RESUMO
Autosomal recessive mutations in RAB27A are associated with Griscelli syndrome type 2 (GS2), characterized by hypopigmentation and development of early-onset, potentially fatal hemophagocytic lymphohistiocytosis (HLH). We describe a 35-year old male who presented with recurrent fever, was diagnosed with Epstein-Barr virus-driven chronic lymphoproliferation, fulfilled clinical HLH criteria, and who carried a novel homozygous RAB27A c.551G > A p.(R184Q) variant. We aimed to evaluate the contribution of the identified RAB27A variant in regard to the clinical phenotype as well as cellular and biochemical function. The patient displayed normal pigmentation as well as RAB27A expression in blood-derived cells. However, patient NK and CD8+ T cell exocytosis was low. Ectopic expression of the RAB27A p.R184Q variant rescued melanosome distribution in mouse Rab27a-deficient melanocytes, but failed to increase exocytosis upon reconstitution of human RAB27A-deficient CD8+ T cells. Mechanistically, the RAB27A p.R184Q variant displayed reduced binding to SLP2A but augmented binding to MUNC13-4, two key effector proteins in immune cells. MUNC13-4 binding was particularly strong to an inactive RAB27A p.T23N/p.R184Q double mutant. RAB27A p.R184Q was expressed and could facilitate melanosome trafficking, but did not support lymphocyte exocytosis. The HLH-associated RAB27A variant increased Munc13-4 binding, potentially representing a novel mode of impairing RAB27A function selectively in hematopoietic cells.
Assuntos
Infecções por Vírus Epstein-Barr , Linfo-Histiocitose Hemofagocítica , Adulto , Humanos , Masculino , Linfócitos T CD8-Positivos , Herpesvirus Humano 4 , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/genética , Ligação Proteica , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismoRESUMO
TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.
Assuntos
Inflamação/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Fator de Necrose Tumoral alfa/farmacologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Autoimunidade/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Feminino , Células HEK293 , Homozigoto , Humanos , Quinase I-kappa B/metabolismo , Imunofenotipagem , Inflamação/patologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Mutação com Perda de Função/genética , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptor 3 Toll-Like/metabolismo , Transcriptoma/genética , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologiaRESUMO
Transcription factors specialized to limit the destructive potential of inflammatory immune cells remain ill-defined. We discovered loss-of-function variants in the X-linked ETS transcription factor gene ELF4 in multiple unrelated male patients with early onset mucosal autoinflammation and inflammatory bowel disease (IBD) characteristics, including fevers and ulcers that responded to interleukin-1 (IL-1), tumor necrosis factor or IL-12p40 blockade. Using cells from patients and newly generated mouse models, we uncovered ELF4-mutant macrophages having hyperinflammatory responses to a range of innate stimuli. In mouse macrophages, Elf4 both sustained the expression of anti-inflammatory genes, such as Il1rn, and limited the upregulation of inflammation amplifiers, including S100A8, Lcn2, Trem1 and neutrophil chemoattractants. Blockade of Trem1 reversed inflammation and intestine pathology after in vivo lipopolysaccharide challenge in mice carrying patient-derived variants in Elf4. Thus, ELF4 restrains inflammation and protects against mucosal disease, a discovery with broad translational relevance for human inflammatory disorders such as IBD.
Assuntos
Proteínas de Ligação a DNA/genética , Doenças Hereditárias Autoinflamatórias/genética , Doenças Inflamatórias Intestinais/genética , Macrófagos/imunologia , Fatores de Transcrição/genética , Animais , Calgranulina A/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Doenças Hereditárias Autoinflamatórias/imunologia , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Lipocalina-2/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th17/imunologia , Transcrição Gênica/genética , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismoRESUMO
The Netherlands launched a nationwide implementation study on non-invasive prenatal testing (NIPT) as a first-tier test offered to all pregnant women. This started on April 1, 2017 as the TRIDENT-2 study, licensed by the Dutch Ministry of Health. In the first year, NIPT was performed in 73,239 pregnancies (42% of all pregnancies), 7,239 (4%) chose first-trimester combined testing, and 54% did not participate. The number of trisomies 21 (239, 0.33%), 18 (49, 0.07%), and 13 (55, 0.08%) found in this study is comparable to earlier studies, but the Positive Predictive Values (PPV)-96% for trisomy 21, 98% for trisomy 18, and 53% for trisomy 13-were higher than expected. Findings other than trisomy 21, 18, or 13 were reported on request of the pregnant women; 78% of women chose to have these reported. The number of additional findings was 207 (0.36%); these included other trisomies (101, 0.18%, PPV 6%, many of the remaining 94% of cases are likely confined placental mosaics and possibly clinically significant), structural chromosomal aberrations (95, 0.16%, PPV 32%,) and complex abnormal profiles indicative of maternal malignancies (11, 0.02%, PPV 64%). The implementation of genome-wide NIPT is under debate because the benefits of detecting other fetal chromosomal aberrations must be balanced against the risks of discordant positives, parental anxiety, and a potential increase in (invasive) diagnostic procedures. Our first-year data, including clinical data and laboratory follow-up data, will fuel this debate. Furthermore, we describe how NIPT can successfully be embedded into a national screening program with a single chain for prenatal care including counseling, testing, and follow-up.
Assuntos
Síndrome de Down/diagnóstico , Testes Genéticos/métodos , Genoma Humano , Implementação de Plano de Saúde , Diagnóstico Pré-Natal/métodos , Síndrome da Trissomia do Cromossomo 13/diagnóstico , Síndrome da Trissomía do Cromossomo 18/diagnóstico , Adolescente , Adulto , Aberrações Cromossômicas , Síndrome de Down/epidemiologia , Síndrome de Down/genética , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Gravidez , Primeiro Trimestre da Gravidez , Prognóstico , Síndrome da Trissomia do Cromossomo 13/epidemiologia , Síndrome da Trissomia do Cromossomo 13/genética , Síndrome da Trissomía do Cromossomo 18/epidemiologia , Síndrome da Trissomía do Cromossomo 18/genética , Adulto JovemRESUMO
The SETD2-related overgrowth syndrome is also called "Luscan-Lumish syndrome" (OMIM 616831) with the clinical characteristics of intellectual disability, speech delay, macrocephaly, facial dysmorphism, and autism spectrum disorders. We report on two novel patients a 4.5-year-old boy and a 23-year-old female adolescent with a speech and language developmental delay, autism spectrum disorder and macrocephaly, who were both diagnosed with SETD2-related overgrowth syndrome due to de novo frameshift mutations in the SETD2 gene. Features not previously described which were present in either one of our patients were nasal polyps, a large tongue with creases, a high pain threshold, constipation, and undescended testicles. These features may be related to the syndrome and may need special attention in future patients. Additionally, prevention of obesity should be an important point of attention for patients diagnosed with a SETD2-related overgrowth syndrome.
Assuntos
Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Histona-Lisina N-Metiltransferase/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Megalencefalia/diagnóstico , Megalencefalia/genética , Fenótipo , Pré-Escolar , Variações do Número de Cópias de DNA , Fácies , Feminino , Mutação da Fase de Leitura , Estudos de Associação Genética , Heterozigoto , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Síndrome , Adulto JovemRESUMO
BACKGROUND: The Wnt/ß-catenin pathway plays a central role in the pathogenesis of most hepatoblastomas (HBs), that is, up to 60-80% carry activating CTNNB1 mutations. HBs can however also be the first manifestation of familial adenomatous polyposis (FAP). As this is a severe disease, it is important for the patient and related family members to firmly exclude FAP at an early stage. Current diagnosis largely depends on APC germline mutation detection on genomic DNA, which is associated with 10-20% false-negative results. Here, we establish and validate a tissue-based ß-catenin gene and immunohistochemical analysis, which complements germline mutation screening to exclude the diagnosis of FAP among HB patients. METHODS: Tumor tissues of 18 HB patients, including three FAP cases were subjected to CTNNB1 exon 3 mutational analysis and immunohistochemistry comparing staining patterns for total and exon 3 specific ß-catenin antibodies. RESULTS: Our novel tissue-based method reliably identified all three FAP patients. Their tumors were characterized by a wild-type exon 3 sequence and a comparable nuclear staining for both antibodies. In contrast, the non-FAP tumors carried missense CTNNB1 mutations combined with a clearly reduced staining for the exon 3 antibody, or complete loss of staining in case of lesions with exon 3 deletions. CONCLUSION: We have successfully established and validated a novel ß-catenin gene and immunohistochemical diagnostic method, which, when combined with routine germline DNA testing, allows the exclusion of the diagnosis of FAP among HB patients.
Assuntos
Polipose Adenomatosa do Colo/diagnóstico , Mutação em Linhagem Germinativa , Hepatoblastoma/diagnóstico , Neoplasias Hepáticas/diagnóstico , Mutação de Sentido Incorreto , beta Catenina/genética , beta Catenina/metabolismo , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Diagnóstico Diferencial , Feminino , Seguimentos , Hepatoblastoma/complicações , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , PrognósticoRESUMO
BACKGROUND: Recently a novel syndromic form of overgrowth with intellectual disability and distinct facial features was identified caused by constitutional mutations in the epigenetic regulator DNA-methyltransferase 3A (DNMT3A), referred to as Tatton-Brown-Rahman syndrome (TBRS). Somatically acquired mutations in DNMT3A occur in haematological malignancies and are frequently present in acute myeloid leukaemia (AML) affecting in more than 50% the arginine residue at position 882 (R882). To date, additional cases with TBRS have been published but so far none of the reported cases with TBRS developed AML. METHODS AND RESULTS: Here we present the first case of TBRS who developed AML at the age of 15 years. Whole-exome sequencing identified a constitutional heterozygous DNMT3A R882C mutation. Our case exhibits macrocephaly, intellectual disability, distinct facial dysmorphism and other recurrent features fitting with the TBRS phenotype. The AML of the myelomonocytic subtype harboured only few additional somatically acquired mutations, that is, an aberrant karyotype and a recurrent PTPN11 mutation. DISCUSSION: The peculiarity of the specific R882 mutation in contrast to other DNMT3A mutations is discussed, including the hypothesis of the more aggressive nature of this variant.Our case represents the first evidence of the possible increased risk of the development of haematological malignancies in particular AML in cases with TBRS.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/genética , Mutação , DNA Metiltransferase 3A , Fácies , Genótipo , Humanos , Deficiência Intelectual/diagnóstico , Cariótipo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Síndrome , Sequenciamento do Exoma , Adulto JovemRESUMO
In acute myeloid leukemia (AML), aberrant expression and mutations of transcription factors have been correlated with disease outcome. In the present study, we performed expression and mutation screening of GATA2, which is an essential transcription factor for regulation of myeloid lineage determination, in de novo pediatric AML patients. GATA2 mutations were detected in 5 of 230 patients, representing a frequency of 2.2% overall and 9.8% in cytogenetically normal AML. GATA2 expression analysis demonstrated that in 155 of 237 diagnostic samples (65%), GATA2 expression was higher than in normal BM. In complete remission, normalization of GATA2 expression was observed, whereas GATA2 expression levels stayed high in patients with resistant disease. High GATA2 expression at diagnosis was an independent poor prognostic factor for overall survival (hazard ratio [HR] = 1.7, P = .045), event-free survival (HR = 2.1, P = .002), and disease-free survival (HR = 2.3, P = .004). The prognostic impact of GATA2 was particularly evident in specific AML subgroups. In patients with French-American-British M5 morphology, inv(16), or high WT1 expression, significant differences in survival were observed between patients with high versus normal GATA2 expression. We conclude that high GATA2 expression is a novel poor prognostic marker in pediatric AML, which may contribute to better risk-group stratification and risk-adapted therapy in the future.
Assuntos
Fator de Transcrição GATA2/genética , Expressão Gênica , Leucemia Mieloide Aguda/genética , População Branca , Criança , Pré-Escolar , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Lactente , Cariotipagem , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Masculino , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Recidiva , Fatores de Risco , Análise de Sequência de DNA , Análise de Sobrevida , Resultado do Tratamento , Proteínas WT1/genéticaAssuntos
Síndrome de Down/complicações , Dosagem de Genes , Leucemia Mieloide/genética , Doença Aguda , Pré-Escolar , Aberrações Cromossômicas , Feminino , Amplificação de Genes , Deleção de Genes , Predisposição Genética para Doença , Humanos , Lactente , Cariotipagem , Leucemia Megacarioblástica Aguda/etiologia , Leucemia Megacarioblástica Aguda/genética , Leucemia Mieloide/etiologia , Masculino , RNA/genética , Telomerase/genética , Telômero/ultraestruturaRESUMO
Translocations involving nucleoporin 98kD (NUP98) on chromosome 11p15 occur at relatively low frequency in acute myeloid leukemia (AML) but can be missed with routine karyotyping. In this study, high-resolution genome-wide copy number analyses revealed cryptic NUP98/NSD1 translocations in 3 of 92 cytogenetically normal (CN)-AML cases. To determine their exact frequency, we screened > 1000 well-characterized pediatric and adult AML cases using a NUP98/NSD1-specific RT-PCR. Twenty-three cases harbored the NUP98/NSD1 fusion, representing 16.1% of pediatric and 2.3% of adult CN-AML patients. NUP98/NSD1-positive AML cases had significantly higher white blood cell counts (median, 147 × 109/L), more frequent FAB-M4/M5 morphology (in 63%), and more CN-AML (in 78%), FLT3/internal tandem duplication (in 91%) and WT1 mutations (in 45%) than NUP98/NSD1-negative cases. NUP98/NSD1 was mutually exclusive with all recurrent type-II aberrations. Importantly, NUP98/NSD1 was an independent predictor for poor prognosis; 4-year event-free survival was < 10% for both pediatric and adult NUP98/NSD1-positive AML patients. NUP98/NSD1-positive AML showed a characteristic HOX-gene expression pattern, distinct from, for example, MLL-rearranged AML, and the fusion protein was aberrantly localized in nuclear aggregates, providing insight into the leukemogenic pathways of these AMLs. Taken together, NUP98/NSD1 identifies a previously unrecognized group of young AML patients, with distinct characteristics and dismal prognosis, for whom new treatment strategies are urgently needed.
Assuntos
Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas Nucleares/fisiologia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genes Homeobox , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/metabolismo , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prognóstico , Adulto JovemRESUMO
BACKGROUND: Several studies of pediatric acute myeloid leukemia have described the various type-I or type-II aberrations and their relationship with clinical outcome. However, there has been no recent comprehensive overview of these genetic aberrations in one large pediatric acute myeloid leukemia cohort. DESIGN AND METHODS: We studied the different genetic aberrations, their associations and their impact on prognosis in a large pediatric acute myeloid leukemia series (n=506). Karyotypes were studied, and hotspot regions of NPM1, CEPBA, MLL, WT1, FLT3, N-RAS, K-RAS, PTPN11 and KIT were screened for mutations of available samples. The mutational status of all type-I and type-II aberrations was available in 330 and 263 cases, respectively. Survival analysis was performed in a subset (n=385) treated on consecutive acute myeloid leukemia Berlin-Frankfurt-Munster Study Group and Dutch Childhood Oncology Group treatment protocols. RESULTS: Genetic aberrations were associated with specific clinical characteristics, e.g. significantly higher diagnostic white blood cell counts in MLL-rearranged, WT1-mutated and FLT3-ITD-positive acute myeloid leukemia. Furthermore, there was a significant difference in the distribution of these aberrations between children below and above the age of two years. Non-random associations, e.g. KIT mutations with core-binding factor acute myeloid leukemia, and FLT3-ITD with t(15;17)(q22;q21), NPM1- and WT1-mutated acute myeloid leukemia, respectively, were observed. Multivariate analysis revealed a 'favorable karyotype', i.e. t(15;17)(q22;q21), t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22). NPM1 and CEBPA double mutations were independent factors for favorable event-free survival. WT1 mutations combined with FLT3-ITD showed the worst outcome for 5-year overall survival (22±14%) and 5-year event-free survival (20±13%), although it was not an independent factor in multivariate analysis. CONCLUSIONS: Integrative analysis of type-I and type-II aberrations provides an insight into the frequencies, non-random associations and prognostic impact of the various aberrations, reflecting the heterogeneity of pediatric acute myeloid leukemia. These aberrations are likely to guide the stratification of pediatric acute myeloid leukemia and may direct the development of targeted therapies.
Assuntos
Aberrações Cromossômicas , Heterogeneidade Genética , Leucemia Mieloide Aguda/genética , Adolescente , Fatores Etários , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Cariótipo , Leucemia Mieloide Aguda/mortalidade , Masculino , Mutação , Nucleofosmina , Prognóstico , Fatores Sexuais , Análise de SobrevidaRESUMO
BACKGROUND: Pediatric acute myeloid leukemia is a heterogeneous disease characterized by non-random genetic aberrations related to outcome. The genetic subtype is currently detected by different diagnostic procedures which differ in success rate and/or specificity. DESIGN AND METHODS: We examined the potential of gene expression profiles to classify pediatric acute myeloid leukemia. Gene expression microarray data of 237 children with acute myeloid leukemia were collected and a double-loop cross validation approach was used to generate a subtype-predictive gene expression profile in the discovery cohort (n=157) which was then tested for its true predictive value in the independent validation cohort (n=80). The classifier consisted of 75 probe sets, representing the top 15 discriminating probe sets for MLL-rearranged, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q21;q22) and t(7;12)(q36;p13)-positive acute myeloid leukemia. RESULTS: These cytogenetic subtypes represent approximately 40% of cases of pediatric acute myeloid leukemia and were predicted with 92% and 99% accuracy in the discovery and independent validation cohort, respectively. However, for NPM1, CEBPA, MLL(-PTD), FLT3(-ITD), KIT, PTPN11 and N/K-RAS gene expression signatures had limited predictive value. This may be caused by a limited frequency of these mutations and by underlying cytogenetics. This latter is exemplified by the fact that different gene expression signatures were discovered for FLT3-ITD in patients with normal cytogenetics and in those with t(15;17)(q21;q22)-positive acute myeloid leukemia, which pointed to HOXB-upregulation being specific for FLT3-ITD(+) cytogenetically normal acute myeloid leukemia. CONCLUSIONS: In conclusion, gene expression profiling correctly predicted the most prevalent cytogenetic subtypes of pediatric acute myeloid leukemia with high accuracy. In clinical practice, this gene expression signature may replace multiple diagnostic tests for approximately 40% of pediatric acute myeloid leukemia cases whereas only for the remaining cases (predicted as 'acute myeloid leukemia-other') are additional tests indicated. Moreover, the discriminative genes reveal new insights into the biology of acute myeloid leukemia subtypes that warrants follow-up as potential targets for new therapies.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Adulto , Biomarcadores Tumorais/metabolismo , Criança , Análise Citogenética , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide , Nucleofosmina , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Dysfunctioning of CCAAT/enhancer binding protein α (C/EBPα) in acute myeloid leukemia can be caused, amongst others, by mutations in the encoding gene (CEBPA) and by promoter hypermethylation. CEBPA-mutated acute myeloid leukemia is associated with a favorable outcome, but this may be restricted to the case of double mutations in CEBPA in adult acute myeloid leukemia. In pediatric acute myeloid leukemia, data on the impact of these mutations are limited to one series, and data on promoter hypermethylation are lacking. Our objective was to investigate the characteristics, gene expression profiles and prognostic impact of the different CEBPA aberrations in pediatric acute myeloid leukemia. DESIGN AND METHODS: We screened a large pediatric cohort (n=252) for CEBPA single and double mutations by direct sequencing, and for promoter hypermethylation by methylation-specific polymerase chain reaction. Furthermore, we determined the gene-expression profiles (Affymetrix HGU133 plus 2.0 arrays) of this cohort (n=237). RESULTS: Thirty-four mutations were identified in 20 out of the 252 cases (7.9%), including 14 double-mutant and 6 single-mutant cases. CEBPA double mutations conferred a significantly better 5-year overall survival compared with single mutations (79% versus 25%, respectively; P=0.04), and compared with CEBPA wild-type acute myeloid leukemia excluding core-binding factor cases (47%; P=0.07). Multivariate analysis confirmed that the double mutations were an independent favorable prognostic factor for survival (hazard ratio 0.23, P=0.04). The combination of screening for promoter hypermethylation and gene expression profiling identified five patients with silenced CEBPA, of whom four cases relapsed. All cases characteristically expressed T-lymphoid markers. Moreover, unsupervised clustering of gene expression profiles showed a clustering of CEBPA double-mutant and silenced cases, pointing towards a common hallmark of abrogated C/EBPα-functioning in these acute myeloid leukemias. CONCLUSIONS: We showed the independent favorable outcome of patients with CEBPA double-mutant acute myeloid leukemia in a large pediatric series. This molecular marker may, therefore, improve risk-group stratification in pediatric acute myeloid leukemia. For the first time, CEBPA-silenced cases are suggested to confer a poor outcome in pediatric acute myeloid leukemia, indicating that further investigation of this aberration is needed. Furthermore, clustering of gene expression profiles provided insight into the biological similarities and diversities of the different aberrations in CEBPA in pediatric acute myeloid leukemia.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Adolescente , Adulto , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Criança , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Estudos de Associação Genética , Marcadores Genéticos , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Masculino , Proteínas de Neoplasias/metabolismo , Reação em Cadeia da Polimerase , Prognóstico , Regiões Promotoras Genéticas , Medição de Risco , Taxa de SobrevidaRESUMO
Although virtually all pediatric patients with acute myeloid leukemia (AML) achieve a complete remission after initial induction therapy, 30%-40% of patients will encounter a relapse and have a dismal prognosis. To prevent relapses, personalized treatment strategies are currently being developed, which target specific molecular aberrations. To determine relevance of established AML type I/II mutations that may serve as therapeutic targets, we assessed frequencies of these mutations and their persistence during disease progression in a large group (n = 69) of paired diagnosis and relapse pediatric AML specimens. In 26 of 42 patients (61%) harboring mutations at either stage of the disease, mutation status changed between diagnosis and relapse, particularly in FLT3, WT1, and RAS genes. Presence or gain of type I/II mutations at relapse was associated with a shorter time to relapse (TTR), whereas absence or loss correlated with longer TTR. Moreover, an adverse outcome was found for patients with activating mutations at relapse, which was statistically significant for FLT3/ITD and WT1 mutations. These findings suggest that mutational shifts affect disease progression. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based on status of type I/II mutations both at initial diagnosis and during follow-up.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Medicina de Precisão , Adolescente , Sequência de Bases , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Primers do DNA/genética , DNA de Neoplasias/genética , Feminino , Seguimentos , Genes do Tumor de Wilms , Genes ras , Humanos , Lactente , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Masculino , Prognóstico , Recidiva , Fatores de Tempo , Resultado do Tratamento , Tirosina Quinase 3 Semelhante a fms/genéticaAssuntos
Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas WT1/genética , Alelos , Biomarcadores Tumorais/genética , Criança , Feminino , Regulação Leucêmica da Expressão Gênica , Alemanha , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Mutação/genética , Países Baixos , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/análise , Taxa de SobrevidaRESUMO
Mixed-lineage leukaemia (MLL)-partial tandem duplications (PTDs) are found in 3-5% of adult acute myeloid leukaemia (AML), and are associated with poor prognosis. In adult AML, MLL-PTD is only detected in patients with trisomy 11 or internal tandem duplications of FLT3 (FLT3-ITD). To date, studies in paediatric AML are scarce, and reported large differences in the frequency of MLL-PTD, frequently utilising mRNA RT-PCR only to detect MLL-PTDs. We studied the frequency of MLL-PTD in a large cohort of paediatric AML (n=276) and the results from two different methods, i.e. mRNA RT-PCR, and multiplex ligation-dependent probe amplification (MLPA), a method designed to detect copy number differences of specific DNA sequences. In some patients with an MLL-rearrangement, MLL-PTD transcripts were detected, but were not confirmed by DNA-MLPA, indicating that DNA-MLPA can more accurately detect MLL-PTD compared to mRNA RT-PCR. In paediatric AML, MLL-PTD was detected in 7/276 patients (2.5%). One case had a trisomy 11, while the others had normal cytogenetics. Furthermore 4 of the 7 patients revealed a FLT3-ITD, which was significantly higher compared with the other AML cases (p=0.016). In conclusion, using DNA-MLPA as a novel screenings technique in combination with mRNA RT-PCR a low frequency of MLL-PTD in paediatric AML was found. Larger prospective studies are needed to further define the prognostic relevance of MLL-PTD in paediatric AML.
Assuntos
Duplicação Gênica , Testes Genéticos/métodos , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Sequências de Repetição em Tandem/genética , Sequência de Bases , Criança , Feminino , Rearranjo Gênico , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Wilms tumor 1 (WT1) mutations have recently been identified in approximately 10% of adult acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) and are associated with poor outcome. Using array-based comparative genome hybridization in pediatric CN-AML samples, we detected a WT1 deletion in one sample. The other WT1 allele was mutated. This prompted us to further investigate the role of WT1 aberrations in childhood AML. Mutations were found in 35 of 298 (12%) diagnostic pediatric AML samples. In 19 of 35 (54%) samples, more than one WT1 aberration was found: 15 samples had 2 different mutations, 2 had a homozygous mutation, and 2 had a mutation plus a WT1 deletion. WT1 mutations clustered significantly in the CN-AML subgroup (22%; P < .001) and were associated with FLT3/ITD (43 vs 17%; P < .001). WT1 mutations conferred an independent poor prognostic significance (WT1 mutated vs wild-type patients: 5-year probability of overall survival [pOS] 35% vs 66%, P = .002; probability of event-free survival 22% vs 46%, P < .001; and cumulative incidence of relapse or regression 70% vs 44%, P < .001). Patients with both a WT1 mutation and a FLT3/ITD had a dismal prognosis (5-year pOS 21%). WT1 mutations occur at a significant rate in childhood AML and are a novel independent poor prognostic marker.