Successful Pregnancy and Delivery in a Patient with Chronic Myeloid Leukemia while on Dasatinib Therapy

Here we report the case of an 18-year-old woman with chronic myeloid leukemia (CML) who became pregnant while undergoing treatment with dasatinib. Before pregnancy, she received imatinib mesylate therapy but could not tolerate the treatment. The regimen was then changed to dasatinib at a dose of 70 mg b.i.d. While she was in hematological remission and on dasatinib therapy, she became pregnant. The unplanned pregnancy was identified after the patient had experienced four weeks of amenorrhea. Because the patient elected to continue the pregnancy to term, dasatinib was stopped immediately. Meanwhile, CML hematological relapse occurred and then she was treated with interferon-α (IFN-α) (9 million IU/day) throughout the pregnancy without a complete hematological response. She successfully gave birth to a male baby at 33 weeks by cesarean section delivery with no sequelae or malformations. Although this experience is limited to a single patient, it provides a useful contribution for counselling patients inadvertently exposed to dasatinib during pregnancy

Evaluation of the expression of genes and miRNAs related to DNA damage repair in patients with chronic myeloid leukemia at diagnosis and blast crisis

A leucemia mieloide crônica (LMC) é caracterizada pela translocação (9;22) que dá origem ao gene quimérico BCR-ABL. Este gene codifica uma proteína com atividade tirosina quinase p210 constitutivamente ativa. Os três mecanismos envolvidos na patogênese da LMC são o aumento da proliferação celular, alteração da adesão celular ao estroma e matriz medular e inibição da apoptose. A introdução dos inibidores de tirosina quinase (ITQ) mudou profundamente o curso natural da LMC, reduzindo drasticamente o risco de progressão para a crise blástica, porém uma parcela dos pacientes progride mesmo com o tratamento sucessivo com diferentes drogas e o manejo desses pacientes continua sendo um grande desafio para os clínicos. Embora a proteína BCR-ABL1 seja o principal agente na patogênese da LMC, várias outras vias podem contribuir para a progressão da doença. O acúmulo de anormalidades cromossômicas e mutações é uma característica da progressão da LMC e são mais frequentes nos pacientes na crise blástica do que nos em fase crônica e podem ocorrer tanto pelo aumento da taxa de dano ao DNA como pela alteração das vias de reparo e expressão de miRNAs, levando as células a um quadro de instabilidade genética. Objetivo: Avaliar a expressão de genes e miRNAs relacionados com as vias de reparo do dano ao DNA em pacientes com LMC ao diagnóstico (LMC-FC ao diagnóstico), na crise blástica (LMC-CB) e em doadores saudáveis e avaliar o potencial destes como possíveis marcadores para a progressão da LMC e para a resposta ao tratamento com inibidores de tirosina quinase. Casuística e métodos: As expressões dos genes GADD45G, ATM, TP53, RAD9A e LIG1 e dos miRNAs miR-17-5p, miR-34a, miR-150, miR-155, miR-221 e miR-222 foram avaliadas em 90 amostras sendo 58 de pacientes com LMC em fase crônica ao diagnóstico, 17 de pacientes como LMC em crise blástica e 15 amostras de doadores saudáveis pela técnica de RT-QPCR utilizando-se o sistema Taqman de sondas de hibridização e quatificação relativa 2-Ct. Resultados: Os resultados deste estudo mostraram que tanto a expressão de genes envolvidos no sistema de reparo como miRNAs estão alterados nas amostras de pacientes em crise blástica. A expressão dos genes ATM, TP53 e ERCC1 apresentam fold change de 1,34 (p = 0,030), 1,58 (p = 0,007) e 0,65 (p < 0,000), respectivamente, quando comparamos amostras de pacientes com LMC-FC ao diagnóstico e amostras de pacientes LMC-CB. A expressão dos miRNAs também apresentou alteração de expressão, especialmente do miR-17-5p, miR-34a e miR-155, com fold change de 1,76 (p < 0,000), 0,098 (p < 0,000) e 0,25 (p < 0,000) respectivamente. A expressão do gene ERCC1 e do miR-155 apresentaram correlação com a sobrevida livre de eventos e a do gene ATM e miR-150 apresentaram correlação com a resposta molecular aos 6 e 12 meses de tratamento com ITQ. Conclusões: A expressão de diferentes genes moduladores da resposta ao dano DNA, como ATM e TP53 estão alteradas nos pacientes com LMC-CB, prejudicando a cascata de sinalização dependente desses genes. A maior expressão do gene ERCC1 contribui para o surgimento de alterações cromossômicas adicionais através do mecanismo de anelamento de fita simples podendo aumentar a ocorrência de alterações cromossômicas adicionais. Já a maior expressão do miR-155 contribui para a redução do mecanismo de pareamento incompatível, reduzindo a expressão dos genes MLH1, MSH2 e MSH6 podendo ocasionar aumento do número de mutações como por exemplo, as mutações do domínio tirosina quinase. Além disso, a maior expressão do 24 gene ERCC1 e do miR-155 podem ser utilizados como marcadores preditivos de surgimento de eventos e a maior expressão do gene ATM e miR-150, como marcadores da resposta molecular ao tratamento com ITQ aos 6 e 12 mesesChronic myeloid leukemia (CML) is characterized by translocation (9; 22) that gives rise to the chimeric BCR-ABL gene. This gene encodes a p210 protein with constitutively active tyrosine kinase activity. The three mechanisms involved in CML pathogenesis are the increase in cell proliferation, alteration of cell adhesion to the stroma and bone marrow matrix, and apoptosis inhibition. The introduction of tyrosine kinase inhibitors (TKI) has profoundly changed CML\'s natural course, drastically reducing blast crisis progression. However, a portion of patients progresses even with successive treatment with different drugs, and the management of these patients remains a significant challenge for clinicians. Although the BCR-ABL1 protein is the principal-agent in CML\'s pathogenesis, several other pathways can contribute to its progression. The accumulation of chromosomal abnormalities and mutations is a characteristic of the progression of CML and is more frequent in patients in blast crisis than in those in chronic phase and can occur both by increasing the rate of DNA damage and by altering the repair and expression pathways of miRNAs, leading the cells to a picture of genetic instability. Objective: To evaluate the expression of genes and miRNAs related to DNA damage repair pathways in patients with CML at diagnosis (CML-CP at diagnosis), blast crisis (CML-BC), and in healthy donors and assess their potential as possible markers for CML progression and response to treatment with tyrosine kinase inhibitors. Casuistry and methods: The expressions of the GADD45G, ATM, TP53, RAD9A, and LIG1 genes and miRNAs miR-17-5p, miR-34a, miR-150, miR-155, miR-221, and miR-222 were evaluated in 90 samples 58 from patients in CML-CP at diagnosis, 17 from patients with CML-BC, and 15 samples from healthy donors using the RT-qPCR technique using the Taqman probes and relative quantification 2 -Ct. Results: This study showed that both, the expression of genes involved in the repair system and miRNAs, are altered in samples from patients in blast crisis. The expression of the ATM, TP53, and ERCC1 genes has a fold change of 1.34 (p = 0.030), 1.58 (p = 0.007), and 0.65 (p < 0.001), respectively, when comparing samples from patients with CML- CP at diagnosis and samples from LMC-BC. The expression of miRNAs also showed changes in expression, especially of miR-17-5p, miR-34a, and miR-155, with a fold change of 1.76 (p < 0.001), 0.098 (p < 0.001) and 0.25 (p < 0.001), respectively. The expression of the ERCC1 gene and miR-155 correlated with event-free survival and that of the ATM and miR-150 gene correlated with the molecular response at 6 and 12 months of treatment with ITQ. Conclusions: Different genes modulating the response to DNA damage, such as ATM and TP53, are altered in patients with CML-BC, impairing the signaling cascade dependent on these genes. The greater expression of the ERCC1 gene contributes to the appearance of additional chromosomal changes through a single-strand annealing mechanism. The higher expression of miR-155 contributes to the reduction of the incompatible pairing mechanism, reducing the expression of the MLH1, MSH2 and MSH6 genes thus contributing to BCR-ABL1 kinase domain mutations. Furthermore, the greater expression of the ERCC1 gene and miR-155 can be used as predictive markers of the free event survival and the greater expression of the ATM gene and miR-150, as markers of the molecular response to treatment with ITQ at 6 and 12 months

Molecular monitoring of patients with chronic myeloid leukemia treated with imatinib mesylate and evaluation of treatment resistance mechanisms: mutation of BCR-ABL and expression of MDR1 and BCRP genes

A leucemia mielóide crônica (LMC) é caracterizada pela translocação (9;22) que dá origem ao gene quimérico BCR-ABL. Este gene codifica uma proteína com atividade tirosina quinase, p210, constitutivamente ativa. O três mecanismos envolvidos na patogênese da LMC são o aumento da proliferação celular, alteração da adesão celular ao estroma e matriz medular e inibição da apoptose. A introdução do mesilato de imatinibe (MI), um inibidor de tirosina quinase, revolucionou o tratamento da LMC levando pacientes em fase crônica a remissões duráveis, porém uma parcela destes não responde ou perde a resposta ao longo do tratamento. Os mecanismos de resistência ao MI podem ser classificados como independentes de BCR-ABL (a1- glicoproteína ácida e genes de resistência a múltiplas drogas) ou dependentes de BCR-ABL (superexpressão de BCR-ABL e mutações do domínio quinase do gene ABL). Objetivo: avaliar a presença de mutações no domínio quinase do gene ABL e a expressão dos genes de resistência a múltiplas drogas MDR1 e BCRP em amostras pré-tratamento com MI, acompanhar estes pacientes mensalmente através da quantificação de transcritos BCR-ABL e quando ocorrer resistência reavaliar a presença de mutações do domínio quinase do ABL e a expressão dos genes de resistência a múltiplas drogas. Material e Métodos: Foram avaliados 61 pacientes com LMC em fase crônica. A pesquisa de mutações do domínio quinase foi realizada pela técnica de seqüenciamento direto e a expressão relativa dos genes de resistência a múltiplas drogas foi avaliada por PCR em tempo real. A quantificação absoluta do número de transcritos BCR-ABL foi realizada pela técnica de PCR em tempo real utilizando-se o sistema Taqman de sondas de hibridização. Resultados: Nas amostras pré-tratamento dos 61 pacientes estudados não foram detectadas mutações. Quando relacionamos o aumento da expressão dos genes MDR1 e BCRP à resposta citogenética completa aos 12 meses de tratamento não houve diferença estatística significativa (p>0,05). Quanto ao número de transcritos BCR-ABL, observamos que os pacientes que apresentaram menos de 1% pela escala internacional aos 3 meses de tratamento atingiram a RMM em período menor (7 meses) do que os que apresentaram mais de 1% (12 meses) com diferença estatística significativa (p = 0,03). Conclusões: As mutações do domínio quinase do gene BCR-ABL nas amostras pré-tratamento não foram detectadas ou pela sensibilidade da técnica de seqüenciamento direto (10%) ou porque tais mutações são mais freqüentes nas fases acelerada e blástica. A expressão dos genes de resistência a múltiplas drogas (MDR1) e BCRP) em pacientes com LMC-FC ao diagnóstico não apresentou correlação com o aparecimento de resistência secundária ao MI. Além disso a quantificação mensal dos transcritos BCR-ABL aos 3 meses pode ser considerada um marcador com valor prognóstico.Chronic myeloid leukemia is characterized by t(9;22) translocation. The chimeric gene BCR-ABL encodes a p210BCRABL protein with constitutive tyrosine kinase activity which is directly related to CML pathogenesis. The imatinib mesylate, a tyrosine kinase inhibitor, is the first-choice treatment for patients in chronic phase but some patients show primary resistance or relapse after initial response. The mechanisms of resistance to the imatinib mesylate treatment are BCR-ABL dependent (amplification of BCR-ABL and mutation of kinase domain of BCR-ABL) or independent of BCR-ABL (1-acid glycoprotein and expression of multidrug resistance genes). Objective: The objective of this work was to evaluate the mechanisms of resistance (kinase domain mutation and MDR1 and BCRP genes expression) to imatinib mesylate in pretreatment samples, quantify of BCR-ABL transcript on a monthly follow up plan, and to re-evaluate the mechanisms of resistance in the absence or loss of treatment response. Patients and Methods: We have evaluated 61 pretreatment samples derived from chronic phase CML patients. The number of BCR-ABL transcripts was quantified by RTQ-PCR with taqman probes and MDR1 and BCRP expression were evaluated by RTQ-PCR with Syber Green. Mutations within the BCR-ABL kinase domain were screened by direct sequencing and we also have screened the T315I mutation in pretreatment samples by allele-specific PCR. Results:We detected no mutations in the 61 pretreatment samples. The correlation analysis between the expression of MDR1/BCRP genes and the cytogenetic response at 12 months of treatment revealed no significant statistical difference (p = > 0.05). The results of BCR-ABL quantification in the follow up of our cohort indicated that patients who had transcripts 1% (median of 12 months) (p = 0,03). Conclusions: As expected, the kinase domain mutations of BCR-ABL in pretreatment samples of CML chronic phase patients are not detectable by direct sequencing because of the sensitivity of the assay (10%) and also because these mutations are more common in accelerated phase and blast crisis. About the expression of multidrug resistance genes MDR1 and BCRP, they showed no correlation with secondary resistance to imatinib mesylate. And finally the number of BCR-ABL transcripts at 3 months of treatment can be considered a marker with prognostic value

Dasatinib Overrides Imatinib Resistance Mediated by the F359I Residue Mutation in Two Patients with Chronic Myeloid Leukemia

Despite the beneficial effects of imatinib mesylate, some patients may either not respond or respond suboptimally. Here, we report two chronic myelogenous leukemia patients; one had a suboptimal response according to European LeukemiaNet criteria (a major molecular response was not achieved after 18 months of standard-dose imatinib therapy) and the other had failure with a standard dose of imatinib. At the time of the suboptimal response in patient 1 and the failure in patient 2, we were able to detect the F359I mutation in the BCR-ABL tyrosine kinase domain using DNA sequencing in both patients. Therefore, it was decided to change the therapeutic regimen to dasatinib at a dose of 100 mg once daily in both patients. This change resulted in the achievement of complete cytogenetic remission in patient 1 after 4 months and a major molecular response within 2 and 3 months in both patients. Detection of the F359I mutation in our two cases likely explains the suboptimal response to imatinib in case 1 and the failure in case 2. This implies that in such cases dasatinib should be considered to effectively suppress the mutated clones. Copyright (C) 2011 S. Karger AG, BaselFundacao Maria Cecilia Souto VidigalFundacao Maria Cecilia Souto Vidiga

Evaluation of Long-Term Outcomes, Cytogenetic and Molecular Responses with Imatinib Mesylate in Early and Late Chronic-Phase Chronic Myeloid Leukemia: A Report from a Single Institute

Here we compare the management and survival outcomes of chronic myeloid leukemia (CML) patients who had early or late imatinib mesylate (IM) therapy. the cytogenetic and molecular responses of 189 CML patients were analyzed. of this group, 121 patients were classified as the early chronic phase (ECP) group and started IM within 12 months of diagnosis. the other 68 patients were classified as the late chronic phase (LCP) group who had been treated with interferon (IFN)-alpha-2 and crossed over to IM more than 12 months after diagnosis. the overall rates of complete cytogenetic response (CCyR) and major molecular response (MMR) at last follow-up were 83.6 and 78.1% in the ECP and LCP groups, respectively. the CCyR rates were 89.3 (for ECP patients) versus 73.5% (for LCP patients; p < 0.0001). At last follow-up, 82.4% ECP and 64.2% LCP patients had achieved an MMR (p < 0.0001). No significant differences were noted between the two groups with regard to survival outcomes. Our experience reveals that IM is an effective rescue therapy in most CML LCP patients who are intolerant or in whom IFN-alpha therapy fails. Such therapeutic options should be considered in LCP patients, particularly in countries where IM may not be available. Copyright (C) 2012 S. Karger AG, BaselFoundation Maria Cecilia Souto Vidigal, São Paulo, BrazilUniv São Paulo, Sch Med, Dept Hematol, Tumor Biol Lab,Fac Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Fac Med, Cytogenet Lab, BR-05403000 São Paulo, BrazilUniv São Paulo, Fac Med, Discipline Hematol, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo Inst Canc, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSão Paulo Inst Trop Med, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo Inst Canc, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilWeb of Scienc

Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

Objectives: A mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS), High-Resolution Melting analysis (HRM), and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP). Design and methods: We used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68). Results: Comparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP. Conclusions: Our results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures. Keywords: Myeloproliferative, JAK2 V617F, Mutation, Wild type, Screenin

Determination of serum levels of imatinib mesylate in patients with chronic myeloid leukemia: validation and application of a new analytical method to monitor treatment compliance

OBJECTIVE: The goal of this study was to monitor imatinib mesylate therapeutically in the Tumor Biology Laboratory, Department of Hematology and Hemotherapy, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP). A simple and sensitive method to quantify imatinib and its metabolite (CGP74588) in human serum was developed and fully validated in order to monitor treatment compliance. METHODS: The method used to quantify these compounds in serum included protein precipitation extraction followed by instrumental analysis using high performance liquid chromatography coupled with mass spectrometry. The method was validated for several parameters, including selectivity, precision, accuracy, recovery and linearity. RESULTS: The parameters evaluated during the validation stage exhibited satisfactory results based on the Food and Drug Administration and the Brazilian Health Surveillance Agency (ANVISA) guidelines for validating bioanalytical methods. These parameters also showed a linear correlation greater than 0.99 for the concentration range between 0.500 µg/mL and 10.0 µg/mL and a total analysis time of 13 minutes per sample. This study includes results (imatinib serum concentrations) for 308 samples from patients being treated with imatinib mesylate. CONCLUSION: The method developed in this study was successfully validated and is being efficiently used to measure imatinib concentrations in samples from chronic myeloid leukemia patients to check treatment compliance. The imatinib serum levels of patients achieving a major molecular response were significantly higher than those of patients who did not achieve this result. These results are thus consistent with published reports concerning other populations

Pretherapeutic Expression of the hOCT1 Gene Predicts a Complete Molecular Response to Imatinib Mesylate in Chronic-Phase Chronic Myeloid Leukemia

In this retrospective study we evaluated the pretherapeutic mRNA expression of the hOCT1 (human organic cation transporter 1) gene in patients with chronic-phase (CP) chronic myeloid leukemia (CML) who varied in terms of their response to imatinib (IM). hOCT1 mRNA was quantified by real-time PCR. Patients were classified as expressing either high (n = 44) or low hOCT1 mRNA (n = 44). the complete cytogenetic response rates observed at 6, 12 and 18 months were 47.7, 84.1 and 91%, respectively, in patients with high hOCT1 mRNA and 47.5, 81.8 and 86.3%, respectively, in patients with low hOCT1 transcripts. the major molecular response rates were not significantly different between patients with high and low hOCT1 mRNA after 6 months of therapy (22.7 vs. 9.1%; p = 0.07), but they were significantly different after 12 months (54.5 vs. 31.8%; p = 0.026) and 18 months (77.2 vs. 56.8%; p = 0.034). Complete molecular responses were observed in 5 patients with low and 17 patients with high hOCT1 mRNA (p = 0.003). the 5-year event-free and overall survival analyses revealed no significant differences between the groups. These data imply that knowledge of the pretherapeutic level of hOCT1 could be a useful marker to predict IM therapy outcome in treatment-naive CP CML patients. Copyright (C) 2012 S. Karger AG, BaselMaria Cecilia Souto Vidigal Foundation, São Paulo, BrazilUniv São Paulo, Dept Hematol, Sch Med, Tumor Biol Lab,Fac Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Cytogenet Lab, Fac Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Discipline Hematol, Fac Med, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSão Paulo Inst Trop Med, São Paulo, BrazilSão Paulo Inst Canc, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilWeb of Scienc