RESUMO
Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfoma de Burkitt/tratamento farmacológico , Ciclo Celular , Humanos , Quimioterapia de Indução , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RecidivaRESUMO
Monitoring of NPM1 mutant (NPM1mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients who are treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as an MRD transcript level <1% to 2% with a <1-log change between any 2 positive samples collected after the end of treatment (EOT). Because the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received ≥2 cycles of intensive chemotherapy were included if bone marrow was NPM1mut MRD positive at the EOT, and they were not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year, either spontaneously achieving complete molecular remission (CRMRD-; 30%) or retaining a low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow-up). Forty percent met the criteria for MP-LCN. Preemptive salvage therapy significantly prolonged relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-internal tandem duplication at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT.
Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Neoplasia Residual , Proteínas Nucleares/genética , Indução de RemissãoRESUMO
Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1mut measurable residual disease (MRD) using off-label venetoclax in combination with low-dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CRMRD- ) without transplantation. Six of seven patients with molecular relapse/progression achieved CRMRD- after 1-2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax-based therapy to reduce the relapse risk in patients with persistent or rising NPM1mut MRD.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Leucemia Mieloide Aguda , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Sulfonamidas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Nucleofosmina , Estudos RetrospectivosRESUMO
Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.
Assuntos
Evolução Clonal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Adolescente , Adulto , Alelos , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Reação em Cadeia da Polimerase Multiplex , Mutação , Proteínas de Fusão Oncogênica/metabolismo , PTEN Fosfo-Hidrolase/genética , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Análise de Célula Única , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Adulto JovemRESUMO
BACKGROUND: Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice. METHODS: We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes. RESULTS: We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials. CONCLUSIONS: The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Adulto , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Análise Mutacional de DNA , Epistasia Genética , Fusão Gênica , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Nucleofosmina , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Splicing de RNA , Análise de SobrevidaRESUMO
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 ÚnicaRESUMO
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, â¼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica/genética , Rearranjo Gênico/genética , Variação Genética , Proteínas de Homeodomínio/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Recombinação Genética/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Variações do Número de Cópias de DNA/genética , Biblioteca Gênica , Genes Supressores de Tumor , Humanos , Dados de Sequência Molecular , Proteínas Repressoras , Análise de Sequência de DNA , Deleção de Sequência/genética , Fatores de Transcrição/genética , Recombinação V(D)J/genéticaRESUMO
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 ÚnicoRESUMO
Few studies on the biologic and molecular properties of pediatric glioblastoma have been performed. Until now, differential genomic analysis of CD133(+)ve and CD133(-)ve fractions has not been described in pediatric glioma. We hypothesize not only that the presence of CD133 could be the source of tumor resistance but also that maintenance of this molecule by hypoxia dictates cellular and molecular behavior. From a series of human glioblastoma biopsies investigated, only one, IN699 (from a pediatric glioblastoma), generated greater than 4% of the total cell volume as CD133(+)ve cells. Using this pediatric glioblastoma, containing unprecedented high levels of the putative brain tumor stem cell marker CD133, as a study model, we report biologic and molecular characteristics of the parent culture and of CD133(+)ve and CD133(-)ve populations derived therefrom under atmospheric and hypoxic culture conditions. Immunocytochemistry and flow cytometry were performed with antigenic markers known to characterize neural stem cells and associated glioma behavior. Behavioral analysis was carried out using proliferation, adhesion, migration, and invasion assays. Cell cycle analysis and array comparative genomic hybridization were used to assess copy number profiles for parental cells and CD133(+)ve and CD133(-)ve fractions, respectively. With regard to invasion and proliferation, CD133(+)ve and CD133(-)ve fractions were inversely proportional, with a significant increase in invasive propensity within the CD133(-)ve cells (P < .005) and a significant increase in proliferation within CD133(+)ve cells (P < .005). Our observations indicate identical genomic imbalances between CD133(+)ve and CD133(-)ve fractions. Furthermore, our research documents a direct link between decreasing oxygen tension and CD133 expression.
RESUMO
We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 4 , Evolução Clonal/genética , Heterogeneidade Genética , Mieloma Múltiplo/genética , Translocação Genética/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 4/genética , Ensaios Clínicos como Assunto , Evolução Clonal/fisiologia , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Humanos , Perda de Heterozigosidade/genética , Masculino , Análise em Microsséries , Modelos Biológicos , Mutação/fisiologia , Transdução de Sinais/genética , Estudos de Validação como AssuntoRESUMO
The heterogeneity of tumours and uncertainties surrounding derived short-term cell cultures and established cell lines fundamentally challenge the research and understanding of tumour growth and development. When tumour cells are cultured, changes are inevitably induced due to the artificial growth conditions. Several recent studies have questioned how representative established cell lines or derived short-term cell cultures are of the tumour in situ. We have characterised gene expression changes induced by short-term culture in astrocytoma in order to determine whether derived short-term cell cultures are representative of the tumour in situ. In comparison to the majority of studies, paired biopsies and derived short-term cultures were investigated to reduce the effects of long-term culture and inter-tumour variability when comparing biopsies and derived cultures from tumours with the same histology from different individuals. We have used the Affymetrix GeneChip U133A to generate gene expression profiles of 6 paediatric pilocytic astrocytoma (PA) biopsies and derived short-term cell cultures and 3 adult glioblastoma multiforme (GBM) biopsies and derived short-term cultures. Significant differential gene expression is induced by short-term culture. However, when the biopsy and derived short-term cell culture samples were grouped according to tumour type (PA and GBM) a molecular signature of 608 genes showed significant differential expression between the groups. This gene cohort can distinguish PA and GBM tumours, regardless of the sample source, suggesting that astrocytoma derived short-term cultures do retain key aspects of the global tumour expression profile and are representative of the tumour in situ. Furthermore, these genes are involved in pathways and functions characteristic of adult GBM including VEGF signalling, hypoxia and TP53 signalling.