RESUMO
Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome, such as AML with translocation t(7;12)(q36;p13) leading to an MNX1::ETV6 fusion along with high expression of MNX1. We have identified the transforming event in this AML and possible ways of treatment. Retroviral expression of MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, were prevented by pre-treatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.
Assuntos
Histonas , Leucemia Mieloide Aguda , Criança , Lactente , Humanos , Animais , Camundongos , Metiltransferases , Cromatina , S-Adenosilmetionina , Leucemia Mieloide Aguda/genética , Metilação , Fatores de Transcrição , Proteínas de Homeodomínio/genéticaRESUMO
Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio , Células-Tronco Pluripotentes Induzidas , Leucemia Mieloide Aguda , Células Progenitoras de Megacariócitos e Eritrócitos , Fatores de Transcrição , Diferenciação Celular/genética , Criança , Expressão Gênica/genética , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia Mieloide Aguda/genética , Células Progenitoras de Megacariócitos e Eritrócitos/fisiologia , Megacariócitos/fisiologia , Fatores de Transcrição/genética , Translocação GenéticaRESUMO
Polycythaemia vera (PV) patients have an overall comparatively favourable prognosis, but disease progression is very heterogeneous and life-threatening thrombosis and bleedings are frequent complications in untreated disease. Moreover, transformation to more severe secondary myelofibrosis and acute myeloid leukaemia can occur. The aim of this study was to identify gene mutations that could be used together with clinical data as prognostic markers to guide treatment decisions in PV patients. A well-characterized WHO-defined cohort of PV patients was used. Clinical data and blood values were evaluated and a myeloid sequencing panel was used to screen for additional mutations other than the diagnostic JAK2 V617F and JAK2 exon 12 mutations. In 78% of the PV patients, at least one mutation additional to JAK2 V617F was detected. Additional mutations in genes coding for epigenetic modifiers, like TET2, DNMT3A and ASXL1, were most frequent. When correlated to overall survival, mutations in ASXL1 were significantly associated with inferior survival. In an attempt to obtain prognostic guidance in a larger number of patients, the presence of ASXL1 mutations was combined with age and vascular complications prior to diagnosis. Based on these data we were able to define three risk groups that predicted survival.
Assuntos
Policitemia Vera/mortalidade , Proteínas Repressoras/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Comorbidade , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases , Progressão da Doença , Feminino , Humanos , Janus Quinase 2/genética , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Policitemia Vera/complicações , Policitemia Vera/genética , Proteínas Proto-Oncogênicas/genética , Tromboembolia/epidemiologiaRESUMO
External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.
Assuntos
Janus Quinase 2/genética , Mutação de Sentido Incorreto , Patologia Molecular/normas , Garantia da Qualidade dos Cuidados de Saúde , Reação em Cadeia da Polimerase em Tempo Real/normas , Substituição de Aminoácidos , Feminino , Humanos , MasculinoRESUMO
The t(7;12)(q36;p13) (MNX1/ETV6) is not included in the WHO classification but has been described in up to 30% of acute myeloid leukemia (AML) in children <2 years and associated with a poor prognosis. We present the clinical and cytogenetics characteristics of AML cases with t(7;12)(p36;p13). A literature review identified 35 patients with this translocation, published between 2000 and 2015. Outcome data were available in 22 cases. The NOPHO-AML (Nordic Society for Pediatric Hematology and Oncology) database contained 651 patients with AML from 1993 to 2014 and seven (1.1%) had the translocation. The t(7;12) was only present in patients <2 years of age (median age 6 months) but none was diagnosed as newborn. These patients constituted 4.3% of the patients <2 years of age. There was a strong association with trisomy 19 (literature: 86%, NOPHO: 100%) and +8 (literature: 19%, NOPHO: 14%). Seventeen of 22 patients from the literature with t(7;12) and four of seven patients from the NOPHO database suffered from relapse. The patients with t(7;12) had a 3-year event free survival of 24% (literature) vs. 43% (NOPHO) and a 3-year overall survival of 42% (literature) vs. 100% (NOPHO). None of the NOPHO patients was treated with hematopoietic stem cell transplantation (HSCT) in first complete remission. Relapse was frequent but the salvage rate using HSCT was high. We conclude that t(7;12)(q36;13) is a unique subgroup of childhood AML with presentation before 2 years of age with most cases being associated with +19.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 7 , Leucemia Mieloide Aguda/genética , Translocação Genética , Trissomia , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Masculino , Recidiva , Análise de SobrevidaRESUMO
Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.
Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/etiologia , MicroRNAs/antagonistas & inibidores , Proteína Meis1/farmacologia , Animais , Carcinogênese/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , MicroRNAs/metabolismoRESUMO
BACKGROUND: Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. STUDY DESIGN AND METHODS: Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. RESULTS: No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. CONCLUSION: The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.
Assuntos
Aldeído Desidrogenase/metabolismo , Apoptose , Protocolos Clínicos/normas , Sangue Fetal/citologia , Antígenos CD34/sangue , Armazenamento de Sangue/métodos , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Sangue Fetal/enzimologia , HumanosRESUMO
Next-generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow-up samples. The aim of this study was to identify leukemia-specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence-activated cell sorting. Exome sequencing of these cells identified 240 leukemia-specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow-up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia-specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high-sensitivity quantification of minimal residual disease in a patient-tailored manner.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Neoplasia Residual/diagnóstico , Adolescente , Adulto , Idoso , Biomarcadores Tumorais , Criança , Pré-Escolar , Aberrações Cromossômicas , Exoma , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Mutação , Nucleofosmina , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Adulto JovemRESUMO
BACKGROUND: The stem cell content in cord blood (CB) units is routinely assessed regarding nucleated cells, CD34+ cell count, and number of colony-forming units (CFUs). Efforts are made toward finding better ways of defining stemness of CB units. Side population (SP) phenotype and activity of aldehyde dehydrogenase (ALDH) are functional markers of stemness that can be assayed using flow cytometry. STUDY DESIGN AND METHODS: We have developed a protocol for simultaneous determination of CD34+, SP, and ALDH+ populations in relation to immature white blood cells (CD45dim) in CB. Viable nucleated cells were consecutively stained for SP and ALDH activity and with antibodies against the CD45, CD34, and CD117 antigens. RESULTS: The SP and ALDH+ populations could reliably be measured simultaneously. The median sizes of the SP and the ALDH+ populations were 0.85 and 3.3% of CD45dim cells, respectively. There was no overlap between the SP and ALDH+ populations. Cells that were ALDH+ expressed CD34 and CD117, but SP cells were negative for these markers. The ALDH+ cell content correlated with CD34+ cell content (p < 0.001) and with CFU-granulocyte-macrophage (GM; p = 0.03) but not with total CFUs. SP did not correlate with CD34+, CFU-GM, or total CFU. CONCLUSIONS: We show that simultaneous detection of the CD34, SP, and ALDH+ cells is clearly feasible using only small amounts of CB. In CB, ALDH+, and CD34+ cells are overlapping populations distinctly separated from the SP population. The difference in relation to the capacity for colony growth between ALDH+ and SP underlines that they define different cell populations.
Assuntos
Aldeído Desidrogenase/sangue , Antígenos CD34/sangue , Sangue Fetal/citologia , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/classificação , Células da Side Population/classificação , Benzimidazóis , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Dactinomicina/análogos & derivados , Corantes Fluorescentes , Células-Tronco Hematopoéticas/química , Humanos , Recém-Nascido , Proteínas Proto-Oncogênicas c-kit/sangue , Estudos de Amostragem , Células da Side Population/química , Coloração e Rotulagem/métodosRESUMO
Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.
Assuntos
Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Calreticulina/genética , Éxons , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mutação , Transtornos Mieloproliferativos/metabolismo , Garantia da Qualidade dos Cuidados de Saúde , Receptores de Trombopoetina/genéticaRESUMO
Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi-drug transporter P-glycoprotein, and overall survival (OS) in normal karyotype (NK)-AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK-AML patients (n = 201) were analysed for 1199G>A, 1236C>T, 2677G>T/A and 3435C>T, and correlations to outcome were investigated. FLT3 wild-type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0·017. There was also an inferior outcome in FLT3 wild-type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0·039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236C>T and 2677G>T may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild-type group, which may contribute to future individualizing of treatment strategies.
Assuntos
Alelos , Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Polimorfismo de Nucleotídeo Único , Tirosina Quinase 3 Semelhante a fms/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Fatores de Risco , Taxa de Sobrevida , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Dysfunction of T cells and natural killer (NK) cells has been proposed to determine the course of disease in acute myeloid leukemia (AML), but only limited information is available on the mechanisms of lymphocyte inhibition. We aimed to evaluate to what extent human malignant AML cells use NADPH oxidase-derived reactive oxygen species (ROS) as an immune evasion strategy. We report that a subset of malignant myelomonocytic and monocytic AML cells (French-American-British [FAB] classes M4 and M5, respectively), recovered from blood or BM of untreated AML patients at diagnosis, expressed the NADPH oxidase component gp91(phox). Highly purified FAB M4/M5 AML cells produced large amounts of ROS on activation and triggered poly-[ADP-ribose] polymerase-1-dependent apoptosis in adjacent NK cells, CD4(+) T cells, and CD8(+) T cells. In contrast, immature (FAB class M1) and myeloblastic (FAB class M2) AML cells rarely expressed gp91(phox), did not produce ROS, and did not trigger NK or T-cell apoptosis. Microarray data from 207 AML patients confirmed a greater expression of gp91(phox) mRNA by FAB-M4/M5 AML cells than FAB-M1 cells (P < 10(-11)) or FAB-M2 cells (P < 10(-9)). Our data are suggestive of a novel mechanism by which monocytic AML cells evade cell-mediated immunity.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Apoptose , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Linfócitos T/patologia , Medula Óssea/patologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/enzimologia , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , NADPH Oxidase 2 , Poli(ADP-Ribose) Polimerase-1 , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: In children, T-cell acute lymphoblastic leukemia (T-ALL) has inferior prognosis compared with B-cell precursor ALL. In order to improve survival, individualized treatment strategies and thus risk stratification algorithms are warranted, ideally already at the time of diagnosis. PROCEDURE: We analyzed the frequency and prognostic implication of mutations in NOTCH1 and FBXW7 in 79 cases of Swedish childhood T-ALL treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-1992 and ALL-2000 protocols. In a subgroup of patients, we also investigated the functional relevance of NOTCH1 mutations measured as expression of the HES1, MYB, and MYC genes. RESULTS: Forty-seven of the cases (59%) displayed mutations in NOTCH1 and/or FBXW7. There was no difference in overall (P = 0.14) or event-free survival (EFS) (P = 0.10) in patients with T-ALL with mutation(s) in NOTCH1/FBXW7 compared with patients with T-ALL without mutations in any of these genes. T-ALL carrying NOTCH1 mutations had increased HES1 and MYB mRNA expression (HES1 9.2 ± 1.9 (mean ± SEM), MYB 8.7 ± 0.8 (mean ± SEM)) compared to T-ALL with wild-type NOTCH1 (HES1 1.8 ± 0.7, MYB 5.1 ± 1.2, P = 0.02 and 0.008, respectively). In cases of T-ALL with high HES1 expression, improved overall (P = 0.02) and EFS (P = 0.028) was seen. CONCLUSIONS: Increased NOTCH activity, reflected by increased HES1 expression, is associated with improved outcome in pediatric T-ALL, but its role as a diagnostic tool or a therapeutic target in future clinical treatment protocols remains to be elucidated.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criança , Pré-Escolar , Proteína 7 com Repetições F-Box-WD , Feminino , Genes myb , Proteínas de Homeodomínio/genética , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Prognóstico , Fatores de Transcrição HES-1RESUMO
Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking may be a more widespread consequence of translocations where no oncofusion product was identified, including e.g. t(1;3) or t(4;12) AML.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , NADPH Oxidase 2/fisiologia , Animais , Células da Medula Óssea/metabolismo , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/metabolismo , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , NADPH Oxidase 2/deficiência , Transplante de Neoplasias , Análise de SobrevidaRESUMO
The innate immune system and, in particular, activation of the multi-protein complex known as the inflammasome complex are involved in ischemic injury in myocardial cells. The nucleotide-binding leucine-rich repeat-containing pyrin receptor 3 (NLRP3) inflammasome has been linked to inflammation and NLRP3 is especially important for increased inflammation in atherosclerosis, which may lead to myocardial infarction. Here we investigated how inflammasome molecules are affected in human ischemic heart tissue. Surprisingly the important member of the inflammasome complex, NLRP3, displayed markedly decreased levels in human ischemic heart tissue compared with non ischemic control heart tissue. However, subsequent gene analysis revealed mutations in NLRP3 in human ischemic heart tissues but not in non-ischemic control tissue. Gene polymorphisms in the NLRP3 inflammasome have been shown to be associated with increased IL-1ß and IL-18 production and severe inflammation. The autoinflammatory disorder familial Mediterranean fever (FMF) is associated with decreased expression of the Mediterranean fever gene (MEFV) and increased inflammation. We also observed reduced expression of MEFV in ischemic versus non-ischemic heart tissue. Further analyses showed a mutation in MEFV in human ischemic heart tissue but not in non-ischemic control tissue. Our data show that defects in the inflammasome and associated proteins may be involved in promoting ischemic heart disease.
Assuntos
Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Inflamassomos/genética , Isquemia Miocárdica/genética , Regulação para Baixo , Humanos , Mutação , Miocárdio/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pirina , RNA Mensageiro/biossínteseRESUMO
Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.
Assuntos
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Sequências de Repetição em Tandem/genética , Tirosina Quinase 3 Semelhante a fms/genética , Adolescente , Idade de Início , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Regulação Leucêmica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/epidemiologia , Mutação , Nucleofosmina , Prognóstico , Regulação para Cima/genéticaRESUMO
Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.
Assuntos
RNA Helicases DEAD-box/metabolismo , Leucemia Mieloide Aguda/genética , MicroRNAs , Células Progenitoras Mieloides/metabolismo , Hibridização de Ácido Nucleico/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Ribonuclease III/metabolismo , Transdução de Sinais , Adolescente , Adulto , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/genética , DNA Complementar/análise , DNA Complementar/biossíntese , Genes Reporter , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Luciferases/análise , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Células Progenitoras Mieloides/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Receptores de Superfície Celular/genética , Retroviridae , Ribonuclease III/genética , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/análise , Taxa de Sobrevida , TransfecçãoRESUMO
Between 1992 and 2008, 713 high hyperdiploid acute lymphoblastic leukemias in children aged 1-15 years were diagnosed and treated according to the Nordic Society for Pediatric Hematology and Oncology acute lymphoblastic leukemia 1992/2000 protocols. Twenty (2.8%) harbored t(1;19), t(9;22), der(11q23), or t(12;21). The median age of patients with "classic" high hyperdiploidy was lower than that of patients with translocation-positive high hyperdiploidy (P<0.001). Cases with triple trisomies (+4, +10, +17), comprising 50%, had higher modal numbers than the triple trisomy-negative cases (P<0.0001). The probabilities of event-free survival and overall survival were lower for those with white blood cell counts ≥ 50 × 10(9)/L (P=0.017/P=0.009), ≥ 5% bone marrow blasts at day 29 (P=0.001/0.002), and for high-risk patients (P<0.001/P=0.003), whereas event-free, but not overall, survival, was higher for cases with gains of chromosomes 4 (P<0.0001), 6 (P<0.003), 17 (P=0.010), 18 (P=0.049), and 22 (P=0.040), triple trisomies (P=0.002), and modal numbers >53/55 (P=0.020/0.024). In multivariate analyses, modal number and triple trisomies were significantly associated with superior event-free survival in separate analyses with age and white blood cell counts. When including both modal numbers and triple trisomies, only low white blood cell counts were significantly associated with superior event-free survival (P=0.009). We conclude that high modal chromosome numbers and triple trisomies are highly correlated prognostic factors and that these two parameters identify the same subgroup of patients characterized by a particularly favorable outcome.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Células Precursoras de Linfócitos B/fisiologia , Trissomia/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Vigilância da População/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Estudos Prospectivos , Taxa de Sobrevida/tendências , Resultado do TratamentoRESUMO
De novo acute myeloid leukemia with normal karyotype (NK-AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single-nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and -451C>T rs532545), 5'-nucleotidase (cN-II 7A>G rs10883841), and deoxycytidine kinase (DCK 3'UTR 948T>C rs4643786) in 205 de novo NK-AML patients. In FLT3-internal tandem duplication (ITD)-positive patients, the CDA 79C/C and -451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, P < 0.001 and 5 vs. 23 months, P = 0.015, respectively), and this was most pronounced in FLT3-ITD-positive/NPM1-positive patients. We observed altered in vitro sensitivity to topoisomerase inhibitory drugs, but not to nucleoside analogues, and a decrease in global DNA methylation in cells carrying both CDA variant alleles. A shorter survival was also observed for the cN-II variant allele, but only in FLT3-ITD-negative patients (25 vs. 31 months, P = 0.075). Our results indicate that polymorphisms in genes related to nucleoside analog drug metabolism may serve as prognostic markers in de novo NK-AML.