RESUMO
EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.
Assuntos
Leucemia Mieloide , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genética , Cromatina , Proteína do Locus do Complexo MDS1 e EVI1/genética , Leucemia Mieloide/genética , Proto-OncogenesRESUMO
BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.
Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Feminino , Humanos , Adulto , Proteínas de Fusão bcr-abl/genética , Estudos Retrospectivos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Hibridização in Situ Fluorescente , Translocação Genética , DNA Polimerase Dirigida por RNA/genéticaRESUMO
Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.
Assuntos
Carcinogênese/genética , Ciclina D1/genética , Ciclina D2/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mieloma Múltiplo/genética , Transcriptoma , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de SobrevidaRESUMO
Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Humanos , Células Matadoras Naturais , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , MicroRNAs/genética , Células-Tronco Neoplásicas , Inibidores de Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Microambiente Tumoral/genéticaRESUMO
In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.
Assuntos
Linhagem da Célula/genética , Dano ao DNA/genética , Leucemia/genética , Células Precursoras de Linfócitos B/patologia , Transformação Genética/genética , Animais , Células Cultivadas , Expressão Gênica/genética , Leucemia/patologia , Linfócitos/patologia , Camundongos , Oncogenes/genética , Fenótipo , Transcrição Gênica/genéticaRESUMO
BACKGROUND: Pregnancies affected by non-molar chromosomal abnormality may sometimes demonstrate abnormal chorionic villous morphology that is similar to partial hydatidiform mole. Determination of the underlying aetiology may be difficult in such cases. CASE PRESENTATION: This report describes a case referred to the regional trophoblastic disease unit as a possible hydatidiform mole that demonstrated both villous dysmorphology and abnormal p57(KIP2) expression. Molecular genotyping revealed that while most chromosomes in the villous tissue were diploid and biparental, chromosomes 3, 7 and 8 were trisomic with an additional paternally derived chromosome. In contrast chromosome 11 showed uniparental disomy of paternal origin a situation more usually associated with complete hydatidiform moles. This unusual case highlights that exceptions may occur to the general rules of both histological morphology and immunoprofile, and that these can be resolved by detailed molecular genetic investigations. CONCLUSION: The findings confirm that trisomic pregnancies may demonstrate morphological villous features similar to hydatidiform mole, and that loss of p57(KIP2) expression occurs due to an absence of maternally transcribed genes on chromosome 11 and can therefore be independent of androgenetic complete hydatidiform mole.
Assuntos
Vilosidades Coriônicas/patologia , Mola Hidatiforme/diagnóstico , Complicações na Gravidez/diagnóstico , Trissomia , Dissomia Uniparental , Neoplasias Uterinas/diagnóstico , Adulto , Biópsia , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 8 , Diagnóstico Diferencial , Feminino , Predisposição Genética para Doença , Humanos , Mola Hidatiforme/genética , Mola Hidatiforme/patologia , Hibridização in Situ Fluorescente , Técnicas de Diagnóstico Molecular , Fenótipo , Valor Preditivo dos Testes , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologiaRESUMO
The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativadores de Enzimas/farmacologia , Cloridrato de Fingolimode , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Humanos , Janus Quinase 2/metabolismo , Células K562 , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/enzimologia , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
The phenotype and function of cells enriched in tumor-propagating activity and their relationship to the phenotypic architecture in multiple myeloma (MM) are controversial. Here, in a cohort of 30 patients, we show that MM composes 4 hierarchically organized, clonally related subpopulations, which, although phenotypically distinct, share the same oncogenic chromosomal abnormalities as well as immunoglobulin heavy chain complementarity region 3 area sequence. Assessed in xenograft assays, myeloma-propagating activity is the exclusive property of a population characterized by its ability for bidirectional transition between the dominant CD19(-)CD138(+) plasma cell (PC) and a low frequency CD19(-)CD138(-) subpopulation (termed Pre-PC); in addition, Pre-PCs are more quiescent and unlike PCs, are primarily localized at extramedullary sites. As shown by gene expression profiling, compared with PCs, Pre-PCs are enriched in epigenetic regulators, suggesting that epigenetic plasticity underpins the phenotypic diversification of myeloma-propagating cells. Prospective assessment in paired, pretreatment, and posttreatment bone marrow samples shows that Pre-PCs are up to 300-fold more drug-resistant than PCs. Thus, clinical drug resistance in MM is linked to reversible, bidirectional phenotypic transition of myeloma-propagating cells. These novel biologic insights have important clinical implications in relation to assessment of minimal residual disease and development of alternative therapeutic strategies in MM.
Assuntos
Resistencia a Medicamentos Antineoplásicos/imunologia , Modelos Teóricos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Animais , Separação Celular , Citometria de Fluxo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Transplante HeterólogoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Medula Óssea/patologia , Daunorrubicina/efeitos adversos , Etoposídeo/efeitos adversos , Leucemia Mieloide Aguda/induzido quimicamente , Segunda Neoplasia Primária/induzido quimicamente , Pancitopenia/induzido quimicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cromossomos Humanos Par 6/ultraestrutura , Cromossomos Humanos Par 9/ultraestrutura , Terapia Combinada , Irradiação Craniana , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Eritropoese/efeitos dos fármacos , Etoposídeo/administração & dosagem , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Quimioterapia de Manutenção/efeitos adversos , Mercaptopurina/administração & dosagem , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Pessoa de Meia-Idade , Segunda Neoplasia Primária/diagnóstico , Segunda Neoplasia Primária/genética , Proteínas de Fusão Oncogênica/genética , Pancitopenia/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/radioterapia , Translocação Genética , Vincristina/administração & dosagemAssuntos
Medula Óssea/patologia , Ciclina D1/genética , Linfócitos/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Translocação Genética , Medula Óssea/metabolismo , Tamanho Celular , Ciclina D1/metabolismo , Feminino , Humanos , Linfócitos/metabolismo , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Plasmócitos/metabolismo , Plasmócitos/patologiaAssuntos
Células da Medula Óssea/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Idoso , Transtornos Cromossômicos , Humanos , Masculino , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Trombocitopenia/etiologiaRESUMO
Activation of the EVI-1 oncogene has been reported in acute myeloid leukemia, chronic myeloid leukemia (CML) in blast crisis, and less commonly, in chronic-phase CML patients. We screened an unselected cohort of 75 chronic-phase CML patients who had failed imatinib for expression of EVI-1 and sought a correlation with subsequent outcome on the second-generation tyrosine kinase inhibitors dasatinib (n = 61) or nilotinib (n = 14). The 8 patients (10.7%) who expressed EVI-1 transcripts detectable by real-time polymerase chain reaction had significantly lower event-free survival, progression-free survival, and overall survival than patients with undetectable transcript. The predictive value of EVI-1 expression was validated in an independent cohort. In a multivariate analysis, EVI-1 expression status and the best cytogenetic response obtained on imatinib were the only independent predictors for overall survival, progression-free survival, and event-free survival. Our data suggest that screening for EVI-1 expression at the time of imatinib failure may predict for response to second-line TKI therapy and consequently aid clinical management.
Assuntos
Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide de Fase Crônica/mortalidade , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proto-Oncogenes/genética , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Fatores de Transcrição/genética , Benzamidas , Crise Blástica , Proteínas de Ligação a DNA/metabolismo , Dasatinibe , Feminino , Humanos , Mesilato de Imatinib , Hibridização in Situ Fluorescente , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/genética , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Terapia de Salvação , Taxa de Sobrevida , Fatores de Transcrição/metabolismoRESUMO
AIMS: Asteroid B cells are a component of normal thymus. It is currently unclear whether these cells are identifiable in T cell lymphoblastic leukaemia/lymphoma (T-ALL/LBL) of the thymus. The aim of this study was to identify asteroid B cells both in thymic and extrathymic tissue involved by T-ALL/LBL. METHODS AND RESULTS: Thymic, lymph node (LN) and bone marrow trephine biopsy (BMTB) samples from eight patients with T-ALL/LBL were reviewed. All had been investigated by immunohistochemistry and one by fluorescent in situ hybridization (FISH). The BMTB samples of two of eight T-ALL/LBLs and LN sample in one of them showed the presence of asteroid-shaped B cells with dendritic cytoplasmic processes. These B cells also expressed CD23 and the features were akin to the unique thymic asteroid B cells. Both patients had aggressive/resistant disease. Cytogenetic analysis in one showed a complex translocation involving the T cell receptor beta (TCRB) gene at 7q35 and a distal region of 9q known to harbour the NOTCH1 gene. CONCLUSION: This is the first report of T-ALL/LBL documenting the presence of an asteroid B cell-rich microenvironment at bone marrow and LN sites. In this small subset, T-ALL/LBL cells are possibly dependent upon asteroid B cells, and whether targeting of asteroid B cells with anti-CD20 monoclonal antibody in such cases will result in clinical benefit remains to be determined.
Assuntos
Linfócitos B/patologia , Medula Óssea/patologia , Linfonodos/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Timo/patologia , Linfócitos B/imunologia , Medula Óssea/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Humanos , Hibridização in Situ Fluorescente , Linfonodos/imunologia , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Timo/imunologiaRESUMO
In recent years it has become increasingly evident that MYC rearrangements are not confined to classical Burkitt lymphoma (BL), but also occur in diffuse large B-cell lymphoma (DLBCL) and in the new subtype, "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (BCLU), which was recently described in the 2008 revision of the World Health Organization classification. The accurate identification of MYC rearrangements in these three subtypes of high-grade lymphoma is becoming increasingly critical both in terms of diagnosis of classical BL and in light of the prognostic implications in cases of DLBCL and BCLU. We describe three cases of high-grade lymphoma in which cryptic insertion events, resulting in clinically significant IGH-MYC rearrangements, were detectable using an IGH/MYC three-color, dual-fusion fluorescence in situ hybridization (FISH) probe set, but were not detected using break-apart MYC FISH probes, thus highlighting the limitations of using break-apart probes as a stand-alone test, particularly with the increased use of interphase FISH analysis of formalin-fixed, paraffin-embedded tissue sections in the diagnostic work-up of these patients.
Assuntos
Genes de Cadeia Pesada de Imunoglobulina , Genes myc , Linfoma não Hodgkin/genética , Translocação Genética , Idoso , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Sondas MolecularesRESUMO
Karyotyping is currently the "gold standard" test for the detection of human chromosome abnormalities. Over the past 40 years, changes in techniques have improved the band definition of chromosomes; however, very little has changed with respect to improvements through automation. In this study, we compare chromosome analysis by traditional microscopy with semi-automatic karyotyping using robotic equipment from MetaSystems (Altlussheim, Germany). Analysis using MetaSystems was significantly quicker than using the microscope with an average reduction in analysis time of 26.5 minutes; for the average analyst, this equates to a reduction of 27 percent. Analysis checking times using MetaSystems showed even greater improvement with an average reduction in checking time of 11.4 minutes; for the average checker, this equates to a reduction of 48 percent. The MetaSystems semi-automatic karyotyping equipment offers increased throughput of cases for karyotype analysis while maintaining accuracy.