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1.
Stem Cell Reports ; 4(1): 7-15, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25533637

RESUMO

Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure. This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Variação Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Fenótipo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Progressão da Doença , Estudo de Associação Genômica Ampla , Genômica , Glioblastoma/patologia , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Polimorfismo de Nucleotídeo Único , Análise de Célula Única
2.
Genome Res ; 23(12): 2115-25, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24056532

RESUMO

The development of cancer is a dynamic evolutionary process in which intraclonal, genetic diversity provides a substrate for clonal selection and a source of therapeutic escape. The complexity and topography of intraclonal genetic architectures have major implications for biopsy-based prognosis and for targeted therapy. High-depth, next-generation sequencing (NGS) efficiently captures the mutational load of individual tumors or biopsies. But, being a snapshot portrait of total DNA, it disguises the fundamental features of subclonal variegation of genetic lesions and of clonal phylogeny. Single-cell genetic profiling provides a potential resolution to this problem, but methods developed to date all have limitations. We present a novel solution to this challenge using leukemic cells with known mutational spectra as a tractable model. DNA from flow-sorted single cells is screened using multiplex targeted Q-PCR within a microfluidic platform allowing unbiased single-cell selection, high-throughput, and comprehensive analysis for all main varieties of genetic abnormalities: chimeric gene fusions, copy number alterations, and single-nucleotide variants. We show, in this proof-of-principle study, that the method has a low error rate and can provide detailed subclonal genetic architectures and phylogenies.


Assuntos
Evolução Clonal , Genômica/métodos , Mutação , Neoplasias/genética , Filogenia , Análise de Célula Única , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase Multiplex , Polimorfismo de Nucleotídeo Único
3.
Methods Mol Biol ; 878: 149-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22674132

RESUMO

Cytogenetic analysis of tumour material has been greatly enhanced over the past 30 years by the application of a range of techniques based around fluorescence in situ hybridization (FISH). Fluorescence detection for in situ hybridization has the advantage of including the use of a multitude of fluorochromes to allow simultaneous specific detection of multiple probes by virtue of their differential labelling and emission spectra. FISH can be used to detect structural (translocation/inversion) and numerical (deletion/gain) genetic aberrations. This chapter will deal with FISH methods to detect and localize one or more complementary nucleic acid sequences (probes) within a range of different cellular targets including metaphase chromosomes, nuclei from cell suspension, and formalin-fixed paraffin-embedded FFPE tissue sections. Methods for the efficient localization of probes to FFPE tissue cores in tissue microarrays (TMAs) are also described.


Assuntos
Hibridização in Situ Fluorescente/métodos , Neoplasias/genética , Neoplasias/patologia , Biotina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos Humanos/genética , Sondas de DNA/metabolismo , Digoxigenina/metabolismo , Marcadores Genéticos/genética , Humanos , Processamento de Imagem Assistida por Computador , Interfase/genética , Metáfase/genética , Inclusão em Parafina , Análise Serial de Tecidos , Fixação de Tecidos
4.
Blood ; 117(23): 6247-54, 2011 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-21482711

RESUMO

B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1(+) acute lymphoblastic leukemia.


Assuntos
Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 6/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Deleção de Sequência , Translocação Genética , Criança , Pré-Escolar , Cromossomos Humanos Par 16/metabolismo , Cromossomos Humanos Par 6/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Ciclina C , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Humanos , Masculino , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Recidiva
6.
Nature ; 469(7330): 356-61, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21160474

RESUMO

Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6-RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or 'driver' copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγ(null) mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.


Assuntos
Células Clonais/patologia , Variação Genética/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Células Clonais/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Progressão da Doença , Genótipo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética
7.
Blood ; 115(17): 3553-8, 2010 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-20061556

RESUMO

Chimeric fusion genes are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and are mostly prenatal, early genetic events in the evolutionary trajectory of this cancer. ETV6-RUNX1-positive ALL also has multiple ( approximately 6 per case) copy number alterations (CNAs) as revealed by genome-wide single-nucleotide polymorphism arrays. Recurrent CNAs are probably "driver" events contributing critically to clonal diversification and selection, but at diagnosis, their developmental timing is "buried" in the leukemia's covert natural history. This conundrum can be resolved with twin pairs. We identified and compared CNAs in 5 pairs of monozygotic twins with concordant ETV6-RUNX1-positive ALL and 1 pair discordant for ETV6-RUNX1 positive ALL. We compared, within each pair, CNAs classified as potential "driver" or "passenger" mutations based upon recurrency and, where known, gene function. An average of 5.1 (range 3-11) CNAs (excluding immunoglobulin/T-cell receptor alterations) were identified per case. All "driver" CNAs (total of 32) were distinct within each of the 5 twin pairs with concordant ALL. "Driver" CNAs in another twin with ALL were all absent in the shared ETV6-RUNX1-positive preleukemic clone of her healthy co-twin. These data place all "driver" CNAs secondary to the prenatal gene fusion event and most probably postnatal in the sequential, molecular pathogenesis of ALL.


Assuntos
Dosagem de Genes , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Gêmeos Monozigóticos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Humanos , Masculino , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
8.
Blood ; 114(13): 2688-98, 2009 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-19641190

RESUMO

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.


Assuntos
Transformação Celular Neoplásica/genética , Linfócitos/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Receptores de Citocinas/genética , Adolescente , Adulto , Idoso , Animais , Células Cultivadas , Criança , Pré-Escolar , Cromossomos Humanos Par 14 , Embrião de Mamíferos , Deleção de Genes , Regulação Leucêmica da Expressão Gênica , Humanos , Lactente , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores de Citocinas/metabolismo , Translocação Genética , Adulto Jovem
9.
Methods Mol Biol ; 538: 57-70, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19277577

RESUMO

Fluorescence in situ hybridization (FISH) provides one of the few ways of analysing the genotype of individual cells, an important consideration for mixed cell populations such as those found in leukaemia. A more sophisticated variation combines fluorescence immunophenotyping and FISH for specific leukaemia-associated chromosome rearrangements. Combined immunophenotyping and FISH is a powerful tool to identify the cell lineage in which the leukaemia-specific chromosome rearrangement occurs and has been used to identify putative pre-leukaemic cells in normal cord blood. Another valuable FISH-based research technique is multi-fluor FISH (M-FISH). This multicolour approach is effectively a molecular karyotype of individual cells and has a range of applications, from chromosome breakage studies and characterising mouse models of leukaemia, to providing a perfect complementary approach to the emerging genomic microarray technologies.


Assuntos
Aberrações Cromossômicas , Hibridização in Situ Fluorescente/métodos , Leucemia/genética , Hibridização de Ácido Nucleico/métodos , Humanos , Imunofenotipagem/métodos , Cariotipagem/métodos , Leucemia/diagnóstico
10.
Blood ; 113(3): 646-8, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18927438

RESUMO

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.


Assuntos
Síndrome de Down/genética , Janus Quinase 2/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Sequência de Bases , Análise Mutacional de DNA , Síndrome de Down/complicações , Deleção de Genes , Humanos , Camundongos , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
11.
Genes Chromosomes Cancer ; 48(1): 98-107, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18932267

RESUMO

Acute leukemia is considered to be a two- or multiple-step process. Although there is a considerable knowledge regarding the character of the "first hit," the nature of the "second hit" remains unanswered in most of the cases including leukemias with MLL gene rearrangement. We demonstrate here a striking sequence of events, which include a covert, protracted preleukemic phase characterized by a dominant MLL/FOXO3A clone with intact myeloid differentiation and the subsequent acquisition of a secondary genetic abnormality, leading to overt lymphoblastic leukemia. Backtracking of the secondary acute lymphoblastic leukemia (sALL) with the MLL rearrangement showed no blasts in the bone marrow (BM) during the protracted preleukemic phase. However, at the same time (more than 1 year before the sALL diagnosis) the MLL/FOXO3A was present in up to 90% of BM cells including myeloid lineage, suggesting that the fusion arose in a multipotent progenitor. To identify potential "second hit" precipitating sALL we compared DNA in preleukemic versus fully leukemic samples. The analysis revealed a 10 Mb gain on 19q13.32 in the sALL, absent in the preleukemic specimen. These data provide insight into the dynamics of leukemogenesis in secondary leukemia with MLL rearrangement.


Assuntos
Fusão Gênica , Leucemia Promielocítica Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Pré-Leucemia/genética , Adolescente , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cromossomos Humanos Par 19/genética , Análise Citogenética , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Leucemia Promielocítica Aguda/terapia , Células Mieloides/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia
12.
Br J Haematol ; 142(6): 934-45, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18557744

RESUMO

Twenty-five cases of B-cell precursor acute lymphoblastic leukaemia (ALL) from Down syndrome (DS) patients were analyzed using array comparative genomic hybridization (aCGH) and compared with two other subgroups of non-DS patients with ALL; five cases with high-hyperdiploidy (HH) and nine cases with ETV6-RUNX1 positive clones. Seven cases of DS-acute megakaryoblastic leukaemia (AMKL) were also included, DS-ALL cases showed relatively stable karyotypes with cryptic losses and gains that most frequently involved chromosomes X, 1, 2, 9, 11, 16, and 17. The most consistent change involved a deletion in 2p, spanning region Chr2:88273220-91084234, which in some cases appeared to be homozygous. ALL from non-DS patients showed a similar overall karyotypic stability, although gains of chromosome 21 were infrequent in the ETV6-RUNX1 positive cases. The most consistent change in this group involved a 12p deletion, where Chr12:10383878-16017619 defined the common region of overlap. All HH-ALL karyotypes showed variable gains of chromosome 21. This overall analysis supports the suggestion that, although constitutional trisomy 21 predisposes to ALL/AMKL, the cytogenetic changes associated with DS-ALL in particular, are most similar to those found in non-DS ETV6-RUNX1 positive ALL. The HH-ALL group, however, undergoes distinct karyotypic evolution not dependent on chromosome translocation/deletion events.


Assuntos
Aberrações Cromossômicas , Síndrome de Down/genética , Leucemia Megacarioblástica Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Hibridização Genômica Comparativa , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Humanos , Cariotipagem , Masculino , Proteínas de Fusão Oncogênica/genética , Ploidias , Adulto Jovem
13.
Genes Chromosomes Cancer ; 47(4): 333-40, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18181181

RESUMO

In a small fraction ( approximately 2%) of cases of childhood acute lymphoblastic leukemia (ALL) clinical presentation of leukemia is preceded, some 2-9 months earlier, by a transient, remitting phase of nonclassical aplastic anemia, usually in connection with infection. The potential "preleukemic" nature of this prodromal phase has not been fully explored. We have retrospectively analyzed the blood and bone marrow of a child who presented with aplastic anemia 9 months before the development of ETV6-RUNX1 fusion gene positive ALL. High resolution SNP genotyping arrays identified 11 regions of loss of heterozygosity, with and without concurrent copy number changes, at the presentation of ALL. In all cases of copy number change, the deletion or gain identified by single nucleotide polymorphism (SNP) analysis was confirmed in the ALL blasts by FISH. Retrospective analysis of aplastic phase bone marrow showed that the ETV6-RUNX1 fusion was present along with all of the additional genetic changes assessed, albeit subclonal to ETV6-RUNX1. These data identify for the first time the leukemic genotype of an aplasia preceding clinical ALL and indicate that multiple secondary genetic abnormalities can contribute to a dominant subclone several months before a diagnosis of ALL. These data have implications for the biology of ALL and for management of similar patients.


Assuntos
Anemia Aplástica/complicações , Anemia Aplástica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pré-Leucemia/genética , Anemia Aplástica/patologia , Medula Óssea/patologia , Pré-Escolar , Genótipo , Humanos , Hibridização in Situ Fluorescente , Masculino , Análise em Microsséries , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Pré-Leucemia/patologia , Estudos Retrospectivos
14.
Genes Chromosomes Cancer ; 45(8): 754-60, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16688745

RESUMO

MLL fusion genes are a predominant feature of acute leukemias in infants and in secondary acute myeloid leukemia (AML) associated with prior chemotherapy with topo-II poisons. The former is considered to possibly arise in utero via transplacental chemical exposure. A striking feature of these leukemias is their malignancy and remarkably brief latencies implying the rapid acquisition of any necessary additional mutations. We have suggested that these coupled features might be explained if MLL fusion gene encoded proteins rendered cells more vulnerable to further DNA damage and mutation in the presence of chronic exposure to the agent(s) that induced the MLL fusion itself. We have tested this idea by exploiting a hormone regulated MLL-ENL (MLLT1) activation system and show that MLL-ENL function in normal murine progenitor cells substantially increases the incidence of chromosomal abnormalities in proliferating cells that survive exposure to etoposide VP-16. This phenotype is associated with an altered pattern of cell cycle arrest and/or apoptosis.


Assuntos
Aberrações Cromossômicas , Dano ao DNA , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Linhagem Celular Transformada , Dano ao DNA/genética , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Humanos , Hibridização in Situ Fluorescente , Lactente , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Tempo de Reação/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia
15.
Genes Chromosomes Cancer ; 45(6): 554-64, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16523483

RESUMO

Human leukemia-derived cell lines containing characteristic chromosomal translocations and inversions have been instrumental in identifying fusion genes implicated in the pathogenesis of the corresponding leukemia. Although chimeric fusion genes usually provide early and essential steps in the development of leukemia, they are not in themselves sufficient, requiring additional genetic events. The nature of these secondary, cooperating genetic events is not known. The advent of genome wide microarray-based methods for assessing copy number changes made it possible to search for cytogenetically invisible regions of chromosome imbalance. We used BAC microarrays with a resolution of 1 Mb to determine whether cryptic regions of deletion or gain were associated with specific leukemia-associated fusion genes in a series of cell lines. To complement the array analysis, we also applied 24-color karyotyping by M-FISH. This revealed cryptic chromosomal translocations and regions of loss or gain in all the cell lines studied. The chromosomal origin of previously unidentified marker chromosomes was revealed. In all cases, chromosomes described as monosomic were shown to be involved in unbalanced translocations with concurrent loss and/or gain of chromosomal material. The extent of these amplified and deleted regions was more accurately defined. Finally, small regions of deletion and amplification, often including genes known to be involved in leukemia progression (for example MYC, TP53, CDKN2A, and KIT), were identified.


Assuntos
Aberrações Cromossômicas , Fusão Gênica , Leucemia/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular Tumoral , Deleção Cromossômica , Genômica/métodos , Humanos , Cariotipagem , Leucemia/metabolismo , Modelos Genéticos , Translocação Genética
16.
Chromosoma ; 114(4): 286-94, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16003502

RESUMO

Cytogenetics has played a pivotal role in haematological malignancy, both as an aid to diagnosis and in identifying recurrent chromosomal rearrangements, an essential prerequisite to identifying genes involved in leukaemia and lymphoma pathogenesis. In the late 1980s, a series of technologies based around fluorescence in situ hybridisation (FISH) revolutionised the field. Interphase FISH, multiplex-FISH (M-FISH, SKY) and comparative genomic hybridisation (CGH) have emerged as the most significant of these. More recently, microarray technologies have come to prominence. In the acute leukaemias, the finding of characteristic gene expression signatures corresponding to biological subgroups has heralded gene expression profiling as a possible future alternative to current cytogenetic and morphological methods for diagnosis. In the lymphomas, high-resolution array CGH has successfully identified new regions of deletion and amplification, providing the prospect of disease-specific arrays.


Assuntos
Análise Citogenética/métodos , Neoplasias Hematológicas/genética , Criança , Humanos , Hibridização in Situ Fluorescente , Hibridização de Ácido Nucleico , Poliploidia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética
17.
Genes Chromosomes Cancer ; 41(3): 191-202, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15334542

RESUMO

The functional consequences of a high-hyperdiploid karyotype, found in up to one-third of cases of acute lymphoblastic leukemia (ALL), are unknown. Using the technique of comparative expressed sequence hybridization (CESH), we sought to address the question of whether increased chromosome copies in hyperdiploid ALL lead to increased gene expression. Relative expression of hyperdiploid ALL blasts versus peripheral blood mononuclear cells was analyzed in 18 patients. Common regions of overexpression corresponding to the presence of tri-/tetrasomies included: Xp22.1-22.2, 4q28, 6q14-15, 6q24, 10p13, 14q23-24, 17q21, 18q12, and 21q21, identified in 28-89% of cases. However, increased expression without underlying trisomy occurred at 3p21.3, 7q11.2, 8p21, and 8q24.1 in 39-90% of cases. High expression at 7q11.2, the most consistent change detected, was confirmed by quantitative PCR. Poor correlation between the presence of tri-/tetrasomy and overexpression was observed for chromosomes 14 and 17. Two cases were reanalyzed versus (i) B cells, (ii) transformed B cells, and (iii) CD34+19+ cells (the putative counterpart of the leukemic cell). A reduction in the number of relatively overexpressed regions was observed with CD34+19+ cells. In particular, the peak at 7q11.2 disappeared, suggesting up-regulation of genes from this region in the early ontology of normal B-cell development. In conclusion, we have shown that tri-/tetrasomies in hyperdiploid ALL lead to an increase in the expression of associated sequences. The choice of a biologically relevant reference is crucial for data interpretation.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Antígenos CD19/biossíntese , Antígenos CD34/biossíntese , Linfócitos B/metabolismo , Pré-Escolar , Cromossomos Humanos Par 7 , Primers do DNA/química , Diploide , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Cariotipagem , Masculino , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA/metabolismo , Análise de Sequência de DNA
19.
Genes Chromosomes Cancer ; 38(2): 191-200, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12939747

RESUMO

The t(7;12)(q36;p13) is a recurrent chromosome abnormality in infant leukemia. In these cases, the involvement of ETV6, with disruption of the gene consistently at its 5' end, has been reported by several groups. A fusion transcript between ETV6 and HLXB9 has been detected in some, but not all, reported cases of t(7;12). We report here a study based on fluorescence in situ hybridization (FISH) mapping of the translocation breakpoints in seven patients and detailed molecular studies using Southern blotting on two of these patients. The FISH studies have shown a cluster of breakpoints within a cosmid contig proximal to the HLXB9 gene. Southern blotting analysis enabled us to define two distinct breakpoints within the area covered by the cosmid contig in two patients. The analysis of an unusual case of t(7;12)(q22;p13) [full karyotype: 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36)] also revealed a break in 7q36, although in a region proximal to the overlapping cosmids. 5' RACE PCR in one patient has shown a rearrangement involving the ETV6 allele not involved in the t(7;12), suggesting that no functional ETV6 allele might be present in this case. These data show some heterogeneity in the distribution of breakpoints in 7q36, indicating that the generation of a fusion gene might not be the mechanism responsible for leukemogenesis in the t(7;12), at least in some cases.


Assuntos
Quebra Cromossômica/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 7/genética , Proteínas de Ligação a DNA/genética , Heterogeneidade Genética , Leucemia/genética , Proteínas Repressoras/genética , Translocação Genética/genética , Sequência de Bases , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Aguda/genética , Masculino , Dados de Sequência Molecular , Síndromes Mielodisplásicas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETS
20.
Genes Chromosomes Cancer ; 37(4): 406-11, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12800152

RESUMO

Previous studies on concordant acute lymphoblastic leukemia (ALL) in identical twins have identified the leukemia as monoclonal with MLL or ETV6-RUNX1 gene fusion as early or initiating events in utero. In the latter case, postnatal latency is associated with secondary genetic events such as ETV6 deletion. We describe here a pair of infant twins with concordant acute monoblastic leukemia (AML). They are a unique pair in that their leukemia blasts display extensive intraclonal chromosomal diversity. Comparison of the leukemic cells between the two twins by karyotype and fluorescence in situ hybridization identifies a common or shared stem line and extensive subclonal diversity for which the twins' leukemic populations are divergent. This case of leukemia illustrates in utero initiation with early imposition of chromosomal instability, the progressively divergent evolution of which can be mapped in the twins into pre- and postnatal periods.


Assuntos
Doenças em Gêmeos/embriologia , Doenças em Gêmeos/genética , Leucemia Monocítica Aguda/embriologia , Leucemia Monocítica Aguda/genética , Gêmeos Monozigóticos/genética , Aberrações Cromossômicas , Deleção Cromossômica , Células Clonais , Proteínas de Ligação a DNA/genética , Humanos , Lactente , Masculino , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Variante 6 da Proteína do Fator de Translocação ETS
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