RESUMO
Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.
Assuntos
Linfócitos T CD8-Positivos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva , Receptor de Morte Celular Programada 1 , Humanos , Mesilato de Imatinib/uso terapêutico , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Idoso , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Adulto JovemRESUMO
Adult acute lymphoblastic leukemia (ALL) is associated with poor outcomes. ALL is initiated by primary aberrations, but secondary genetic lesions are necessary for overt ALL. In this study, we reassessed the value of primary and secondary aberrations in intensively treated ALL patients in relation to mutator enzyme expression. RT-PCR, genomic PCR, and sequencing were applied to evaluate primary aberrations, while qPCR was used to measure the expression of RAG and AID mutator enzymes in 166 adult ALL patients. Secondary copy number alterations (CNA) were studied in 94 cases by MLPA assay. Primary aberrations alone stratified 30% of the patients (27% high-risk, 3% low-risk cases). The remaining 70% intermediate-risk patients included BCR::ABL1pos subgroup and ALL lacking identified genetic markers (NEG ALL). We identified three CNA profiles: high-risk bad-CNA (CNAhigh/IKZF1pos), low-risk good-CNA (all other CNAs), and intermediate-risk CNAneg. Furthermore, based on RAG/AID expression, we report possible mechanisms underlying the CNA profiles associated with poor outcome: AID stratified outcome in CNAneg, which accompanied most likely a particular profile of single nucleotide variations, while RAG in CNApos increased the odds for CNAhigh/IKZF1pos development. Finally, we integrated primary genetic aberrations with CNA to propose a revised risk stratification code, which allowed us to stratify 75% of BCR::ABL1pos and NEG patients.
RESUMO
Non-small cell lung cancer is the predominant form of lung cancer and is associated with a poor prognosis. MiRNAs implicated in cancer initiation and progression can be easily detected in liquid biopsy samples and have the potential to serve as non-invasive biomarkers. In this study, we employed next-generation sequencing to globally profile miRNAs in serum samples from 71 early-stage NSCLC patients and 47 non-cancerous pulmonary condition patients. Preliminary analysis of differentially expressed miRNAs revealed 28 upregulated miRNAs in NSCLC compared to the control group. Functional enrichment analyses unveiled their involvement in NSCLC signaling pathways. Subsequently, we developed a gradient-boosting decision tree classifier based on 2588 miRNAs, which demonstrated high accuracy (0.837), sensitivity (0.806), and specificity (0.859) in effectively distinguishing NSCLC from non-cancerous individuals. Shapley Additive exPlanations analysis improved the model metrics by identifying the top 15 miRNAs with the strongest discriminatory value, yielding an AUC of 0.96 ± 0.04, accuracy of 0.896, sensitivity of 0.884, and specificity of 0.903. Our study establishes the potential utility of a non-invasive serum miRNA signature as a supportive tool for early detection of NSCLC while also shedding light on dysregulated miRNAs in NSCLC biology. For enhanced credibility and understanding, further validation in an independent cohort of patients is warranted.
RESUMO
Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.
Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Neoplasia Residual/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We report on long-term outcomes of 267 patients (pts) with chronic myeloid leukemia (CML) treated with imatinib administered as initial therapy. The median follow-up time was 11.4 years. 38 (14.2%) pts attempted to discontinue imatinib therapy after achieving sustained deep molecular response (≥MR4), 15 of them achieved treatment-free remission (TFR). 144 pts (53.9%) have been switched to other TKI during the follow-up period. The estimated OS for 267 pts for 10, 15 and 18 years was 98.8%, 75.6% and 52.1%. According to prognostic scores (Sokal, Euro, ELTS), OS and PFS were significantly better in low-risk pts than in high-risk pts. According to ELTS, low-risk pts have a better chance of achieving MR4 than both intermediate (p = 0.03) and high-risk (p = 0.04) groups. It suggests that a specific ELTS-adapted treatment could help to optimize treatment results. In the study population, imatinib has demonstrated sustained efficacy and an acceptable rate of late toxic effects.
Assuntos
Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Antineoplásicos/efeitos adversos , Progressão da Doença , Humanos , Mesilato de Imatinib/efeitos adversos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Resultado do TratamentoRESUMO
This study aimed to investigate the association between selected variants of genes related to dopamine metabolism pathways and the risk of and progression of Parkinson's disease (PD). This prospective cohort study was conducted in one academic teaching hospital. The study was conducted on 126 patients diagnosed with idiopathic Parkinson's disease. Blood samples were collected to conduct a genotyping of MAOB, DRD1, DRD2, and DDC genes. Genotype and allele frequencies of MAOB (rs1799836) variants were not associated with the course of PD. Genotype and allele frequencies of DRD2 (rs2283265) variants were associated with risk of dementia (p = 0.001) and resulted in parts II and III of the UPDRS scale (p = 0.001). Genotype and allele frequencies of DRD2 (rs1076560) variants were associated with risk of dementia (p = 0.001) and resulted in parts II and III of the UPDRS scale (p = 0.001). Genotype and allele frequencies of DDC (rs921451) variants were not associated with the course of PD.
Assuntos
Demência , Doença de Parkinson , Proteínas de Transporte/genética , Dopa Descarboxilase/genética , Genótipo , Humanos , Monoaminoxidase/genética , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Receptores de Dopamina D2/genéticaRESUMO
PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.
Assuntos
Desaminases APOBEC/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas Musculares/metabolismo , Fator de Transcrição PAX7/metabolismo , Desaminases APOBEC/genética , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , DNA Metiltransferase 3BRESUMO
Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients' outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37-0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.
Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Idoso , Cladribina/uso terapêutico , Citarabina/uso terapêutico , Daunorrubicina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Variantes Farmacogenômicos , Polônia/epidemiologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population. METHODS: In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers. RESULTS: We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired. CONCLUSIONS: Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.
Assuntos
Células Satélites de Músculo Esquelético , Teratoma , Animais , Diferenciação Celular , Camundongos , Células-Tronco Embrionárias Murinas , Desenvolvimento Muscular/genética , Músculo Esquelético , Fatores de Transcrição NFI , Fator de Transcrição PAX7/genética , Teratoma/genéticaRESUMO
Philadelphia-negative myeloproliferative neoplasms (MPNs) are a diverse group of diseases whose common feature is the presence of V617F mutation of the JAK2 gene. In the era of novel therapeutic strategies in MPNs, such as JAK-inhibitor therapy, there is a growing need for establishing high sensitive quantitative methods, which can be useful not only at diagnosis but also for monitoring therapeutic outcomes, such as minimal residual disease (MRD). In this study, we compared the qPCR and ddPCR methods and their clinical utility for diagnosis, prognostication, and treatment monitoring of MPNs with JAK2 V617F mutation in 63 MPN patients of which 6 were subjected to ruxolitinib treatment. We show a high conformance between the two methods (correlation coefficient r = 0.998 (p < 0.0001)). Our experiments revealed high analytical sensitivity for both tests, suggesting that they are capable of detecting the JAK2 V617F mutation at diagnosis of MPN with a limit of detection (LoD) of 0.12% for qPCR and 0.01% for ddPCR. The alterations of JAK2 V617F allele burden in patients treated with ruxolitinib were measured by both methods with equal accuracy. The results suggest an advantage of ddPCR in monitoring MRD because of allele burdens below the LoD of qPCR. Overall, the clinical utility of qPCR and ddPCR is very high, and both methods could be recommended for the routine detection of the V617F mutation at diagnosis, though ddPCR will probably supersede qPCR in the future due to cost-effectiveness.
Assuntos
Alelos , Neoplasias Hematológicas/genética , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Cromossomo Filadélfia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Idoso , Substituição de Aminoácidos , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Janus Quinase 2/sangue , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/tratamento farmacológico , Rituximab/administração & dosagemAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Duplicação Gênica , Cariótipo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Cladribina/administração & dosagem , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Humanos , Leucemia Mieloide Aguda/mortalidade , Resultado do TratamentoRESUMO
Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Variações do Número de Cópias de DNA , Cariótipo , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Cromatina/genética , Pontos de Quebra do Cromossomo , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Regulação Leucêmica da Expressão Gênica , Loci Gênicos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutagênese , Mutação , Motivos de Nucleotídeos , RNA Mensageiro/genética , Adulto JovemRESUMO
More than 95% of patients with detected translocation t(9;22), is characterized by the fusion between exons e13 or e14 of BCR gene, which are located in major breakpoint cluster region (M-bcr) and exon a2 of ABL gene. These fusions are described as b2a2 (e13a2) and b3a2 (e14a2). Other fusions of exons e1, e6, e8, e12, e19, e20 of BCR gene with exons a2 or a3 of ABL gene occur very rarely and lead to formation of so called unusual fusion BCR-ABL genes. The aim of this study is to describe long-term observations of the occurrence and routine procedure in the diagnosis of atypical variants of the fusion gene BCR-ABL in a population of patients with chronic myeloid leukemia (CML). It was found that the vast majority of patients with detected BCR-ABL transcripts were b3a2 and b2a2. Other detected variants, which are described as rare were: e1a2, b2a3, b3a3, c3a2, e6a2, e6a3. At the stage of diagnosis as well as during monitoring of the effects of treatment, molecular methods which are based on polymerase chain reaction were used (multiplex RT-PCR, nested RT-PCR, RQ-PCR). Multiplex RT-PCR reaction gave possibility to detect variants of the fusion BCR-ABL gene in one reaction and was crucial in the selection of appropriate test used for further monitoring of the disease and the effectiveness of treatment. This paper proposes a scheme for dealing with the diagnosis and monitoring of minimal residual disease (MRD) in patients with CML treated with tyrosine kinase inhibitors (TKIs) in the presence of rare fusion of the BCR and ABL genes.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Proteínas Tirosina Quinases/antagonistas & inibidores , Adulto JovemRESUMO
Introduction of tyrosine kinase inhibitors (TKIs) in the therapy of chronic myeloid leukemia have been significantly improved the results of the treatment and prognosis of CML patients. Despite of high efficacy of TKIs therapy, resistance is developing in substantial percentage of patients, which accounts for up to 40% after several years of treatment. There are several identified mechanisms of resistance to TKIs. The presence of ABL kinase domain point mutation, which could be detected by molecular methods is one of them. The aim of the study was to screen 60 CML patients resistant to TKI therapy for the presence of ABL point mutation. ABL mutation was detected in 19 (31,6%) patients. In four cases with detected mutation the disease has progressed to blast crisis. Investigation of ABL mutation occurrence can help in finding the cause of resistance to TKI therapy in some patients suffering from CML.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
Beta carotene (BC) is a nutritional compound widespread in foods which can influence vital cellular functions--differentiation, proliferation and apoptosis of normal and cancer cells. However its role in the carcinogenesis remains controversial. We performed a microarray expression analysis in three human acute leukemia cell lines (HL-60, U937 and TF-1) exposed to 10mM BC and found that BC stimulated the apoptosis in all studied cell lines. This effect was most evident in the HL-60 cell line and correlated with increased expression of proapoptotic BAX and CAPN2 genes. The micro-array findings were replicated by the quantitative BAX and CAPN2 expression analysis using real-time PCR and by Western Blot on protein level. The biological tests (TUNEL method) for apoptosis showed consistent proapoptotic effects in all studied cell lines. In this paper the stimulatory effect of BC on apoptosis (enhanced expression of proapoptotic genes and proteins) in human acute myeloid leukemia cells was confirmed. The most potent activation of apoptosis in the HL-60 cells is in line with other investigators observations suggesting distinct molecular mechanism of apoptosis stimulation by BC in different human acute myeloid leukemia cells.
Assuntos
Apoptose/genética , Calpaína/genética , Proteína X Associada a bcl-2/genética , beta Caroteno/metabolismo , Expressão Gênica/fisiologia , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais Cultivadas , Células U937RESUMO
Chronic myeloid leukemia is a clonal disorder caused by formation of chimeric BCR/ABL gene and bcr/abl protein with abnormally high tyrosine kinase activity. The use of imatinib--the first tyrosine kinase inhibitor results in achievement of hematologic, cytogenetic and molecular response in majority of patients. However despite its high efficacy not all patients respond to imatinib, whereas others lose an initial response. Imatinib is a substrate of human organic cation transporter-1 (hOCT1), which actively delivers the drug into the cells, and efflux transporters. To identify potential imatinib failures, we investigated the expression of hOCT1 using real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR) in 155 CML patients. Patients with low pretreatment hOCT1 expression had inferior major and complete molecular response (MMR and CMR) rates (p = 0.0001, p = 0.0001) achieved any time or at 18 months of imatinib treatment (p = 0.023, p = 0.022). The expression of hOCT1 is important in determining the clinical response to imatinib. The analysis of hOCT1 expression by RQ-PCR is convenient and clinically available, and the results could help in introduction of optimal first line therapy in CML patients.
Assuntos
Biomarcadores Tumorais/análise , Marcadores Genéticos/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Transportador 1 de Cátions Orgânicos/genética , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Benzamidas , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-IdadeRESUMO
Allogeneic hematopoietic stem cell transplantation (alloSCT) is a curative treatment for many patients suffering from malignant and non-malignant hematological disorders. Successful transplantation is a process that requires the engraftment of transplanted pluripotent hematopoietic stem cells which re-establish normal hematological and immunological systems. Distinguishing between host and donor origin of bone marrow and blood cells is vitally important for monitoring of the engraftment process. One of the most useful tools for engraftment monitoring is the assessment of hematopoietic chimerism. Which occurs after alloHSCT and describes the percentage of donor hematopoietic and lymphoid cells in a transplant recipient. 38 adult patients, after alloSCT performed in Katedra i Klinika Hematologii Collegium Medicum UJ entered the study and the total number of transplantations was 43. The evaluation of hematopoietic chimerism was based on PCR amplification of polymorphic non-coding DNA sequences--short tandem repeats (STR-PCR). The main tool was a semiquantitative method--fragment length analysis. The product of amplification was analyzed using the sequencer. The second method was based on a quantitative Real Time PCR technique (RQ-PCR) based on SYBRgreen chemistry. There were performed amplification of biallelic non-coding DNA sequences with short insertions or deletions. Hematopoietic chimerism evaluations were performed on +30, +60, +90, +120, +150, +180, +270 and +360 day and then every 6 months post alloSCT on peripheral blood or bone marrow samples. STR-PCR and RQ-PCR chimerism assays were compared and results evidenced the greater sensitivity of RQ-PCR method. There were not crucial differences in the results of chimerism evaluation obtained by means of these two methods. The analysis of chimerism kinetics after allogeneic stem cell transplantation allowed to modify the post-transplantation-treatment in 3 patients after alloNMSCT leading to increase of donor-origin hematopoiesis in transplant recipients (in 2 pts decision of DLI, 1 of them withdrawal of immunosuppression, 1 pt giving G-CSF). The results of chimerism monitoring confirmed that the failure of achieving a CC or lost of CC can predict the relapse of the disease.
Assuntos
Transplante de Medula Óssea/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Quimeras de Transplante/genética , Adulto , Sequência de Bases , Hematopoese/genética , Células-Tronco Hematopoéticas/química , Humanos , Reação em Cadeia da Polimerase/métodos , Subpopulações de Linfócitos T/patologiaRESUMO
Chronic Myeloid Leukemia (CML), belonging to mieloproliferative syndromes, is one of the myeloproliferative clonal hyperplasia. It is caused by the Philadelphia chromosome resulting from the reciprocal translocation, t(9;22) between the long arms of chromosomes 9 and 22. This results in the production of fusion BCR-ABL transcript and chimeric protein--tyrosine kinase activity. This protein leads to increased proliferation, resistance to apoptosis, and worse adhesion of CML cells. Molecular analysis are very important in the era treatment of CML by tyrosine kinase inhibitors (TKI). Constant monitoring of the level of BCR-ABL transcript aimed at monitoring response to medical treatment as well as early detection of resistance to TKI therapy. The most common causes of resistance are point mutations ABL kinase domain of the BCR-ABL gene. In this aim, the biological material used (peripheral blood) derived from 58 patients of the Department of Hematology, Jagiellonian University Collegium Medicum. The isolated RNA was performed in successive stages: RT-PCR, RQ-PCR to a semi-nested PCR. In order to detect point mutations ABL kinase domain technique used direct sequencing of the product obtained in response to a semi-nested PCR. Using this technique allow in do not only a rapid detection of point mutations but also identification of its position in the ABL domain, type of mutation (e.g., T3151), as well as nucleotide and the amino acid substitution. The most common point mutations detected were T3151 and M244V.
Assuntos
Resistência a Medicamentos/genética , Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual , Proteínas Tirosina Quinases/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Monitoring of chronic myeloid leukemia treatment efficacy requires very sensitive methods of BCR-ABL gene detection based on polymerase chain reaction (PCR). The lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories was the cause of an international multicenter trial initiation with the participation of 133 laboratories in 24 European countries cooperating within the "EUTOS for CML" project. Pracownia Diagnostyki Molekularnej Kliniki Hematologii is taking part in standardisation rounds organised since 2005. The compatibility of methodology used in Pracownia with European Leukemia Net (ELN) standards was confirmed, and correction factor for the expression of RQ-PCR results in an international scale was calculated. Pracownia was charge by ELN with a task of conducting the standardisation in polish molecular biology laboratories. Test probes were prepared and sent to eight cooperating laboratories. The results obtained in six laboratories were concordant with results from laboratory in Krakow after conversion to international scale, therefore it was possible to calculate individual correction factors. The participation of polish laboratories in international standardization process created the opportunity for unification of BCR-ABL quantification methodologies with recommendations of international experts, and showed that the quality of analyses performed in majority of them was satisfactory enough to calculate correction factor and to express the RQ-PCR results in widely accepted international scale.