DNA Circular , DNA de Neoplasias , Humanos , Modelos Genéticos , Mutação , Neuroblastoma/genética , Sequenciamento Completo do Genoma
6.
Cancers (Basel) ; 13(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34503173

RESUMO

BACKGROUND: To identify variables predicting outcome in neuroblastoma patients assigned to the high-risk group solely by the presence of MYCN oncogene amplification (MNA). METHODS: Clinical characteristics, genomic information, and outcome of 190 patients solely assigned to high-risk neuroblastoma by MNA were analyzed and compared to 205 patients with stage 4 neuroblastoma aged ≥18 months with MNA (control group). RESULTS: Event-free survival (EFS) and overall survival (OS) at 10 years were 47% (95%-CI 39-54%) and 56% (95%-CI 49-63%), respectively, which was significantly better than EFS and OS of the control group (EFS 25%, 95%-CI 18-31%, p < 0.001; OS 32% 95%-CI 25-39%, p < 0.001). The presence of RAS-/p53-pathway gene alterations was associated with impaired 10-year EFS and OS (19% vs. 55%, and 19% vs. 67%, respectively; both p < 0.001). In time-dependent multivariable analyses, alterations of RAS-/p53-pathway genes and the extent of the best primary tumor resection were the only independent prognostic variables for OS (p < 0.001 and p = 0.011, respectively). CONCLUSIONS: Neuroblastoma patients attributed to high risk solely by MYCN amplification have generally a more favorable outcome. Mutations of genes of the RAS and/or p53 pathways and incomplete resection are the main risk factors predicting poor outcome.

7.
Pediatr Blood Cancer ; 68(8): e29038, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33826231

RESUMO

INTRODUCTION: The survival of children with stage 4(M) neuroblastoma without MYCN amplification and below the age of 18 months is considered better than the still dismal outcome of older high-risk neuroblastoma patients. This study analyzes the impact of clinical and molecular characteristics on the long-term outcome. PATIENTS AND METHODS: Clinical presentation, survival, and recurrence patterns of patients enrolled onto trials NB90, NB97, and NB2004 were retrospectively analyzed. Gene expression signatures based on RNA microarrays (TH10) were investigated if tumor material was available. RESULTS: Between 1990 and 2015, 177 patients with stage 4(M) MYCN nonamplified neuroblastoma aged less than 18 months at diagnosis were eligible. After a median follow-up of 9.7 years (IQR 5.0, 13.4), the proportions of 10-year event-free survival (EFS) and overall survival (OS) were 73% (95% confidence interval [CI] 67-79%) and 86% (95% CI 80-92%), respectively. Of the 27 neuroblastoma recurrences, 44% occurred in more than one site. Four additional patients presented histologically mature ganglioneuroma at recurrence. Six patients developed a secondary malignancy. The secondary 5-year EFS and OS of the 27 patients with neuroblastoma recurrence were 44% and 59%, respectively. TH10 gene expression signature was not prognostically predictive in the investigated subcohort. CONCLUSION: The outcome of patients with stage 4(M) neuroblastoma aged less than 18 months is favorable when treated with high-risk or otherwise intensive therapy. The development of secondary malignancies and the potential of maturation to ganglioneuroma call for a controlled stepwise reduction of treatment intensity.


Assuntos
Ganglioneuroma , Neuroblastoma , Intervalo Livre de Doença , Ganglioneuroma/genética , Ganglioneuroma/patologia , Amplificação de Genes , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc/genética , Estadiamento de Neoplasias , Neuroblastoma/genética , Neuroblastoma/patologia , Prognóstico , Estudos Retrospectivos
8.
Nat Commun ; 12(1): 1269, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627664

RESUMO

Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas (n = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome.


Assuntos
Sequenciamento Completo do Genoma/métodos , Western Blotting , Éxons/genética , Citometria de Fluxo , Humanos , Proteoma/metabolismo , Estudos Retrospectivos , Análise de Sequência de RNA/métodos , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética
9.
Nat Commun ; 11(1): 5823, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199677

RESUMO

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.


Assuntos
Cromossomos Humanos/genética , Elementos Facilitadores Genéticos/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Acetilação , Sequência de Bases , Linhagem Celular Tumoral , Metilação de DNA/genética , DNA Circular/genética , Epigênese Genética , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Lisina/metabolismo , Sequenciamento por Nanoporos
11.
Nat Genet ; 52(1): 29-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31844324

RESUMO

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 Cultivadas
12.
Science ; 362(6419): 1165-1170, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30523111

RESUMO

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/genética
13.
Neoplasia ; 19(12): 982-990, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29091799

RESUMO

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.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/mortalidade , Fatores Etários , Biomarcadores Tumorais , Criança , Pré-Escolar , Biologia Computacional/métodos , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Masculino , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco
14.
Genome Biol ; 16: 133, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26109056

RESUMO

BACKGROUND: Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model. RESULTS: We generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models. CONCLUSIONS: We demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.


Assuntos
Perfilação da Expressão Gênica , Neuroblastoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA , Adolescente , Adulto , Criança , Pré-Escolar , Determinação de Ponto Final , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Genéticos , Neuroblastoma/classificação , Neuroblastoma/diagnóstico , Células Tumorais Cultivadas , Adulto Jovem
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