RESUMEN
Patients with ALK-driven neuroblastoma may respond to tyrosine kinase inhibitors, but resistance to treatment occurs and methods currently used for detection of residual disease have limited sensitivity. Here we present a national unselected cohort of five patients with relapsed or refractory ALK-driven neuroblastoma treated with lorlatinib as monotherapy, and test the potential of targeted circulating tumor DNA (ctDNA) analysis as a guide for treatment decisions in these patients. We developed a sequencing panel for ultrasensitive detection of ALK mutations associated with neuroblastoma or resistance to tyrosine kinase inhibitors and used it for ctDNA analysis in 83 plasma samples collected longitudinally from the four patients who harbored somatic ALK mutations. All four patients with ALK p.R1275Q experienced major responses and were alive 35-61 months after starting lorlatinib. A fifth patient with ALK p.F1174L initially had a partial response but relapsed after 10 months of treatment. In all cases, ctDNA was detected at the start of lorlatinib single agent treatment and declined gradually, correlating with clinical responses. In the two patients exhibiting relapse, ctDNA was increased nine and three months before clinical detection of disease progression, respectively. In one patient harboring HRAS p.Q61L in the relapsed tumor, retrospective ctDNA analysis showed that the mutation appeared de novo after eight months of lorlatinib treatment. We conclude that some patients with relapsed or refractory high-risk neuroblastoma show durable responses to lorlatinib as monotherapy, and targeted ctDNA analysis is effective for evaluation of treatment and early detection of relapse in ALK-driven neuroblastoma.
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Neuroblastoma (NB) is a heterogeneous childhood cancer with a slightly higher incidence in boys than girls, with the reason for this gender disparity unknown. Given the growing evidence for the involvement of loss of the Y chromosome (LoY) in male diseases including cancer, we investigated Y chromosome status in NB. Male NB tumor samples from a Swedish cohort, analyzed using Cytoscan HD SNP-microarray, were selected. Seventy NB tumors were analyzed for aneuploidy of the Y chromosome, and these data were correlated with other genetic, biological, and clinical parameters. LoY was found in 21% of the male NB tumors and it was almost exclusively found in those with high-risk genomic profiles. Furthermore, LoY was associated with increased age at diagnosis and enriched in tumors with 11q-deletion and activated telomere maintenance mechanisms. In contrast, tumors with an MYCN-amplified genomic profile retained their Y chromosome. The understanding of LoY in cancer is limited, making it difficult to conclude whether LoY is a driving event in NB or function of increased genomic instability. Gene expression analysis of Y chromosome genes in male NB tumors showed low expression of certain genes correlating with worse overall survival. KDM5D, encoding a histone demethylase stands out as an interesting candidate for further studies. LoY has been shown to impact the epigenomic layer of autosomal loci in nonreproductive tissues, and KDM5D has been reported as downregulated and/or associated with poor survival in different malignancies. Further studies are needed to explore the mechanisms and functional consequences of LoY in NB.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Cromosomas Humanos Y , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patología , Masculino , Cromosomas Humanos Y/genética , Cromosomas Humanos Par 11/genética , Lactante , Preescolar , Femenino , Homeostasis del Telómero/genética , Niño , Histona Demetilasas/genética , Telómero/genética , Proteína Proto-Oncogénica N-Myc/genética , Suecia/epidemiologíaRESUMEN
Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.
Asunto(s)
Metiltransferasas , Neuroblastoma , ARN Largo no Codificante , Humanos , Adenina/análogos & derivados , Metiltransferasas/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estructuras R-Loop , ARN Largo no Codificante/metabolismo , Telómero/genética , Homeostasis del TelómeroRESUMEN
Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.
RESUMEN
Neuroblastoma (NB) is a childhood malignancy of the sympathetic nervous system. NB is mainly driven by copy number alterations, such as MYCN amplification, large deletions of chromosome arm 11q and gain of chromosome arm 17q, which are all markers of highrisk disease. Genes targeted by recurrent, smaller, focal alterations include CDKN2A/B, TERT, PTPRD and ATRX. Our previous study on relapsed NB detected recurrent structural alterations centered at limbic systemassociated membrane protein (LSAMP; HUGO Gene Nomenclature Committee: 6705; chromosomal location 3q13.31), which is a gene frequently reported to be deleted or downregulated in other types of cancer. Notably, in cancer, LSAMP has been shown to have tumorsuppressing functions. The present study performed an expanded investigation using whole genome sequencing of tumors from 35 patients, mainly with highrisk NB. Focal duplications or deletions targeting LSAMP were detected in six cases (17%), whereas single nucleotide polymorphismmicroarray analysis of 16 NB cell lines detected segmental alterations at 3q13.31 in seven out of the 16 NB cell lines (44%). Furthermore, low expression of LSAMP in NB tumors was significantly associated with poor overall and eventfree survival. In vitro, knockdown of LSAMP in NB cell lines increased cell proliferation, whereas overexpression decreased proliferation and viability. These findings supported a tumor suppressor role for LSAMP in NB. However, the higher incidence of LSAMP aberrations in cell lines and in relapsed NB tumors suggested that these alterations were a late event predominantly in advanced NB with a poor prognosis, indicating a role of LSAMP in tumor progression rather than in tumor initiation. In conclusion, the present study demonstrated recurrent genomic aberrations of chromosomal region 3q13.31 that targeted the LSAMP gene, which encodes a membrane protein involved in cell adhesion, central nervous system development and neurite outgrowth. The frequent aberrations affecting LSAMP, together with functional evidence, suggested an antiproliferative role of LSAMP in NB.
Asunto(s)
Genes Supresores de Tumor , Neuroblastoma , Niño , Humanos , Línea Celular Tumoral , Aberraciones Cromosómicas , Neuroblastoma/genéticaRESUMEN
Vesicoureteral reflux (VUR) is a common urological problem in children and its hereditary nature is well recognised. However, despite decades of research, the aetiological factors are poorly understood and the genetic background has been elucidated in only a minority of cases. To explore the molecular aetiology of primary hereditary VUR, we performed whole-exome sequencing in 13 large families with at least three affected cases. A large proportion of our study cohort had congenital renal hypodysplasia in addition to VUR. This high-throughput screening revealed 23 deleterious heterozygous variants in 19 candidate genes associated with VUR or nephrogenesis. Sanger sequencing and segregation analysis in the entire families confirmed the following findings in three genes in three families: frameshift LAMC1 variant and missense variants of KIF26B and LIFR genes. Rare variants were also found in SALL1, ROBO2 and UPK3A. These gene variants were present in individual cases but did not segregate with disease in families. In all, we demonstrate a likely causal gene variant in 23% of the families. Whole-exome sequencing technology in combination with a segregation study of the whole family is a useful tool when it comes to understanding pathogenesis and improving molecular diagnostics of this highly heterogeneous malformation.
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Cinesinas , Laminina , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Reflujo Vesicoureteral , Humanos , Heterocigoto , Cinesinas/genética , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/genética , Mutación , Linaje , Reflujo Vesicoureteral/genética , Secuenciación del Exoma , Laminina/genéticaRESUMEN
A preterm infant with central hypoventilation was diagnosed with multifocal neuroblastoma. Congenital anomalies of the autonomic nervous system in association with neuroblastoma are commonly associated with germline mutations in PHOX2B. Further, the ALK gene is frequently mutated in both familial and sporadic neuroblastoma. Sanger sequencing of ALK and PHOX2B, SNP microarray of three tumor samples and whole genome sequencing of tumor and blood were performed. Genetic testing revealed a germline ALK F1174I mutation that was present in all tumor samples as well as in normal tissue samples from the patient. Neither of the patient's parents presented the ALK variant. Array profiling of the three tumor samples showed that two of them had only numerical aberrations, whereas one sample displayed segmental alterations, including a gain at chromosome 2p, resulting in two copies of the ALK-mutated allele. Whole genome sequencing confirmed the presence of the ALK variant and did not detect any aberrations in the coding or promotor region of PHOX2B. This study is to our knowledge the first to report a de novoALK F1174I germline mutation. This may not only predispose to congenital multifocal neuroblastoma but may also contribute to the respiratory dysfunction seen in this patient.
RESUMEN
In neuroblastoma, MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53wild-type cells. Combined CDK4/MDM2 targeting had an additive effect in p53wild-type cell lines, while no or negative additive effect was observed in p53mutated cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms' tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial.
Asunto(s)
Neuroblastoma , Proteínas Proto-Oncogénicas c-mdm2 , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Amplificación de Genes , Humanos , Neuroblastoma/patología , Pronóstico , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Vesicoureteral reflux (VUR) is a congenital malformation carrying a high risk of recurrent urinary tract infections (UTI) and, at worst, chronic renal failure. Familial clustering implies a genetic etiology, but studies during the past few decades have demonstrated a causal gene variant in <10% of patients with VUR. The aim of the present study was to search for fully or partially shared ancestral haplotypes in 14 families from south-western Sweden with at least three affected members. High-density single nucleotide polymorphism microarray was used for genotyping prior to analysis with a compatibility matching method developed in-house, and the analysis of copy number variations (CNV). No single unique haplotype was revealed to be shared by the families, thereby excluding a common ancestry and founder mutations as a probable cause of VUR. After evaluation of haplotypes shared by subsets of families, a haplotype shared by nine families was found to be of particular interest. This haplotype, located at chromosomal region 4q21.21, harbours two tentative candidate genes (bone morphogenetic protein 3 and fibroblast growth factor 5), both expressed in metanephros and with known functions during nephrogenesis. As to CNV, only one family had a specific CNV shared by all affected members. This was a focal deletion at 5q31.1 including follistatin-like 4, a gene without a previous known connection to VUR. These data demonstrated the genetic heterogeneity of VUR and indicated that an interaction of environmental and genetic factors, including non-coding and epigenetic regulators, all contribute to the complexity of VUR.
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Benzamidas/uso terapéutico , Indazoles/uso terapéutico , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasa de Linfoma Anaplásico/fisiología , Cromosomas Humanos Par 2/genética , Citocinas/genética , Variación Genética , Células Germinativas , Humanos , Lactante , Masculino , Neuroblastoma/genética , Neuroblastoma/secundario , Receptor trkA/fisiologíaRESUMEN
High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature" that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, highlighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Diferenciación Celular , Deleción Cromosómica , Cromosomas Humanos Par 11/genética , Guanilato-Quinasas/genética , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Supresoras de Tumor/genética , Adrenérgicos/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Células Cromafines/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Guanilato-Quinasas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos BALB C , Factor de Crecimiento Nervioso/farmacología , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Pronóstico , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Células de Schwann/patología , Factor de Transcripción Sp1/metabolismo , Transcripción Genética/efectos de los fármacos , Resultado del Tratamiento , Tretinoina/farmacología , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Gain of chromosome arm 2p is a previously described entity in neuroblastoma (NB). This genomic address is home to two important oncogenes in NB-MYCN and anaplastic lymphoma kinase (ALK). MYCN amplification is a critical prognostic factor coupled with poor prognosis in NB. Mutation of the ALK receptor tyrosine kinase has been described in both somatic and familial NB. Here, ALK activation occurs in the context of the full-length receptor, exemplified by activating point mutations in NB. ALK overexpression and activation, in the absence of genetic mutation has also been described in NB. In addition, the recently identified ALK ligand ALKAL2 (previously described as FAM150B and AUGα) is also found on the distal portion of 2p, at 2p25. Here we analyze 356 NB tumor samples and discuss observations indicating that gain of 2p has implications for the development of NB. Finally, we put forward the hypothesis that the effect of 2p gain may result from a combination of MYCN, ALK, and the ALK ligand ALKAL2.
RESUMEN
The ALK tyrosine kinase receptor is oncogenically activated in neuroblastoma. Whereas numerous ALK fusion genes have been reported in different malignancies, in neuroblastoma ALK is mainly activated through point mutations. Three hotspot residues (F1174, F1245, and R1275) account for 85% of mutant ALK seen in neuroblastoma. In a cohort of 105 Swedish neuroblastoma cases of all stages, these hotspot regions were re-sequenced (>5000X). ALK mutations were detected in 16 of 105 patients (range of variant allele fraction: 2.7-60%). Mutations at the F1174 and F1245 hotspot were observed in eleven and three cases respectively. ALK mutations were also detected at the I1171 and L1240 codons in one tumor each. No mutations were detected at R1275. Sanger sequencing could confirm ALK status for all mutated samples with variant allele fraction above 15%. Four of the samples with subclonal ALK mutation fraction below this would have gone undetected relying on Sanger sequencing only. No distinct mutation spectrum in relation to neuroblastoma tumours genomic subtypes could be detected although there was a paucity of ALK mutations among 11q-deleted tumors. As ALK mutations status opens up an excellent opportunity for application of small molecule inhibitors targeting ALK, early and sensitive detection of ALK alterations is clinically important considering its potential role in tumour progression.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Biomarcadores de Tumor , Mutación , Neuroblastoma/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Niño , Exones , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Tasa de Mutación , Neuroblastoma/diagnóstico , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/mortalidad , Polimorfismo de Nucleótido Simple , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto JovenRESUMEN
Activation of the RAS-RAF-MEK-ERK signaling pathway is implicated in driving the initiation and progression of multiple cancers. Several inhibitors targeting the RAS-MAPK pathway are clinically approved as single- or polyagent therapies for patients with specific types of cancer. One example is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK-positive, EGFR-activated, or KRAS-mutant lung cancers and neuroblastomas that also contain activating mutations in the RAS-MAPK pathway. In addition, in neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations in ALK, NRAS, and NF1, leading to increased activation of RAS-MAPK signaling. Co-targeting ALK and the RAS-MAPK pathway is an attractive option, because monotherapies have not yet produced effective results in ALK-addicted neuroblastoma patients. We evaluated the response of neuroblastoma cell lines to MEK-ERK pathway inhibition by trametinib. In contrast to RAS-MAPK pathway-mutated neuroblastoma cell lines, ALK-addicted neuroblastoma cells treated with trametinib showed increased activation (inferred by phosphorylation) of the kinases AKT and ERK5. This feedback response was mediated by the mammalian target of rapamycin complex 2-associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling. In xenografts in mice, trametinib inhibited the growth of EML4-ALK-positive non-small cell lung cancer and RAS-mutant neuroblastoma but not ALK-addicted neuroblastoma. Thus, our results advise against the seemingly rational option of using MEK inhibitors to treat ALK-addicted neuroblastoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Neuroblastoma/enzimología , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the pediatric cancer neuroblastoma, analysis of recurrent chromosomal aberrations such as loss of chromosome 1p, 11q, gain of 17q and MYCN amplification are used for patient stratification and subsequent therapy decision making. Different analysis techniques have been used for detection of segmental abnormalities, including fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH)-microarrays and multiplex ligation-dependent probe amplification (MLPA). However, as next-generation sequencing becomes available for clinical use, this technique could also be used for assessment of copy number alterations simultaneously with mutational analysis. In this study we compare genomic profiles generated through exome sequencing data with profiles generated from high resolution Affymetrix single nucleotide polymorphism (SNP) microarrays on 30 neuroblastoma tumors of different stages. Normalized coverage reads for tumors were calculated using Control-FREEC software and visualized through a web based Shiny application, prior to comparison with corresponding SNP-microarray data. The two methods show high-level agreement for breakpoints and copy number of larger segmental aberrations and numerical aneuploidies. However, several smaller gene containing deletions that could not readily be detected through the SNP-microarray analyses were identified through exome profiling, most likely due to difference between spatial distribution of microarray probes and targeted regions of the exome capture. These smaller aberrations included focal ATRX deletion in two tumors and three cases of novel deletions in chromosomal region 19q13.2 causing homozygous loss of multiple genes including the CIC (Capicua) gene. In conclusion, genomic profiles generated from normalized coverage of exome sequencing show concordance with SNP microarray generated genomic profiles. Exome sequencing is therefore a useful diagnostic tool for copy number variant (CNV) detection in neuroblastoma tumors, especially considering the combination with mutational screening. This enables detection of theranostic targets such as ALK and ATRX together with detection of significant segmental aneuploidies, such as 2p-gain, 17q-gain, 11q-deletion as well as MYCN amplification.
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Cromosomas Humanos Par 19/genética , Eliminación de Gen , Dosificación de Gen , Neuroblastoma/genética , Polimorfismo de Nucleótido Simple , Proteínas Represoras/genética , Línea Celular Tumoral , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Exoma , Amplificación de Genes , Homocigoto , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADN , SueciaRESUMEN
Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.
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Reordenamiento Génico , Neuroblastoma/genética , Proteínas Tirosina Quinasas Receptoras/genética , Translocación Genética , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Puntos de Rotura del Cromosoma , Exones , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neuroblastoma/metabolismo , Células PC12 , Polimorfismo de Nucleótido Simple , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
PURPOSE: In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. METHODS: We analyzed ALK mutations in 54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. RESULTS: All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). CONCLUSION: In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.
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Neuroblastoma/enzimología , Neuroblastoma/genética , Mutación Puntual , Proteínas Tirosina Quinasas Receptoras/genética , Adolescente , Adulto , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico , Preescolar , Activación Enzimática , Exones , Humanos , Lactante , Persona de Mediana Edad , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/genética , Adulto JovenRESUMEN
BACKGROUND: Chromosomal instability is a hallmark of human cancer caused by errors in mitotic control and chromosome segregation. STAG2 encodes a subunit of the cohesion complex that participates in mitotic chromatid separation and was recently found to show low expression and inactivating mutations in Ewing's sarcoma, melanoma and glioblastoma.In the childhood tumor neuroblastoma (NB) segmental chromosomal alterations are associated with poor prognosis whereas tumors displaying whole chromosome gains and losses have a much better prognosis. METHOD: As the genetic contribution to aneuploidy is unknown in NB, we investigated the presence of STAG2 mutations through sequence analysis of all 33 coding exons in 37 primary NB tumors. RESULTS AND CONCLUSION: As no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.
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Aneuploidia , Antígenos Nucleares/genética , Neoplasias del Sistema Nervioso/genética , Neuroblastoma/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Exones , Humanos , Neoplasias del Sistema Nervioso/patología , Neuroblastoma/patología , Mutación Puntual , Análisis de Secuencia de ADNRESUMEN
BACKGROUND/AIM: Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients' prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features. METHODS: We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques. RESULTS AND CONCLUSION: This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11/genética , Amplificación de Genes , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Niño , Preescolar , Puntos de Rotura del Cromosoma , Femenino , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Lactante , Masculino , Proteína Proto-Oncogénica N-Myc , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma. RESULTS: In the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material. IN CONCLUSION: This study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.