RESUMO
The extensively employed limited-gene coverage NGS panels lead to clinically inadequate molecular profiling of myeloid neoplasms. The aim of the present investigation was to assess performance and clinical utility of a comprehensive DNA panel for myeloid neoplasms. Sixty-one previously well characterized samples were sequenced using TSO500 library preparation kit on NextSeq550 platform. Variants with a VAF ≥ 5% and a total read depth of >50X were filtered for analysis. The following results were recorded-for clinical samples: clinical sensitivity (97%), specificity (100%), precision (100%) and accuracy (99%) whereas reference control results were 100% for analytical sensitivity, specificity, precision and accuracy, with high intra- and inter-run reproducibility. The panel identified 880 variants across 292 genes, of which, 749 variants were in genes not covered in the 54 gene panel. The investigation revealed 14 variants in ten genes, and at least one was present in 96.2% patient samples that were pathogenic/ likely pathogenic in myeloid neoplasms. Also, 15 variants in five genes were found to be pathogenic/ likely pathogenic in other tumor types. Further, the TMB and MSI scores ranged from 0-7 and 0-9, respectively. The high analytical performance and clinical utility of this comprehensive NGS panel makes it practical and clinically relevant for adoption in clinical laboratories for routine molecular profiling of myeloid neoplasms.
Assuntos
Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide/genética , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Idoso , Análise Custo-Benefício , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Instabilidade de Microssatélites , Mutação , Análise de Sequência de DNA , Fatores de TempoRESUMO
The diagnosis of myeloid neoplasms (MN) has significantly evolved through the last few decades. Next Generation Sequencing (NGS) is gradually becoming an essential tool to help clinicians with disease management. To this end, most specialized genetic laboratories have implemented NGS panels targeting a number of different genes relevant to MN. The aim of the present study is to evaluate the performance of four different targeted NGS gene panels based on their technical features and clinical utility. A total of 32 patient bone marrow samples were accrued and sequenced with 3 commercially available panels and 1 custom panel. Variants were classified by two geneticists based on their clinical relevance in MN. There was a difference in panel's depth of coverage. We found 11 discordant clinically relevant variants between panels, with a trend to miss long insertions. Our data show that there is a high risk of finding different mutations depending on the panel of choice, due both to the panel design and the data analysis method. Of note, CEBPA, CALR and FLT3 genes, remains challenging the use of NGS for diagnosis of MN in compliance with current guidelines. Therefore, conventional molecular testing might need to be kept in place for the correct diagnosis of MN for now.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide/genética , Genes Neoplásicos , Humanos , Mutação/genéticaRESUMO
Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.
Assuntos
Colorimetria/métodos , Proteínas de Fusão bcr-abl/genética , Ouro/química , Leucemia Mieloide/genética , Nanopartículas Metálicas/química , RNA Neoplásico/genética , Análise de Sequência de RNA/métodos , Humanos , Técnicas de Sonda Molecular , Sondas Moleculares/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
OBJECTIVE: Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). METHODS: We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. RESULTS: Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P < 0.001). However, there was no linear relationship between the transcript copies of the BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. CONCLUSION: BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.
Assuntos
Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genética , Doença Aguda , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Humanos , Quimioterapia de Indução , Lactente , Leucemia Mieloide/tratamento farmacológico , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Transcrição Gênica , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Cytogenetically normal acute myeloid leukemia (AML) represents nearly half of newly diagnosed de novo AML cases. XPD is one of the DNA repair proteins, whose genetic polymorphisms are thought to affect their function as regards response to chemotherapeutic drugs and chemotherapy-induced toxicities. SUBJECTS AND METHODS: We investigated the XPD Asp312Asn and Lys751Gln polymorphisms by polymerase chain reaction-restriction fragment length polymorphism in 51 newly diagnosed cytogenetically normal de novo AML patients. The response to the standard induction chemotherapy protocol and chemotherapy-induced toxicities were monitored. RESULTS: The XPD Asp312Asn GG genotype was the most frequent (57%) followed by the GA variant (37%), and the AA variant was the least frequent (6%). As regards the XPD Lys751Gln polymorphism, the AA genotype was the most frequent (49%), followed by the AC (39%) and CC (12%) variants. These variants were not associated with age, sex, FAB subtype, CNS infiltration, chemotherapy-induced hepatotoxicity, nephrotoxicity, or metabolic toxicity. The XPD Lys751Gln CC polymorphic variant was associated with chemotherapy-induced cardiotoxicity and lower chance to achieve response to induction chemotherapy. CONCLUSION: XPD Lys751Gln and not Asp312Asn polymorphism was associated with chemotherapy-induced cardiotoxicity and response to induction chemotherapy in newly diagnosed cytogenetically normal AML patients. Pretreatment assay of XPD Lys751Gln may help to anticipate cardiotoxicity in those at risk. Moreover, it may be considered a prognostic marker in AML cases. However, further large scale research is needed to verify its usefulness.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Leucemia Mieloide/tratamento farmacológico , Polimorfismo Genético , Proteína Grupo D do Xeroderma Pigmentoso/genética , Doença Aguda , Adulto , Substituição de Aminoácidos , Distribuição de Qui-Quadrado , Análise Citogenética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Feminino , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Cardiopatias/induzido quimicamente , Humanos , Quimioterapia de Indução/efeitos adversos , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Método de Monte Carlo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prognóstico , Fatores de Risco , Adulto JovemRESUMO
CD4 count ≤200×10(6) cells/L has been identified as a predictor of short survival in HIV-associated acute myeloid leukemia (HIV-AML), but karyotype, which is the best predictor of survival in AML, has not been evaluated in HIV-AML patients. A retrospective cohort of 31 patients was created from 9 local cases and 22 published cases. HIV-AML karyotypes were heterogeneous and were similar in distribution to those in HIV-negative AML. Among intensively treated patients, most achieved complete remission, but succumbed to infectious complications, mostly non-opportunistic, during consolidation therapy. Median survival for intensively-treated patients with CD4 counts ≤200×10(6) cells/L was 8.5 months, compared to 48 months for those with >200×10(6) CD4 cells/L (p=0.03). In contrast, AML karyotype did not predict survival (p=0.43), albeit with small numbers in each karyotype group. Thus, CD4 count is a strong predictor of short survival in HIV-AML patients regardless of karyotype. Studies evaluating innovative strategies for infection prophylaxis and for improving immune reconstitution are needed.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Infecções por HIV/complicações , HIV , Leucemia Mieloide/tratamento farmacológico , Doença Aguda , Adulto , Idoso , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/virologia , Humanos , Cariótipo , Leucemia Mieloide/genética , Leucemia Mieloide/imunologia , Masculino , Pessoa de Meia-Idade , Medição de Risco , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Gene-expression signature-based disease classification and clinical outcome prediction has not been widely introduced in clinical medicine as initially expected, mainly due to the lack of extensive validation needed for its clinical deployment. Obstacles include variable measurement in microarray assay, inconsistent assay platform, analytical requirement for comparable pair of training and test datasets, etc. Furthermore, as medical device helping clinical decision making, the prediction needs to be made for each single patient with a measure of its reliability. To address these issues, there is a need for flexible prediction method less sensitive to difference in experimental and analytical conditions, applicable to each single patient, and providing measure of prediction confidence. The nearest template prediction (NTP) method provides a convenient way to make class prediction with assessment of prediction confidence computed in each single patient's gene-expression data using only a list of signature genes and a test dataset. We demonstrate that the method can be flexibly applied to cross-platform, cross-species, and multiclass predictions without any optimization of analysis parameters.
Assuntos
Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doença Aguda , Animais , Neoplasias da Mama/genética , Análise por Conglomerados , Regulação Leucêmica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide/genética , Cirrose Hepática/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ratos , Reprodutibilidade dos TestesRESUMO
PRAME is expressed at low levels in normal testes and highly in solid tumor cells and hematological malignancies. The purpose of this study was to investigate PRAME expression levels in children with acute leukemia with real-time PCR analysis. Seventeen children with newly diagnosed or relapsed acute leukemia (11 ALL, 4 AML, 1 acute myeloblastic leukemia secondary to MDS, 1 ALL at relapse) and a control group of seven children were studied. Overexpression of PRAME was found in 52.9% (3 AML, 6 ALL) of the patients studied. No important correlation between PRAME expression and the patients' prognosis was observed. The above findings indicate that PRAME expression in acute leukemia does not seem to be of prognostic significance, whereas it might represent a candidate marker for the monitoring of minimal residual disease.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Leucemia Mieloide/genética , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Doença Aguda , Adolescente , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Humanos , Prognóstico , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.
Assuntos
Dosagem de Genes , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Proteínas Nucleares/genética , Doença Aguda , Análise Mutacional de DNA/métodos , Perfilação da Expressão Gênica , Humanos , Mutação , Nucleofosmina , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
BACKGROUND: Despite a considerable effort, the majority of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The results of some studies suggest the Wilms tumour gene (WT1) as a possible molecular marker of MRD. METHODS AND RESULTS: We measured the expression of WT1 at diagnosis and during treatment of the acute myeloid leukaemia (AML) patients. The expression of WT1 was measured by the quantitative real-time RT-PCR in peripheral leukocytes from 56 AML at diagnosis and 7 patients with AML transformed from myelodysplastic syndromes (MDS). The WT1 expression was significantly elevated (up to 3 orders of magnitude) in peripheral blood samples (PB) of AML patients at diagnosis compared to PB samples of healthy donors (P < 0.0001). The level of WT1 expression depends particularly on FAB AML subtype, with the highest being found in AML patients with subtypes M4, M1, M3 and AML transformed from MDS. Conversely, AML patients with M2 and with the presence of AML1/ET0 at presentation showed a significantly lower expression of the WT1 gene compared to the remaining AML patients at presentation (P = 0,005). Further, sequence samples of 12 AML patients under long-term surveillance were tested for the WT1 expression in parallel with the expression of specific MRD markers--fusion genes: AMLI/ETO, PML/RARalpha and CBFB/MYH11. The levels of WT1 gene expression and the above specific fusion genes significantly correlated. Moreover, 14 patients without the specific MRD marker were tested for the WT1 expression. The results show that haematological relapses were associated with the rise of expression of the specific fusion genes and with the WT1 gene expression. The rise of WT1 expression above the level seen in leucocytes from peripheral blood and/or bone marrow of healthy donors--in four patients under long-term surveillance the "molecular relapse" predicted ongoing haematological relapses as early as 2 months in advance. CONCLUSIONS: Our results, in accordance with some of the previously published ones, show that WT1 expression seems to be a suitable marker of minimal residual disease in AML patients.
Assuntos
Genes do Tumor de Wilms , Leucemia Mieloide/diagnóstico , Doença Aguda , Biomarcadores Tumorais/análise , Subunidade alfa 2 de Fator de Ligação ao Core/análise , Marcadores Genéticos , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/terapia , Proteínas de Neoplasias/análise , Neoplasia Residual , Proteínas de Fusão Oncogênica/análise , Proteínas de Plantas , Proteína 1 Parceira de Translocação de RUNX1RESUMO
Advances in molecular genetics have revolutionized our understanding of acute myeloid and lymphoblastic leukemia. Structural and numerical chromosomal aberrations are common, and their detection is vital for leukemia diagnosis, risk stratification, and monitoring of response to therapy. Fusion proteins resulting from chromosomal translocations are necessary but not sufficient for leukemogenesis, and there is intense research activity to elucidate the cooperating molecular abnormalities that may be suitable targets for novel therapeutic approaches. Candidate gene approaches have identified mutations in kinases and transcription factors in a proportion of patients, but more comprehensive genomic approaches are required. Gene expression profiling accurately classifies known subtypes of acute leukemia and has highlighted potentially leukemogenic abnormalities in gene expression. Newer techniques, such as single-nucleotide polymorphism arrays to analyze changes in gene copy number and zygosity, cancer genome sequencing, and RNA interference, are promising tools to identify mutations, although at present, data from these approaches are limited. This review provides an overview of these techniques in clinical practice and as research tools to develop new therapeutic approaches in pediatric leukemia.
Assuntos
Aberrações Cromossômicas , Perfilação da Expressão Gênica , Leucemia Mieloide/genética , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Doença Aguda , Criança , Análise Mutacional de DNA , Diagnóstico Diferencial , Humanos , Leucemia Mieloide/patologia , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Medição de RiscoRESUMO
PURPOSE: The recognition of a number of leukemia-specific cytogenetic abnormalities and their role as independent prognostic factors have provided considerable insights into leukemia pathogenesis and have paved the way to adopt risk-adapted treatment. However, approximately 50% of newly diagnosed acute myeloid leukemia (AML) have a normal karyotype. There has therefore been much interest in identifying molecular markers that could help to improve the prognostic stratification of patients with normal-karyotype AML. EXPERIMENTAL DESIGN: Consecutive untreated AML patients (n = 67) from a single institution all with normal karyotype were analyzed for the presence of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/enhancer binding protein-alpha), for internal tandem duplications (ITD) of the tyrosine kinase receptor gene FLT3 (for fms-like tyrosine kinase 3), and for expression of the BAALC gene (for brain and acute leukemia, cytoplasmic). RESULTS: 17.9% of normal-karyotype AML had mutations in the CEBPA gene, and 28.4% had FLT3-ITD; 65.7% (44 of 67) had high BAALC expression and 34.3% (23 of 67) had low BAALC expression. Patients with CEBPA mutations had a very favorable course of their disease. Median disease-free survival (DFS) and overall survival (OS) were 33.5 and 45.5 months, respectively, compared with 10 (e.g., 12 months in patients without CEBPA mutations; P = 0.0017; P = 0.0007). AML patients with FLT3-ITD had significantly shorter median DFS (P = 0.0328) and OS (P = 0.0148) than patients without FLT3-ITD. High BAALC expression predicted for a shorter DFS (P = 0.0152) and OS (P = 0.0210) compared with AML with low BAALC expression; 53.7% of normal-karyotype AML had neither FLT3-ITD nor CEBPA mutations. We found that high BAALC expression in normal-karyotype AML with neither FLT3-ITD nor CEBPA mutations (18 of 67) indicates adverse prognosis for both DFS and OS (P = 0.0001; e.g., P = 0.0001) compared with the group with low BAALC expression and absent FLT3-ITD and CEBPA mutations (18 of 67). Thus, BAALC expression represents a novel prognostic marker particularly for normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations. CONCLUSIONS: Assessment of CEBPA mutations, FLT3-ITD, and BAALC expression permits to split normal-karyotype AML into clinically distinct subgroups.
Assuntos
Leucemia Mieloide/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Antígenos CD/análise , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Cariotipagem , Leucemia Mieloide/genética , Leucemia Mieloide/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Prognóstico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Medição de Risco/métodos , Análise de Sobrevida , Tirosina Quinase 3 Semelhante a fmsRESUMO
Quantification of minimal residual disease (MRD) reveals significant prognostic information in patients treated for acute myeloid leukemia (AML). The application of multiparameter flow cytometry (MFC) for MRD assessment has resulted in significant prognostic information in selected cases in previous analyses. We analyzed MRD in unselected patients with AML in complete remission (CR) after induction (n = 58) and consolidation (n = 62) therapies. By using a comprehensive panel of monoclonal antibodies we identified at least one leukemia-associated aberrant immunophenotype (LAIP) in each patient. The degree of reduction between diagnosis and CR in LAIP-positive cells (log difference [LD]) as a continuous variable was significantly related to relapse-free survival (RFS) both after induction (P = .0001) and consolidation (P = .000 08) therapies, respectively. The LD determined after consolidation therapy was the only parameter related to overall survival (OS) (P = .005). Separation of patients based on the 75th percentile of LD after consolidation therapy resulted in groups with highly different RFS (83.3% versus 25.7%, P = .0034) and OS (87.5% versus 51.4%, P = .0507) at 2 years. Multivariate analysis identified LD as an independent prognostic factor for RFS at both checkpoints. MFC-based quantification of MRD reveals important prognostic information in unselected patients with AML in addition to cytogenetics and should be further evaluated and used in clinical trials.
Assuntos
Citometria de Fluxo/métodos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/mortalidade , Neoplasia Residual/diagnóstico , Neoplasia Residual/mortalidade , Doença Aguda , Adolescente , Adulto , Idoso , Antineoplásicos/uso terapêutico , Medula Óssea , Estudos de Coortes , Feminino , Humanos , Imunofenotipagem , Leucemia Mieloide/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neoplasia Residual/genética , Prognóstico , Recidiva , Indução de Remissão , Fatores de RiscoRESUMO
BACKGROUND: The role of allogeneic bone marrow transplantation (BMT) in the consolidation of young adults with acute myeloid leukemia (AML) with matched sibling donors (MSD) is controversial. Although BMT is associated with increased event free survival compared with intensive chemotherapy (CT) consolidation, BMT also is associated with increased treatment-related mortality and likely decreased quality of life and life expectancy in patients who do not develop recurrent disease. METHODS: The authors used decision analysis to compare three strategies for maximizing quality-adjusted life years (QALYs) in patients with AML in first remission with an MSD: BMT All, BMT None (consolidation CT only), or BMT in high-risk patients, as defined by baseline cytogenetic testing (Test strategy). A second decision-analysis tree was then constructed that compared BMT with CT specifically for patients with intermediate cytogenetics. RESULTS: Using expected QALYs as the outcome measure, the Test, BMT All, and BMT None strategies were associated with 20.10 QALYs, 19.63 QALYs, and 18.38 QALYs, respectively. Thus, the Test strategy, with CT for low-risk patients and BMT for intermediate risk and high-risk patients, was expected to be the optimal strategy. In the intermediate cytogenetic decision analysis, although the expected QALY for BMT recipients was higher compared with CT recipients (19.78 QALYs vs. 18.75 QALYs), because of uncertainty in variable estimates, the optimal choice was less clear. CONCLUSIONS: CT consolidation is a reasonable option for patients with AML who have favorable cytogenetics, even if an MSD is available. This model provides a framework from which patients with AML and their physicians can make decisions about consolidation therapy.
Assuntos
Antineoplásicos/uso terapêutico , Transplante de Medula Óssea , Técnicas de Apoio para a Decisão , Leucemia Mieloide/terapia , Doença Aguda , Citogenética , Teste de Histocompatibilidade , Humanos , Leucemia Mieloide/genética , Probabilidade , Anos de Vida Ajustados por Qualidade de Vida , Indução de Remissão , Transplante HomólogoRESUMO
Development of further diagnostic and prognostic tools in myelodysplastic syndromes (MDS) is warranted. In this study we tested two molecular markers in bone marrow of MDS patients: Galpha16, a hematopoiesis-specific G protein alpha subunit serving as an intracellular marker of hematopoietic activity, and 5'-lipoxygenase (5-LO), a putative differentiation marker. The results were correlated with clinical and laboratory features and outcome. Bone marrow mononuclear cells were evaluated by block cycler PCR in 32 patients for Galpha16 and in 25 patients for 5-LO. In 12 patients cDNA analyzed by the block cycler method was quantified by real-time quantitative (RTQ) PCR for both Galpha16 and 5-LO. The results confirmed concordance of the two methods. All FAB and WHO subtypes were positive for at least one of the two genes. Strikingly, only 1 of 11 patients with refractory anemia with ringed sideroblasts was positive for Galpha16. No correlation between Galpha16 or 5-LO and bone marrow cellularity or cytogenetic risk factors (IPSS) was detected. Only combined evaluation of Galpha16 and 5-LO expression showed a correlation with extent of anemia and thrombocytopenia. A relationship between the two markers and preleukemic duration was found. Our findings show that both Galpha16 and 5-LO can be expressed in MDS, reflecting proliferation and differentiation processes in this disease. Different expression patterns of the two molecules indicate that proliferation and differentiation of hematopoietic cells may occur independently. These parameters could constitute a new class of risk factors in MDS. Determination of Galpha16 and 5-LO expression may provide a tool for observing growth and maturation in these diseases.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Síndromes Mielodisplásicas/patologia , Doença Aguda , Idoso , Biomarcadores/análise , Células da Medula Óssea/patologia , Diferenciação Celular/genética , Divisão Celular/genética , Transformação Celular Neoplásica/genética , Distribuição de Qui-Quadrado , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Humanos , Leucemia Mieloide/etiologia , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Masculino , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Resultado do TratamentoRESUMO
BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Citometria de Fluxo/métodos , Células Tumorais Cultivadas/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Anexina A5 , Apoptose , Benzimidazóis , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Corantes Fluorescentes , Humanos , Irinotecano , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Necrose , Propídio , Ácido Tranexâmico , Células Tumorais Cultivadas/patologia , Vincristina/farmacologiaRESUMO
In this report we show the chromosomal changes seen in a group of 303 Mexican patients with de novo Acute Myeloblastic Leukemia (AML). Two hundred forty-two patients were diagnosed and treated at two hospitals affiliated with the Instituto Mexicano del Seguro Social (IMSS). These are the Centro Medico Nacional Siglo XXI and Centro Medico La Raza Hospitals; the remaining 61 patients were diagnosed and treated at the Hospital General de Mexico (HGM). Clonal abnormalities were detected in 75.6% of the patients; this result agrees with what has been reported in other large series of AML studies. The incidence of changes per hospital was similar in patients from the IMSS hospitals (72-75%), while an increase was seen in patients from the HGM (85.2%). The chromosomal changes seen in this study in order of frequency were: t(15;17)[18.8%], t(9;22)[9.2%], miscellaneous chromosomal changes (mainly rearrangements of chromosomes 1,2,3,12y17)[8.2%], abnormalities of 16q22 [7.3%], t(8;21)[6.3%], -7/del(7q)[5.6%], t(6;9)[5.3%], and abnormalities of 11q23 [4.6%]. We reported an increase in the incidence of certain types of chromosomal changes seen in cases of AML, in comparison with reports from other countries. These differences could be due to methodological variations, although ethnic, socioeconomic and nutritional differences must not be disregarded. We support this finding when comparing distribution of changes in the population of patients seen in the IMSS hospitals with those from the HGM; the main difference lies in the socioeconomic level.
Assuntos
Aberrações Cromossômicas , Leucemia Mieloide/genética , Doença Aguda , Adolescente , Adulto , Idoso , Deleção Cromossômica , Cromossomos Humanos Par 15/ultraestrutura , Cromossomos Humanos Par 17/ultraestrutura , Células Clonais/ultraestrutura , Feminino , Hospitais Gerais , Hospitais Públicos , Humanos , Incidência , Leucemia Mieloide/epidemiologia , Leucemia Mieloide/patologia , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/ultraestrutura , Cromossomo Filadélfia , Previdência Social , Fatores Socioeconômicos , Translocação GenéticaRESUMO
Serial peripheral blood specimen from eight adult patients after sex-mismatched bone marrow transplantation (BMT) for Chronic Myeloid Leukemia (CML) (N = 3). Ewing sarcoma (N = 1), Acute Myeloid Leukemia (AML) in second remission (N = 1), Acute Lymphoid Leukemia (ALL) (N = 1), of multiple myeloma (N = 2) were analyzed by the simultaneous immunophenotypic (moAbs/ APAAP-staining) and genotypic analysis (for X and Y chromosomes) of interphase cells to characterize mixed chimerism, residual host cells, and leukemic relapse. Although a stable donor chimerism for T cells, myelomonocytic cells, and granulocytes was developed in seven of the eight patients at Days +21 to +28 post BMT, 0.5 to 1% host cells of different lineages remained continuously in five of the eight patients post BMT (> day 100). In two patients, one with common ALL and the other with multiple myeloma and long-term stable mixed chimerism, a tumor cell relapse was detected first in a sample at Day +176 and confirmed at Day +294. These malignant cells were genotypically of host origin and presented phenotypes identical to those at diagnosis. In the three patients with CML, residual host cells were identified as CD13 (Patient 3) of CD13/CD34 (Patient 4) positive and in one case as CD4/CD8 positive (Patient 7). Since no exclusive antigenic marker is available for this discrimination in these CML patients, normal host hematopoiesis can interfere with the identification of residual disease. Therefore, the identification of the bcr-abl transcripts by a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) was included in this analysis. Patient 3 was bcr-abl positive at [Days +21, +28, +35, and +311, but negative at Days +121 and +400; Patient 4 was bcr-abl positive at only Day +166 post BMT. These results are interpreted as signaling a continuing risk of relapse. In Patient 7, the bcr-abl RT-PCR was negative at Days +142, +166, and +237. Thus, the combination of the simultaneous immunophenotypic and genotypic analysis and the bcr-abl detection by RT-PCR clearly improves the discrimination between malignant cells and normal residual host cells.
Assuntos
Transplante de Medula Óssea , Leucemia/genética , Mieloma Múltiplo/genética , Sarcoma de Ewing/genética , Doença Aguda , Adulto , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Proteínas de Fusão bcr-abl/análise , Genótipo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia/terapia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide/genética , Leucemia Mieloide/terapia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Mieloma Múltiplo/terapia , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Recidiva , Sarcoma de Ewing/terapiaAssuntos
Biomarcadores/análise , Purging da Medula Óssea , Transplante de Medula Óssea , Bussulfano , Ciclofosfamida , Marcadores Genéticos , Interleucina-2 , Leucemia Mieloide/terapia , Mesna , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Doença Aguda , Adolescente , Sequência de Bases , Separação Celular , Criança , Pré-Escolar , Protocolos Clínicos , Terapia Combinada , Resistência a Medicamentos , Estudos de Avaliação como Assunto , Vetores Genéticos , Células-Tronco Hematopoéticas , Humanos , Fatores Imunológicos , Lactente , Consentimento Livre e Esclarecido , Canamicina Quinase , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Proteínas Recombinantes de Fusão , Indução de Remissão , Projetos de Pesquisa , Retroviridae/genética , Segurança , Transplante AutólogoRESUMO
Applying conventional statistical techniques to cytogenetic data usually faces the problem of working with small numbers and many classes. We describe two techniques, one based on a binomial test procedure, the other on Monte Carlo simulations, aimed at studying the non-randomness of chromosomal aberrations. Both techniques were used to study the distribution of breakpoints involved in variant Philadelphia translocations in chronic myeloid leukaemia. The results showed that 28 bands were non-randomly rearranged (P less than or equal to 0.05). Furthermore, the probabilities calculated from the binomial test procedure were close to those calculated from the Monte Carlo simulations.