RESUMO
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.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Y , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Masculino , Cromossomos Humanos Y/genética , Cromossomos Humanos Par 11/genética , Lactente , Pré-Escolar , Feminino , Homeostase do Telômero/genética , Criança , Histona Desmetilases/genética , Telômero/genética , Proteína Proto-Oncogênica N-Myc/genética , Suécia/epidemiologiaRESUMO
Neuroblastoma (NB) is a remarkably heterogenic childhood tumor of the sympathetic nervous system with clinical behavior ranging from spontaneous regression to poorly differentiated tumors and metastasis. MYCN is amplified in 20% of cases and correlates with an undifferentiated, aggressive phenotype and poor prognosis. Estrogen receptor alpha (ERα) and the nerve growth factor (NGF) receptors TrkA and p75NTR are involved in neuronal differentiation and survival. We have previously shown that MYCN, via miR-18a, targets ERα in NB cells. Here, we demonstrate that interference with miR-18a or overexpression of ERα is sufficient to induce NGF signaling and to modulate both basal and NGF-induced neuronal differentiation in MYCN-amplified NB cells. Proteomic analysis confirmed an increase of neuronal features and showed that processes linked to tumor initiation and progression were inhibited upon ERα overexpression. Indeed, ectopic ERα expression was sufficient to inhibit metabolic activity and tumorigenic processes, including glycolysis, oxidative phosphorylation, cell viability, migration, and anchorage independent growth. Importantly, ERα overexpression reduced tumor burden in NB mouse models and high ERα levels were linked to improved survival in patients. In addition to ERα, several other nuclear hormone receptors (NHRs), including the glucocorticoid and the retinoic acid receptors, correlated with clinical markers for favorable and low-stage NB disease. Our data suggest that MYCN targets ERα and thereby NGF signaling to maintain an undifferentiated and aggressive phenotype. Notably, we identified the estrogen-NGF crosstalk, as well as a set of other NHRs, as potential prognostic markers and targets for therapeutic strategies against NB.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Proto-Oncogênica N-Myc/genética , Fator de Crescimento Neural/farmacologia , Neuroblastoma/patologia , Animais , Diferenciação Celular , Receptor alfa de Estrogênio/genética , Amplificação de Genes , Humanos , Camundongos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fenótipo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event-free survival (EFS). METHODS: Bone marrow aspirates and peripheral blood samples from 97 infants/toddlers enrolled in the European High-Risk Neuroblastoma trial were collected at diagnosis in PAXgene™ blood RNA tubes. Samples were analyzed by reverse transcription quantitative polymerase chain reaction according to standardized procedures. RESULTS: Bone marrow tyrosine hydroxylase (TH) or paired-like homeobox 2b (PHOX2B) levels in the highest tertile were associated with worse EFS; hazard ratios, adjusted for age and MYCN status, were 1.5 and 1.8 respectively. Expression of both TH and PHOX2B in the highest tertile predicted worse outcome (p = 0.015), and identified 20 (23%) infants/toddlers with 5-year EFS of 20% (95%CI: 4%-44%). Prognostic significance was maintained after adjusting for over-fitting bias (p = 0.038), age and MYCN status. In peripheral blood, PHOX2B levels in the highest tertile predicted a two-fold increased risk of an event (p = 0.032), and identified 23 (34%) infants/toddlers with 5-year EFS of 29% (95%CI: 12%-48%). Time-dependent receiver operating characteristic analysis confirmed the prognostic value of combined TH and PHOX2B in bone marrow and of PHOX2B in peripheral blood during the first year of follow-up. CONCLUSIONS: High levels of bone marrow TH and PHOX2B and of peripheral blood PHOX2B at diagnosis allow early identification of a group of high-risk infant and toddlers with neuroblastoma who may be candidates for alternative treatments. Integration with additional biomarkers, as well as validation in additional international trials is warranted.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Homeodomínio/análise , Neuroblastoma/mortalidade , Fatores de Transcrição/análise , Tirosina 3-Mono-Oxigenase/análise , Área Sob a Curva , Feminino , Proteínas de Homeodomínio/biossíntese , Humanos , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Neuroblastoma/metabolismo , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , RNA Mensageiro/análise , Curva ROC , Sensibilidade e Especificidade , Fatores de Transcrição/biossíntese , Tirosina 3-Mono-Oxigenase/biossínteseRESUMO
BACKGROUND: Aggressive neuroblastoma remains a significant cause of childhood cancer death despite current intensive multimodal treatment protocols. The purpose of the present work was to characterize the genetic and clinical diversity of such tumors by high resolution arrayCGH profiling. METHODS: Based on a 32K BAC whole-genome tiling path array and using 50-250K Affymetrix SNP array platforms for verification, DNA copy number profiles were generated for 34 consecutive high-risk or lethal outcome neuroblastomas. In addition, age and MYCN amplification (MNA) status were retrieved for 112 unfavorable neuroblastomas of the Swedish Childhood Cancer Registry, representing a 25-year neuroblastoma cohort of Sweden, here used for validation of the findings. Statistical tests used were: Fisher's exact test, Bayes moderated t-test, independent samples t-test, and correlation analysis. RESULTS: MNA or segmental 11q loss (11q-) was found in 28/34 tumors. With two exceptions, these aberrations were mutually exclusive. Children with MNA tumors were diagnosed at significantly younger ages than those with 11q- tumors (mean: 27.4 vs. 69.5 months; p=0.008; n=14/12), and MNA tumors had significantly fewer segmental chromosomal aberrations (mean: 5.5 vs. 12.0; p<0.001). Furthermore, in the 11q- tumor group a positive correlation was seen between the number of segmental aberrations and the age at diagnosis (Pearson Correlation 0.606; p=0.037). Among nonMNA/non11q- tumors (n=6), one tumor displayed amplicons on 11q and 12q and three others bore evidence of progression from low-risk tumors due to retrospective evidence of disease six years before diagnosis, or due to tumor profiles with high proportions of numerical chromosomal aberrations. An early age at diagnosis of MNA neuroblastomas was verified by registry data, with an average of 29.2 months for 43 cases that were not included in the present study. CONCLUSION: MNA and segmental 11q loss define two major genetic variants of unfavorable neuroblastoma with apparent differences in their pace of tumor evolution and in genomic integrity. Other possible, but less common, routes in the development of aggressive tumors are progression of low-risk infant-type lesions, and gene amplifications other than MYCN. Knowledge on such nosological diversity of aggressive neuroblastoma might influence future strategies for therapy.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Perfilação da Expressão Gênica , Neuroblastoma/genética , Deleção de Sequência , Adolescente , Fatores Etários , Sequência de Bases , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Amplificação de Genes , Humanos , Lactente , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/genética , Sistema de Registros , SuéciaRESUMO
Analysis of chromosomal aberrations is used to determine the prognosis of neuroblastomas (NBs) and to aid treatment decisions. MYCN amplification (MNA) alone is an incomplete poor prognostic factor, and chromosome 11q status has recently been included in risk classification. We analyzed 165 NB tumors using high-density SNP microarrays and specifically compared the high-risk groups defined by MNA (n = 37) and 11q-deletion (n = 21). Median patient age at diagnosis was 21 months for MNA tumors and 42 months for 11q-deletion tumors, and median survival time after diagnosis was 16 months for MNA and 40 months for 11q deletion. Overall survival (at 8 years) was approximately 35% in both groups. MNA and 11q deletion were almost mutually exclusive; only one case harbored both aberrations. The numbers of segmental aberrations differed significantly; the MNA group had a median of four aberrations, whereas the 11q-deletion group had 12. The high frequency of chromosomal breaks in the 11q-deletion group is suggestive of a chromosomal instability phenotype gene located in 11q; one such gene, H2AFX, is located in 11q23.3 (within the 11q-deletion region). Furthermore, in the groups with segmental aberrations without MNA or 11q deletion, the tumors with 17q gain have worse prognosis than those with segmental aberrations without 17q gain, which have a favorable outcome. This study has implications for therapy in different risk groups and stresses that genome-wide microarray analyses should be included in clinical management to fully evaluate risk, aid diagnosis, and guide treatment.
Assuntos
Instabilidade Cromossômica , Cromossomos Humanos Par 11 , Neuroblastoma/genética , Histonas/genética , Humanos , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The incidence rates of childhood central nervous system (CNS) tumors in the Nordic countries remain among the highest in the world. Large geographical and temporal variations in the incidence rates of CNS tumors have been reported. Increasing incidence rates would be a public health concern, as they might indicate increased exposure to environmental risk factors. METHODS: All 3,983 children 0-14 years of age registered with a primary CNS tumor in 1985-2006 in the national cancer registries of the Denmark, Finland, Norway, and Sweden were identified. Tumors were classified according to the International Classification of Childhood Cancer version 3 (ICCC-3). Join-point analysis was used to detect changes in trends and to estimate annual changes in incidence rates. RESULTS: The mean annual incidence rate of CNS tumors was 42 per million. No statistically significant change in time trends of incidence rates was observed during 1985-2006. Furthermore, the incidence by birth cohort was relatively stable during the study period. CONCLUSION: The incidence rates of childhood CNS tumors in the Nordic countries remain among the highest in the world. The stable incidence rates during the last 22 years indicate that major changes in environmental risk factors are unlikely.
Assuntos
Neoplasias do Sistema Nervoso Central/epidemiologia , Adolescente , Neoplasias do Sistema Nervoso Central/etiologia , Criança , Pré-Escolar , Meio Ambiente , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Modelos Estatísticos , Fatores de Risco , Países Escandinavos e Nórdicos/epidemiologia , Topografia MédicaRESUMO
Several transcripts have been claimed to be clinically valuable for detecting minimal disease in neuroblastoma, but they have not been prospectively compared in a standardized manner. Tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and GD2 synthase (GD2S) mRNAs were analyzed in 554 blood (PB) and bone marrow (BM) samples from 58 children with neuroblastoma. Samples from 44 children with other diseases served as controls. High transcript concentrations of TH, GD2S or DDC in PB or BM at diagnosis were associated with poor prognosis. TH in BM above median indicated worse outcome for a homogenous cohort with high-risk neuroblastoma (survival probability 91% for TH below median versus 33% for TH above median, p = 0.009). The number of children with localized neuroblastoma with increased results in PB did not differ between the three transcripts. In these children, all without morphologically detectable neuroblastoma in BM, the number of patients with elevated GD2S in BM at diagnosis was significantly higher than for the other transcripts (10/16 elevated, p = 0.012). GD2S was elevated in PB from 10/28 controls without neuroblastoma compared to 1/28 for TH and DDC (p < 0.001). In BM from these children GD2S was significantly elevated. We conclude that high expression of TH and DDC both in PB and BM corresponds to metastatic neuroblastoma at diagnosis, residual disease, and poor outcome. Children with high-risk neuroblastoma and low levels of TH in BM at diagnosis may be cured by current therapy. GD2S is less specific than TH and DDC mRNA for neuroblastoma detection in PB and BM.
Assuntos
Biomarcadores Tumorais/genética , Medula Óssea/enzimologia , Dopa Descarboxilase/genética , N-Acetilgalactosaminiltransferases/genética , Neuroblastoma/enzimologia , Tirosina 3-Mono-Oxigenase/genética , Adolescente , Estudos de Casos e Controles , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Estadiamento de Neoplasias , Neuroblastoma/genética , Neuroblastoma/patologia , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Fatores de Risco , Sensibilidade e Especificidade , Suécia , Resultado do TratamentoRESUMO
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.
Assuntos
Neuroblastoma/enzimologia , Neuroblastoma/genética , Mutação Puntual , Receptores Proteína Tirosina Quinases/genética , Adolescente , Adulto , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Pré-Escolar , Ativação Enzimática , Éxons , Humanos , Lactente , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/genética , Adulto JovemRESUMO
BACKGROUND: The peak incidence of central nervous system (CNS) tumors in childhood indicates that intrauterine or neonatal characteristics are potential risk factors or symptoms of early onset of disease. METHODS: We conducted a registry-based case-control study nested in the childhood populations of Denmark, Finland, Sweden, and Norway on the association between indicators of fetal growth and neonatal stress and childhood CNS tumor risk diagnosed during the period 1985-2006. Each of the 3,443 cases was matched individually on date of birth, sex, and country to five controls sampled randomly from population registries. Information on birth characteristics was obtained from national birth registries. We estimated odds ratios (OR) and 95% confidence intervals (95% CI) by conditional logistic regression analyses. RESULTS: We observed a U-shaped relation between risk for CNS tumors and birthweight, at >4.5 kg (OR, 1.27; 95% CI, 1.03-1.55) and <2.0 kg (OR, 1.50; 95% CI, 1.13-1.99), the latter being attenuated after adjustment for gestational age. Moreover, small-for-gestational age (OR, 1.28; 95% CI, 0.98-1.66) and large-for-gestational age (OR, 1.26; 95% CI, 1.02-1.55) were both associated with CNS tumors. The OR for preterm births was increased per 1-week decrease in gestational age (OR, 1.58; 95% CI, 1.04-2.44). Increased ORs were also observed for head circumference >38 cm (1.80; 95% CI, 1.18-2.74), 5-minute Apgar score <7 (1.44; 95% CI, 0.98-2.12), and breech presentation (1.33; 95% CI, 1.04-1.69). The observed associations varied little by histologic subgroup. CONCLUSIONS: This study supports intrauterine or neonatal onset of childhood CNS tumors. The findings provide insight into the natural history of childhood CNS tumors indicating an early onset or, alternatively, potentially harmful exposures in the neonatal period that might be preventable.
Assuntos
Neoplasias do Sistema Nervoso Central/epidemiologia , Neoplasias do Sistema Nervoso Central/etiologia , Adolescente , Peso ao Nascer , Estudos de Casos e Controles , Criança , Pré-Escolar , Dinamarca/epidemiologia , Feminino , Feto , Finlândia/epidemiologia , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Masculino , Noruega/epidemiologia , Gravidez , Nascimento Prematuro , Sistema de Registros , Fatores de Risco , Suécia/epidemiologiaRESUMO
BACKGROUND: Sensitive monitoring of minimal residual disease may improve the treatment of neuroblastoma in children. To detect and monitor neuroblastoma cells in blood and bone marrow, we developed a quantitative method for the analysis of tyrosine hydroxylase mRNA. METHODS: We used real-time reverse transcription-PCR. The calibrator was constructed from a segment of tyrosine hydroxylase mRNA that included the target. Blood and bone marrow samples from 24 children with neuroblastoma and 1 child with ganglioneuroma were analyzed. Controls were blood samples from the cords of 40 babies, from 58 children 6 months to 15 years of age, and from 34 healthy adults, as well as from 12 children with other diseases. RESULTS: The detection limit was approximately 70 transcripts/mL. All 144 blood controls were below this limit. At diagnosis, blood tyrosine hydroxylase mRNA was higher in children with widespread disease (stage 4/4S; n = 6; range, 203-46,000 transcripts/mL) than in patients with localized disease (stages 1-3; n = 6; =83 transcripts/mL; P = 0.002). Bone marrow from all five children with localized disease had concentrations <72 transcripts/mL, whereas five of six stage 4 patients had increased concentrations (6,000-8,000,000 transcripts/mL; P <0.05). In nine children in whom tyrosine hydroxylase mRNA was measured repeatedly, the results corresponded to the clinical course. CONCLUSION: Quantitative analysis of tyrosine hydroxylase mRNA in blood and bone marrow is reliable and easy to perform and may be used for upfront staging, prognostic assessment, and treatment monitoring of neuroblastoma.
Assuntos
Medula Óssea/enzimologia , Neuroblastoma/diagnóstico , RNA Mensageiro/análise , Tirosina 3-Mono-Oxigenase/sangue , Adolescente , Adulto , Medula Óssea/patologia , Criança , Pré-Escolar , Seguimentos , Humanos , Lactente , Neoplasia Residual , Neuroblastoma/enzimologia , Neuroblastoma/terapia , Reação em Cadeia da Polimerase , RNA Mensageiro/sangue , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Pterin-dependent tyrosine hydroxylase has been described to occur occasionally in melanocytes. It is therefore important to quantify the mRNA of this enzyme in pigment cells to understand whether this enzyme can take an active part in pigment formation. A real-time reverse transcription-polymerase chain reaction method was used to quantify tyrosine hydroxylase mRNA in melanocytes and melanoma cells. The calibrator was obtained by amplification of a segment of cDNA from tyrosine hydroxylase mRNA, which included the target thus allowing enumeration of the number of transcripts per cell. In melanocytes (n = 3), tyrosine hydroxylase mRNA ranged from non-detectable to 0.000492 transcripts/cell and in melanoma cells from non-detectable to 0.005340 transcripts/cell. In neuroblastoma cells, the median tyrosine hydroxylase mRNA number was 0.4 transcripts/cell (range 0.02-25 transcripts/cell). The amount of tyrosine hydroxylase mRNA in the pigment cells was far less than the mRNA concentrations of four melanocyte-specific proteins measured in the same melanocytes and melanoma cells. We conclude that on the average less than 1 of 1000 melanocytes and melanoma cells contains at least one tyrosine hydroxylase mRNA molecule. Consequently, in 999 of 1000 cells translation into the corresponding enzyme protein cannot occur because of the lack of an mRNA template. Thus, in these cells there is no pterin-dependent tyrosine hydroxylase that can contribute to pigment formation by producing priming amounts of l-dopa for proper function of tyrosinase.