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1.
Blood ; 128(7): 911-22, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27229005

RESUMO

Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.


Assuntos
Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Criança , Pré-Escolar , Aberrações Cromossômicas , Estudos de Coortes , Análise Citogenética , Variações do Número de Cópias de DNA/genética , Demografia , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Prognóstico , Recidiva , Fatores de Risco
2.
Blood ; 124(23): 3420-30, 2014 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-25253770

RESUMO

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.


Assuntos
Benzimidazóis/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Genes ras , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Linhagem Celular Tumoral , Criança , Ensaios Clínicos como Assunto , Frequência do Gene , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Recidiva , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Br J Haematol ; 171(4): 595-605, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26310606

RESUMO

Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.


Assuntos
Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinase Quinase 4/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Apoptose/efeitos dos fármacos , Linfócitos B/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Éxons/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Multiplex , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/fisiologia , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em Tandem
4.
Blood ; 117(25): 6848-55, 2011 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-21527530

RESUMO

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.


Assuntos
Cromossomos Humanos Par 21 , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Dosagem de Genes , Humanos , Janus Quinases/genética , Masculino , Mutação , Adulto Jovem
5.
Genes Chromosomes Cancer ; 51(3): 250-6, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22072526

RESUMO

Casitas B-lineage lymphoma (CBL) proteins are RING finger ubiquitin E3 ligases that attenuate the signaling of receptor tyrosine kinases and are mutated in a number of myeloid disorders. In this study, mutational screening of the linker-RING domains of CBL and CBLB was performed by denaturing high performance liquid chromatography in a cohort of diagnostic (n = 180) or relapse (n = 46) samples from children with acute lymphoblastic leukemia. Somatic mutations were identified in three children, giving an overall incidence of 1.7% and involved small deletions affecting the intron/exon boundaries of exon 8, leading to skipping of exon 8 and abolishing E3 ligase function. Mutated primary samples were associated with constitutive activation of the RAS pathway and sensitivity to MEK inhibitors was shown. Thus, mutation of CBL is an alternative route to activate the RAS pathway and may identify children who are candidates for MEK inhibitor clinical trials.


Assuntos
Mutação , Proteína Oncogênica v-cbl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transdução de Sinais/genética , Adolescente , Sequência de Bases , Criança , Pré-Escolar , Cromatografia Líquida , Estudos de Coortes , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Íntrons , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Domínios RING Finger , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Recidiva , Reino Unido
6.
Blood ; 113(1): 100-7, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18838613

RESUMO

Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.


Assuntos
Deleção de Genes , Dosagem de Genes , Regulação Leucêmica da Expressão Gênica , Genes p16 , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Criança , Metilação de DNA , Feminino , Genômica , Hormônio do Crescimento Humano , Humanos , Hibridização in Situ Fluorescente , Incidência , Perda de Heterozigosidade , Masculino , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia
7.
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
8.
Proc Natl Acad Sci U S A ; 105(44): 17050-4, 2008 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-18957548

RESUMO

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.


Assuntos
Quebra Cromossômica , Genes Neoplásicos , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocação Genética/genética , Alelos , Sequência de Bases , Cromossomos Humanos Par 9/genética , Dosagem de Genes , Humanos , Hibridização in Situ Fluorescente , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo
9.
Cancer Med ; 10(14): 4864-4873, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34080325

RESUMO

BACKGROUND: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T-cell acute lymphoblastic leukemia (T-ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T-ALL has a specific biological-molecular profile distinct from pediatric or adult T-ALL. METHODS: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15-26 years with primary or relapsed T-ALL, using a combination of Genome-Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real-time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1-14 years), 439 TYA (15-24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search. RESULTS: Genomic characterization of this large cohort of TYA T-ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation - relapsed T-ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways. CONCLUSIONS: All genetic aberrations in TYA T-ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T-ALL exhibits a transitional genomic profile. Analysis of matched presentation - relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub-clones associated with drug resistance (NT5C2 and TP53 mutations) and re-iterative mutation of known key T-ALL drivers, including NOTCH1.


Assuntos
Perfilação da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Adulto , Fatores Etários , Cromossomos Humanos Par 7 , Evolução Clonal , Humanos , Isocromossomos , Mutação , Polimorfismo de Nucleotídeo Único , Recidiva , Estudos Retrospectivos , Sequenciamento do Exoma , Adulto Jovem
10.
Haematologica ; 95(4): 679-83, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19951974

RESUMO

Flow cytometric minimal residual disease (MRD) monitoring could become more powerful if directed towards the disease-maintaining leukemic stem cell (LSC) compartment. Using a cohort of 48 children with B-lineage acute lymphoblastic leukemia (ALL), we sought the newly proposed candidate-LSC population, CD34(+)CD38(low)CD19(+), at presentation and in end of induction bone marrow samples. We identified the candidate LSC population in 60% of diagnostic samples and its presence correlated with expression of CD38, relative to that of normal B-cell progenitors. In addition, the candidate LSC was not detectable in all MRD positive samples. The absence of the population in 40% of diagnostic and 40% of MRD positive samples does not support the use of this phenotype as a generic biomarker to track LSCs and suggests that this phenotype may be an artifact of CD38 underexpression rather than a biologically distinct LSC population. ClinicalTrials.gov Identifier: NCT00222612.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD19/metabolismo , Antígenos CD34/metabolismo , Neoplasia Residual/diagnóstico , Células-Tronco Neoplásicas/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Criança , Ensaios Clínicos como Assunto , Citometria de Fluxo , Humanos , Imunofenotipagem , Neoplasia Residual/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia
11.
Haematologica ; 94(6): 870-4, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19377076

RESUMO

Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.


Assuntos
Citometria de Fluxo/métodos , Leucemia de Células B/diagnóstico , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Antígenos CD19/análise , Antígenos CD34/análise , Criança , Citometria de Fluxo/normas , Rearranjo Gênico , Humanos , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Neoplasia Residual/genética , Neoplasia Residual/metabolismo , Neprilisina/análise , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prognóstico , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T/genética , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
FASEB J ; 20(14): 2600-2, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17077285

RESUMO

Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Glucocorticoides/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptores de Glucocorticoides/metabolismo , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Glucocorticoides/metabolismo , Humanos , Mutação , Receptores de Glucocorticoides/genética , Fatores de Transcrição/metabolismo
13.
Cancer Res ; 65(21): 9712-8, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16266991

RESUMO

Glucocorticoids are pivotal in the treatment of children with acute lymphoblastic leukemia (ALL) and have significant antileukemic effects in the majority of children. However, clinical resistance is a significant problem. Although cell line models implicate somatic mutations and loss of heterozygosity (LOH) of the glucocorticoid receptor (GR) gene as a mechanism of in vitro glucocorticoid resistance, the relevance of this mechanism as a cause of clinical resistance in children with ALL is not known. Mutational screening of all coding exons of the GR gene and LOH analyses were done in a large cohort of relapsed ALL. We show that somatic mutations and LOH of the GR rarely contribute to relapsed disease in children with ALL. However, we report the second case of ALL with a somatic mutation of the GR involving a 29-bp deletion in exon 8 and resulting in a truncated protein with loss of part of the ligand-binding domain. There was no evidence of a remaining wild-type allele. Allele-specific PCR detected the mutated clone at day 28 after presentation, which persisted at a low level throughout the disease course before relapse several years later. We hypothesize that the mutated allele present in a leukemic subclone at initial diagnosis was selected for during remission induction with glucocorticoids and contributed to the emergence of a glucocorticoid-resistant cell population.


Assuntos
Perda de Heterozigosidade , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/genética , Alelos , Medula Óssea/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Estudos de Coortes , Éxons , Deleção de Genes , Humanos , Repetições de Microssatélites/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisolona/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Clin Biochem ; 37(5): 395-403, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15087256

RESUMO

BACKGROUND: Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome and predict impending relapse. In ALL, the cell surface over expression of 9-O-acetylated sialoglycans (9-OAcSGs) on lymphoblasts and concomitant high antibody titers in patients' sera was reported. OBJECTIVES: The present study was aimed to evaluate whether anti-9-OAcSG titers can be harnessed to monitor the clinical outcome of ALL. DESIGN AND METHODS: Anti-9-OAcSGs were analyzed by ELISA in children receiving either UK ALL X (n = 69, Group I) in India or UK ALL 97 (n = 47, Group II) in UK along with age-matched normal healthy controls at different time points over a period of >2 years. An attempt was also made to investigate subclass distribution of disease-specific IgG. Moreover, 17 patients having a higher sample size were longitudinally monitored. RESULTS: Antibody levels were raised at disease presentation, decreased with remission induction, and importantly, reappeared with clinical relapse. Sera from patients with other hematological disorders and normal controls showed negligible levels of circulating anti-9-OAcSGs. In patients of both Groups I and II, the assay showed high sensitivity (98.92% and 96.77%) and specificity (92.1% and 95.91%), respectively. IgG subclass analyses during different phases of treatment revealed that 9-OAcSG-specific IgG(1) could serve as a better prognostic marker in ALL. CONCLUSIONS: This study demonstrated the potential of this disease-specific antibody as an alternate marker in diagnosis and long-term assessment of ALL patients, suggesting its application in detection and prediction of impending relapse. Therefore, the expression of anti-9-OAcSGs, irrespective of their treatment protocol, may serve as an economical yet effective index for monitoring of childhood ALL.


Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Polissacarídeos/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Ácidos Siálicos/química , Antineoplásicos/uso terapêutico , Autoanticorpos/isolamento & purificação , Biomarcadores Tumorais/economia , Estudos de Casos e Controles , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/economia , Índia/epidemiologia , Masculino , Polissacarídeos/sangue , Polissacarídeos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Estudos Prospectivos , Receptores de Antígenos de Linfócitos B/sangue , Receptores de Antígenos de Linfócitos B/economia , Sensibilidade e Especificidade , Reino Unido/epidemiologia
15.
Leuk Res ; 34(8): 1098-102, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20233627

RESUMO

The mismatch repair (MMR) pathway is a post-replicative DNA repair process and MMR deficiency is a common feature of ALL cell lines. In this study we have investigated MMR deficiency in a large cohort of primary relapsed ALL (n=40) and investigated coding microsatellites (MS) of the lymphoid transcription factors, PAX5 and IKZF1 as downstream target genes. Only one patient showed MMR deficiency, as evidenced by microsatellite instability, which was acquired at relapse and was associated with reduced expression of both MLH1 and MSH2. Coding MS in candidate target genes including PAX5, IKZF1, BAX and TGFBRII were all wild type in this patient but the MMR-deficient cell line REH, was confirmed to have a coding MS in both PAX5 and TGFBRII. Whilst MMR deficiency is not highly prevalent in primary ALL, optimisation of the drug regimen to omit/replace thioguanines should be considered for children with MMR deficiency and/or reduced expression of key pathway components.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Fator de Transcrição Ikaros/genética , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas de Peixe-Zebra/genética , Adolescente , Western Blotting , Criança , Pré-Escolar , Estudos de Coortes , Metilação de DNA , Humanos , Fator de Transcrição Ikaros/metabolismo , Técnicas Imunoenzimáticas , Lactente , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Mutação/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas de Peixe-Zebra/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
16.
Cancer Res ; 68(16): 6803-9, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18701506

RESUMO

Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.


Assuntos
Genes ras/fisiologia , Mutação/genética , Recidiva Local de Neoplasia/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteínas ras/genética , Adolescente , Western Blotting , Sobrevivência Celular , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Inibidores Enzimáticos/farmacologia , Éxons/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Recidiva Local de Neoplasia/patologia , Fragmentos de Peptídeos , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Indução de Remissão , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/genética
17.
Mol Pharmacol ; 62(1): 102-9, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12065760

RESUMO

Although the thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well established agents for the treatment of leukemia, controversies remain regarding their main mode of action. Previous evidence has suggested that although 6-TG exerts a cytotoxic effect through incorporation of 6-thioguanine nucleotides into newly synthesized DNA (DNA-TGN), an important component of the mode of action of 6-MP is inhibition of purine de novo synthesis (PDNS) through the production of S-methyl-thioinosine 5'-monophosphate (MeTIMP), not formed in cells exposed to 6-TG. We have shown that thiopurine methyltransferase (TPMT) modulates this effect. By transfection of the human TPMT gene using an inducible system to produce a 3.8-fold increase in TPMT activity in the ecdysone receptor 293 embryonic kidney cell line, we demonstrated a 4.4-fold increase in sensitivity to 6-MP. This was associated with a rise in intracellular levels of MeTIMP but a decrease in levels of DNA-TGN. In contrast, induction of TPMT produced a 1.6-fold decrease in sensitivity to 6-TG, a decrease in levels of DNA-TGN, and an increase in levels of methylated thioguanosine monophosphate. Exposure of cells to equitoxic doses of drug showed similar incorporation of DNA-TGN for 6-TG but for 6-MP significantly reduced DNA-TGN in TPMT-induced compared with uninduced cells. For equitoxic doses of 6-MP, equivalent levels of MeTIMP correlated with equivalent amounts of PDNS. These observations suggest that intracellular TGN levels do not give an accurate reflection of cytotoxic potential in patients treated with 6-MP, because different levels of DNA-TGN may be associated with equitoxic effects.


Assuntos
Desoxiguanosina/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Mercaptopurina/farmacologia , Metiltransferases/biossíntese , Inibidores da Síntese de Ácido Nucleico/farmacologia , Tioguanina/farmacologia , Linhagem Celular , Células Cultivadas , DNA/biossíntese , DNA/efeitos dos fármacos , Desoxiguanosina/metabolismo , Humanos , Metiltransferases/genética , Tionucleosídeos/metabolismo , Transfecção
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