Apoptosis-related gene expression profile in chronic myeloid leukemia patients after imatinib mesylate and dasatinib therapy.

BACKGROUND/AIMS: We investigated the effects of tyrosine kinase inhibitors (TKIs) on the expression of apoptosis-related genes (BCL-2 and death receptor family members) in chronic myeloid leukemia (CML) patients. METHODS: Peripheral blood mononuclear cells from 32 healthy subjects and 26 CML patients were evaluated before and after treatment with imatinib mesylate (IM) and dasatinib (DAS) by quantitative PCR. RESULTS: Anti-apoptotic genes (c-FLIP and MCL-1) were overexpressed and the pro-apoptotic BIK was reduced in CML patients. Expression of BMF, A1, c-FLIP, MCL-1, CIAP-2 and CIAP-1 was modulated by DAS. In IM-resistant patients, expression of A1, c-FLIP, CIAP-1 and MCL-1 was upregulated, and BCL-2, CIAP-2, BAK, BAX, BIK and FASL expression was downregulated. CONCLUSION: Taken together, our results point out that, in CML, DAS interferes with the apoptotic machinery regulation. In addition, the data suggest that apoptosis-related gene expression profiles are associated with primary resistance to IM.

Relationship between SLCO1B3 and ABCA3 polymorphisms and imatinib response in chronic myeloid leukemia patients.

BACKGROUND: Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients. METHODS: Patients in chronic phase CML (N = 118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334T > G (rs4149117) and c.699G > A (rs7311358), SLCO1A2 c.516A > C (rs11568563) and c.-62-361G > A (rs3764043), and ABCA3 c.1755C > G (rs323043) and c.4548-191C > A (rs150929) polymorphisms was carried out by real-time polymerase chain reaction. RESULTS: SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and non-responders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02-4.64, P = 0.04). Poor CMR for ABCA3 4548-91C > A was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014). CONCLUSIONS: SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis.

BACKGROUND: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. RESULTS: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. CONCLUSIONS: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Acute myeloid leukemia: update in diagnosis and treatment in Brazil.

OBJECTIVE: To identify how the Brazilian hematology centers treated and diagnosed cases of acute myeloid leukemia in 2009. METHODS: An epidemiological observational multicenter study of 11 listed Brazilian centers that treat acute myeloid leukemia and perform bone marrow transplantation. Data were collected from clinical charts of patients with acute myeloid leukemia treated at the said centers between 2005 and 2009. The availability for immunophenotyping and cytogenetic tests was assessed. RESULTS: During 2009, a total of 345 new cases of acute myeloid leukemia were diagnosed. Differences were noted in the tests performed between patients who initiated treatment at the center and those referred for treatment. Of the participating centers, 72% conducted some type of molecular study in acute myeloid leukemia upon diagnosis. CONCLUSION: Treatment for acute myeloid leukemia in Brazil shows significantly inferior results when compared to other centers worldwide.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases.

BACKGROUND: Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. AIMS: To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. SUBJECTS AND METHODS: Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. RESULTS: In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. CONCLUSIONS: The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Leucemia mieloide aguda: atualidade brasileira de diagnóstico e tratamento / Acute myeloid leukemia: update in diagnosis and treatment in Brazil

Objective: To identify how the Brazilian hematology centers treated and diagnosed cases of acute myeloid leukemia in 2009. Methods: An epidemiological observational multicenter study of 11 listed Brazilian centers that treat acute myeloid leukemia and perform bone marrow transplantation. Data were collected from clinical charts of patients with acute myeloid leukemia treated at the said centers between 2005 and 2009. The availability for immunophenotyping and cytogenetic tests was assessed. Results:During 2009, a total of 345 new cases of acute myeloid leukemia were diagnosed. Differences were noted in the tests performed between patients who initiated treatment at the center and those referred for treatment. Of the participating centers, 72% conducted some type of molecular study in acute myeloid leukemia upon diagnosis. Conclusion: Treatment for acute myeloid leukemia in Brazil shows significantly inferior results when compared to other centers worldwide.

Objetivo: Identificar como centros de hematologia brasileiros trataram e diagnosticaram os casos de leucemia mieloide aguda no ano de 2009. Métodos: Estudo epidemiológico, observacional, multicêntrico de 11 centros brasileiros cadastrados para tratamento de leucemia mieloide aguda e transplante de medula óssea. Os dados foram coletados a partir de prontuários de pacientes com leucemia mieloide aguda tratados nos centros citados entre os anos de 2005 e 2009. Foi avaliada a disponibilidade para realização de exames de imunofenotipagem e citogenética nos centros estudados. Resultados: Foram diagnosticados 345 casos novos de leucemia mieloide aguda no ano de 2009. Observaram-se diferenças na realização de exames entre pacientes que iniciaram o tratamento no centro em relação àqueles referenciados para tratamento. Dos centros participantes, 72% realizaram algum tipo de pesquisa molecular em leucemia mieloide aguda ao diagnóstico. Conclusão: O tratamento da leucemia mieloide aguda no Brasil apresenta resultados muito inferiores quando comparado a outros centros mundiais.

Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.

A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença.

Indicações em transplante de células-tronco hematopoéticas em pacientes adultos com leucemia linfoide aguda / Indications for hematopoietic stem cell transplantation in adults in acute lymphoblastic leukemia

Na leucemia linfoide aguda (LLA), a proliferação, acúmulo e infiltração de células imaturas caracterizam uma entidade heterogênea, apresentando ampla diversidade de aspectos clínicos e biológicos. Na LLA do adulto, a concentração de fatores prognósticos de alto risco, como o imunofenótipo B, alterações cromossômicas e, principalmente, a presença do cromossomo Ph positivo. Considerações a respeito da alta morbidade e mortalidade relacionadas ao transplante de células-tronco hematopoéticas (TCTH) têm gerado controvérsias quanto à indicação desta modalidade terapêutica, nos pacientes adultos com LLA em primeira remissão (1ª RC). Os resultados da terapia convencional com quimioterapia, diante dos diferentes grupos de risco em pacientes com LLA, têm sido utilizados para a indicação de TCTH. Apresentamos o algoritmo de indicações do transplante de células-tronco hematopoéticas em pacientes adultos com LLA.

In acute lymphoblastic leukemia, accumulation and proliferation of immature cells infiltration characterise a heterogeneous entity, featuring a wide variety of clinical and biological aspects. In the adult LLA concentration of high-risk prognosis factors such as age, B-cell, chromosomic changes, and chiefly the presence of chromosome positive Ph. Considerations of high morbidity and mortality rates related to haematopoietic stem cell transplantation (TCTH) have generated controversy about this therapeutic modality in adult patients with LLA in first remission (1st RC). The results of conventional therapy with chemotherapy in contrast with different risk groups of patients with LLA, has been used for the indication of TCTH. Thus we present the algorithm indications of haematopoietic stem cell transplantation in adult patients with LLA.

Perspectivas para a leucemia mielóide aguda na infância após a observação de um grupo de pacientes tratados convencionalmente / Pediatric acute myeloid leukemia outcomes after conventional treatment

O tratamento atual das leucemias mielóides agudas (LMA) obedece principalmente aos seguintes critérios: precisa caracterização clínica e biológica, adequada e agressiva quimioterapia, intensa terapia de suporte e possível indicação precoce de transplante de medula óssea. Neste artigo, envolvendo crianças tratadas entre 1986 e 1996, são apresentados os resultados passíveis de obtenção com tratamento quimioterápico convencional e discutidas as bases de implantação de um programa terapêutico moderno e de maior eficácia. Quarenta e três pacientes com LMA "de novo", idades entre 6 meses e 14,5 anos, foram tratados conforme programa terapêutico que incluiu indução e consolidação com quatro drogas: DAUNO, ARA-C, VCR e DEXA e 60 semanas de manutenção com os seguintes ciclos administrados seqüencialmente: ARA-C & ASP, CICLO & ETO, DAUNO (6-TG) & ARA-C & VCR & DEXA e 6-TG & ARA-C & VCR & DEXA. Profilaxia de envolvimento de SNC com ARA-C por via intratecal. Nenhuma criança foi submetida a transplante de medula óssea em primeira instância. 38/43 (84 por cento) crianças alcançaram remissão completa e a sobrevida livre de eventos estimada para três anos foi de 24 ± 7 por cento. A importância da discriminação das LMA em grupos de risco, que levem em consideração particularmente os achados citogenéticos, além de novas modalidades de abordagem terapêutica, é discutida, destacando-se a importância atual dos transplantes alogênicos de medula óssea.

Modern treatment of acute myeloid leukemias follows well defined guidelines: precise clinical and biological characterization, adequate aggressive chemotherapy, intensive supportive care and eventual early indication of bone marrow transplantation. In this report we present the best results obtained with conventional chemotherapy delivered to a group of pediatric patients between 1986 and 1996 and discuss the strategies for contemporary and more efficient regimens. Forty-three patients with "de novo" acute myeloid leukemias with ages between 6 months and 14.5 years, were treated with a 4-drug induction and consolidation program: AUNO, ARA-C, VCR and DEXA, followed by 60 weeks of maintenance with sequential cycles of ARAC & ASP, CICLO & ETO, DAUNO (6-TG) & ARA-C & VCR & DEXA and 6-TG & ARA-C & VCR & DEXA. CNS prophylaxis with intrathecal ARA-C was undertaken. No child underwent bone marrow transplantation in first remission. A total of 38/43 (84 percent) children achieved complete remission and event-free survival at 3 years was estimated at 24 ± 7 percent. The importance of discriminating AML in risk groups with particular attention paid to cytogenetic criteria and new modalities of treatment are discussed. The current relevance of bone marrow transplantations is emphasized.

Experiência brasileira com transplante de medula óssea em leucemias agudas / Bone marrow transplantation in acute leukemias: the Brazilian experience

Os autores sumarizam três experiências brasileiras publicadas ou apresentadas em eventos científicos com a utilização do transplante de medula óssea no tratamento das leucemias agudas, mielóide e linfóide. Estes estudos foram fruto de levantamentos realizados em 16 centros brasileiros de TMO através de questionários padronizados. As análises foram, portanto, retrospectivas. O número de pacientes nestes estudos foi superior a 1000, em torno de 700 na LMA e 400 na LLA. Os resultados, apesar das limitações metodológicas, mostram a factibilidade do procedimento em nossa realidade e respostas de curto e longo prazo adequadas. Os estudos auxiliaram ainda no reconhecimento de limites estruturais, como a pouca disponibilidade da citogenética em nosso meio e a necessidade de estudos prospectivos e controlados neste grupo de pacientes.

Te authors summarized three Brazilian experiences published or presented in scientific meetings, using bone marrow transplantation in the treatment of acute myeloid or lymphoid leukemia. These studies were performed using a standard questionnaire applied in 16 Brazilian centers. The analysis was retrospective. The number of analyzed patients was about of 1000, about 700 AML and 400 ALL. Although the methodological limitation the studies showed the feasibility of procedure in our reality. The responses and long term outcome were acceptable compared to international data. The studies helped us to recognize structural limitation as availability of cytogenetic laboratories. Prospective and controlled studies are needed in this setting of patients in Brazil.

Hematopoese, fatores de crescimento e aplicacao clinica da eritropoetina na anemia da prematuridade

Os autores apresentam inicialmente uma revisao sobre a hematopose enfocando a fisiologia, a circulacao das stem cells e sua relacao com os diversos fatores de crescimento celular nos diversos periodos de formacao das celulas do sangue durante a gestacao. A producao de hemoglobina na sequencia do desenvolvimento da hematopoese, a regulacao e as caracteristicas da eritropoetina sao a seguir descritas. Finalizamos a revisao com a fisiologia das anemias no periodo neonatal do recem-nascido de termo e em prematuros alem de uma breve abordagem terapeutica.