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1.
Mol Cancer ; 12: 142, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24252366

RESUMO

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.


Assuntos
Células Eritroides/citologia , Proteínas de Choque Térmico HSP70/metabolismo , Policitemia Vera/metabolismo , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Eritroides/metabolismo , Feminino , Proteínas de Choque Térmico HSP70/genética , Humanos , Masculino , Pessoa de Meia-Idade , Policitemia Vera/sangue , Policitemia Vera/genética , Proteômica , Trombocitemia Essencial/sangue
2.
Br J Haematol ; 161(5): 667-676, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23560534

RESUMO

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia-negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F-positive cell lines, HEL and Ba/F3 (JAK2V617F EPOR) , and in primary mononuclear and bone marrow CD34-positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50 )(PV)  = 15 nmol/l], as well as sorafenib (IC50 PV=8µmol/l), KNK437 (IC50 PV=100µmol/l ), and perifosine (IC50 PV=15µmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)(PV)  < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2-related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.


Assuntos
Janus Quinases/antagonistas & inibidores , Transtornos Mieloproliferativos/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Compostos Benzidrílicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Dasatinibe , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Feminino , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/patologia , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Nitrilas , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Policitemia Vera/tratamento farmacológico , Policitemia Vera/enzimologia , Policitemia Vera/patologia , Pirazóis/administração & dosagem , Pirimidinas/farmacologia , Pirrolidinonas/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Tiazóis/farmacologia , Trombocitemia Essencial/tratamento farmacológico , Trombocitemia Essencial/enzimologia , Trombocitemia Essencial/patologia , Células Tumorais Cultivadas/efeitos dos fármacos
3.
Cancer Cell Int ; 12(1): 25, 2012 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-22676581

RESUMO

We have investigated the role of erythroid transcription factors mRNA expression in patients with acute myeloid leukemia (AML) in the context of cytogenetic and other prognostic molecular markers, such as FMS-like Tyrosine Kinase 3 (FLT3), Nucleophosmin 1 (NPM1), and CCAAT/enhance-binding protein α (CEBPA) mutations. Further validation of Erythroid Krüppel-like Factor (EKLF) mRNA expression as a prognostic factor was assessed.We evaluated GATA binding protein 1 (GATA1), GATA binding protein 2 (GATA2), EKLF and Myeloproliferative Leukemia virus oncogen homology (cMPL) gene mRNA expression in the bone marrow of 65 AML patients at diagnosis, and assessed any correlation with NPM1, FLT3 and CEBPA mutations. EKLF-positive AML was associated with lower WBC in peripheral blood (P = 0.049), a higher percentage of erythroblasts in bone marrow (p = 0.057), and secondary AMLs (P = 0.036). High expression levels of EKLF showed a trend to association with T-cell antigen expression, such as CD7 (P = 0.057). Patients expressing EKLF had longer Overall Survival (OS) and Event Free Survival (EFS) than those patients not expressing EKLF (median OS was 35.61 months and 19.31 months, respectively, P = 0.0241; median EFS was 19.80 months and 8.03 months, respectively, P = 0.0140). No correlation of GATA1, GATA2, EKLF and cMPL levels was observed with FLT-3 or NPM1 mutation status. Four of four CEBPA mutated AMLs were EKLF positive versus 10 of 29 CEBPA wild-type AMLs; three of the CEBPA mutated, EKLF-positive AMLs were also GATA2 positive. There were no cases of CEBPA mutations in the EKLF-negative AML group. In conclusion, we have validated EKLF mRNA expression as an independent predictor of outcome in AML, and its expression is not associated with FLT3-ITD and NPM1 mutations. EKLF mRNA expression in AML patients may correlate with dysregulated CEBPA.

4.
Gac Sanit ; 25(4): 274-81, 2011.
Artigo em Espanhol | MEDLINE | ID: mdl-21664727

RESUMO

OBJECTIVES: We evaluated the cost-effectiveness of rituximab added to the chemotherapy regimen of fludarabine plus cyclophosphamide (R-FC) versus fludarabine plus cyclophosphamide (FC) for the treatment of patients with previously untreated or relapsed/refractory chronic lymphocytic leukemia (CLL). METHODS: Two Markov models were built, using published results on progression-free survival (PFS) in patients receiving first- or second-line therapy with R-FC vs FC, rates of disease progression and mortality rates in Spain. Patient-elicited utilities were applied to PFS and progressed health states. The cost of drugs, supportive care, and quality-adjusted life years (QALY) were estimated over a 10-year period. Univariate and probabilistic (Monte Carlo) sensitivity analyses were performed. RESULTS: The addition of rituximab to chemotherapy in first- and second-line therapy increased life-years gained (LYG) and QALYs compared with chemotherapy. The incremental cost per LYG and QALY gained was €20,703 and €19,343 for first-line treatment and was €23,183 and €24,781 for second-line treatment. CONCLUSION: In patients with previously untreated or relapsed/refractory CLL, the addition of rituximab to the FC regimen increased life expectancy and quality-adjusted life expectancy. In both types of patient, the treatment was cost-effective.


Assuntos
Anticorpos Monoclonais Murinos/economia , Protocolos de Quimioterapia Combinada Antineoplásica/economia , Ciclofosfamida/economia , Leucemia Linfocítica Crônica de Células B/economia , Vidarabina/análogos & derivados , Anticorpos Monoclonais Murinos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Análise Custo-Benefício , Ciclofosfamida/administração & dosagem , Progressão da Doença , Intervalo Livre de Doença , Custos de Cuidados de Saúde , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Cadeias de Markov , Modelos Teóricos , Método de Monte Carlo , Anos de Vida Ajustados por Qualidade de Vida , Rituximab , Terapia de Salvação/economia , Espanha , Vidarabina/administração & dosagem , Vidarabina/economia
5.
Eur J Haematol ; 85(1): 20-5, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20665989

RESUMO

Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and striking abnormalities of erythroblast morphology. The mutated genes are known for the most frequent types, CDA I and II, but data about their frequency do not exist. The objective of this retrospective study was to estimate the frequency of CDA I and II, based on all cases reported in the last 42 yr in publications and identified registries or surveys. Reports were collected of 124 and 377 confirmed cases of CDA I and CDA II cases, respectively. The cumulated incidence of both types combined varied widely between European regions, with minimal values of 0.08 cases/million in Scandinavia and 2.60 cases/million in Italy. CDA II is more frequent than CDA I, with an overall ratio of approximately 3.2, but the ratio also varied between different regions. The most likely explanations for the differences are both differences in the availability of advanced diagnostic procedures and different levels of the awareness for the diagnosis of the CDAs. The estimations reported here are most probably below the true incidence rates, because of failure to make the correct diagnosis and to underreporting. Limited data do not suggest differing levels of risk in identified ethnic groups.


Assuntos
Anemia Diseritropoética Congênita/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Anemia Diseritropoética Congênita/classificação , Anemia Diseritropoética Congênita/diagnóstico , Anemia Diseritropoética Congênita/genética , Criança , Pré-Escolar , Coleta de Dados , Fatores Epidemiológicos , Etnicidade , Europa (Continente)/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Adulto Jovem
6.
Adv Hematol ; 2009: 476342, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19960060

RESUMO

A Spanish male patient with beta-thalassaemia major was studied. Compound heterozygosity was found for one of the most common beta-globin gene mutations in the Spanish population (codon 39 C --> T) and for a mutation in the TATA box element of the beta-globin gene promoter (-28 A --> C mutation). To our knowledge this is the first report of a CD39 C --> T and -28 A --> C change association and the first report of the -28 A --> C substitution in a Spanish patient.

7.
Haematologica ; 94(10): 1354-61, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19794081

RESUMO

BACKGROUND: Protein 4.1R is an important component of the red cell membrane skeleton. It imparts structural integrity and has transmembrane signaling roles by direct interactions with transmembrane proteins and other membrane skeletal components, notably p55 and calmodulin. DESIGN AND METHODS: Spontaneous and ligation-induced phosphatidylserine exposure on erythrocytes from two patients with 4.1R deficiency were studied, using CD47 glycoprotein and glycophorin C as ligands. We also looked for protein abnormalities in the 4.1R-based multiprotein complex. RESULTS: Phosphatidylserine exposure was significantly increased in 4.1R-deficient erythrocytes obtained from the two different individuals when ligands to CD47 glycoprotein were bound. Spontaneous phosphatidylserine exposure was normal. 4.1R, glycophorin C and p55 were missing or sharply reduced. Furthermore there was an alteration or deficiency of CD47 glycoprotein and a lack of CD44 glycoprotein. Based on a recent study in 4.1R-deficient mice, we found that there are clear functional differences between interactions of human red cell 4.1R and its murine counterpart. CONCLUSIONS: Glycophorin C is known to bind 4.1R, and we have defined previously that it also binds CD47. From our evidence, we suggest that 4.1R plays a role in the phosphatidylserine exposure signaling pathway that is of fundamental importance in red cell turnover. The linkage of CD44 to 4.1R may be relevant to this process.


Assuntos
Antígeno CD47 , Proteínas do Citoesqueleto/deficiência , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Receptores de Hialuronatos , Proteínas de Membrana/deficiência , Fosfatidilserinas/sangue , Adulto , Sequência de Aminoácidos , Antígeno CD47/sangue , Antígeno CD47/genética , Pré-Escolar , Proteínas do Citoesqueleto/sangue , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/genética , Ligantes , Masculino , Proteínas de Membrana/sangue , Dados de Sequência Molecular , Fosfatidilserinas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
8.
Liver Transpl ; 15(6): 581-91, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19479801

RESUMO

We aimed to quantify peripheral donor chimerism (DC) and to analyze its association with graft and recipient outcome. Forty-two liver transplant recipients and their respective donors were studied, providing a total of 148 posttransplantation serum samples. DC was assessed with real-time quantitative polymerase chain reaction (qPCR) to detect polymorphic markers. DC did not decrease with time post-transplantation and was higher in child recipients versus adults and in recipients of deceased donor liver transplants versus recipients of live donor liver transplants. Higher levels of DC were detected in Rh-positive blood group donors, in O blood group recipients versus A blood group recipients, and in recipients with hepatitis C virus versus recipients with alcoholic cirrhosis. High DC was associated with patients with organ damage due to recurrent disease and rejection. Stable, high levels of DC, in the absence of other major clinical events, may thus be a marker of transplantation tolerance, and this knowledge may help to tailor immunosuppressive treatment. In conclusion, qPCR is a useful technique for DC follow-up in liver transplantation, although the evolution of DC levels should be analyzed in accordance with the clinical outcome of the patient.


Assuntos
Quimerismo , Transplante de Fígado/imunologia , Doadores Vivos , Tolerância ao Transplante/genética , Tolerância ao Transplante/imunologia , Sistema ABO de Grupos Sanguíneos/genética , Adolescente , Adulto , Idoso , Alelos , Criança , Pré-Escolar , DNA/sangue , DNA/genética , Feminino , Seguimentos , Genótipo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Humanos , Terapia de Imunossupressão , Lactente , Transplante de Fígado/mortalidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Análise de Sobrevida , Adulto Jovem
9.
J Mol Diagn ; 11(2): 155-61, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19225136

RESUMO

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations.


Assuntos
Análise Mutacional de DNA/métodos , Éxons , Janus Quinase 2/genética , Técnicas de Diagnóstico Molecular/métodos , Transtornos Mieloproliferativos/diagnóstico , Análise Mutacional de DNA/economia , Humanos , Técnicas de Diagnóstico Molecular/economia , Transtornos Mieloproliferativos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Temperatura de Transição
10.
Am J Hematol ; 84(2): 79-86, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19097174

RESUMO

The aim of this study was to evaluate the biological correlation and prognostic impact of Gata-1, Gata-2, EKLF, and c-MPL transcript level in a group of 41 acute myeloid leukemia (AML) patients. Gata-1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata-1 expression on the probability of achieving complete remission has been confirmed. Gata-2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c-MPL was associated with CD34+ AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML.


Assuntos
Fator de Transcrição GATA1/fisiologia , Fator de Transcrição GATA2/fisiologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/fisiologia , Receptores de Trombopoetina/fisiologia , Adolescente , Adulto , Idoso , Medula Óssea/patologia , Aberrações Cromossômicas , Intervalo Livre de Doença , Eritropoese/genética , Fator de Transcrição GATA1/análise , Fator de Transcrição GATA2/análise , Humanos , Fatores de Transcrição Kruppel-Like/análise , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/metabolismo , Segunda Neoplasia Primária/mortalidade , Segunda Neoplasia Primária/patologia , Prognóstico , Receptores de Trombopoetina/análise , Análise de Sobrevida , Adulto Jovem
11.
Ann Hematol ; 87(9): 741-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18575865

RESUMO

Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.


Assuntos
Substituição de Aminoácidos , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Primers do DNA , Humanos , Mutação , Transtornos Mieloproliferativos/sangue , Hibridização de Ácido Nucleico , Policitemia/genética , Reação em Cadeia da Polimerase , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Termodinâmica , Trombocitose/genética
12.
Blood Rev ; 21(5): 267-83, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17611006

RESUMO

Deficiency of glucose-6-phosphate dehydrogenase is a very common X-linked genetic disorder though most deficient people are asymptomatic. A number of different G6PD variants have reached polymorphic frequencies in different parts of the world due to the relative protection they confer against malaria infection. People, usually males, with deficient alleles are susceptible to neonatal jaundice, and acute hemolytic anemia, usually during infection, after treatment with certain drugs or after eating fava beans. Very rarely de novo mutations can arise causing the more severe condition of chronic nonspherocytic hemolytic anemia. Altogether 160 different mutations have been described. The majority of mutations cause red cell enzyme deficiency by decreasing enzyme stability. The polymorphic mutations affect amino acid residues throughout the enzyme and decrease the stability of the enzyme in the red cell, possibly by disturbing protein folding. The severe mutations mostly affect residues at the dimer interface or those that interact with a structural NADP molecule that stabilizes the enzyme.


Assuntos
Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Anemia Hemolítica/etiologia , Cromossomos Humanos X/genética , Suscetibilidade a Doenças , Feminino , Genes Ligados ao Cromossomo X , Genótipo , Glucosefosfato Desidrogenase/sangue , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/enzimologia , Humanos , Malária/metabolismo , Masculino , Fenótipo , Mutação Puntual , Polimorfismo Genético
13.
J Clin Oncol ; 24(22): 3611-8, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16877728

RESUMO

PURPOSE: To study the prognostic significance of the presence of breast cancer-specific mRNA transcripts in peripheral blood (PB), defined by serial analysis of gene expression, in high-risk breast cancer (HRBC) patients undergoing high-dose chemotherapy after receiving adjuvant chemotherapy. METHODS: From 1994 to 2000, 84 HRBC patients (median age, 44 years; > 10 nodes; 74%) received adjuvant chemotherapy (fluorouracil, epirubicin, and cyclophosphamide for six cycles [83%] or doxorubicin and cyclophosphamide followed by paclitaxel) before undergoing one course of cyclophosphamide plus thiotepa plus carboplatin (STAMP V). Radiotherapy or hormone therapy was administered whenever indicated. Aliquots of apheresis-mononuclear blood cells were frozen from each patient. mRNA was isolated using an automatic nucleic acid extractor based on the magnetic beads technology; reverse transcription was performed using random hexamers. Cytokeratin 19, HER-2, P1B, PS2, and EGP2 transcripts were quantified to B-glucuronidase by real-time polymerase chain reaction (RT-PCR) using a linear DNA probe marked with a quencher and reporter fluorophores used in RT-PCR. Presence of PB micrometastases, estrogen receptor and progesterone receptor status, tumor size, age, tumor grade, number of nodes affected, and treatment with paclitaxel were included in the statistical analysis. RESULTS: Median follow-up was 68.3 months (range, 6 months to 103 months). Forty-seven relapses (56%) and 35 deaths (41.7%) were registered. Both tumor size and presence of micrometastases reached statistical significance according to the Cox multivariate model. Relapse hazard ratio (HR) for those patients with PB micrometastases was 269% (P = .006); death HR, 300% (P = .011). Time relapse was 53 months longer for patients without micrometastases: 31.3 v 84.2 months (P = .021). CONCLUSION: PB micrometastases presence after adjuvant chemotherapy predicts both relapse and death more powerful than classical factors in HRBC patients undergoing high-dose chemotherapy. Micrometastases search using a gene panel appears to be a more accurate procedure than classical approaches involving only one or two genes.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transplante de Células-Tronco de Sangue Periférico , Precursores de RNA/sangue , Adulto , Idoso , Antígenos de Superfície/genética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Neoplasias da Mama/terapia , Proteínas de Ligação ao Cálcio/genética , Carboplatina/administração & dosagem , Quimioterapia Adjuvante , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Epirubicina/administração & dosagem , Molécula de Adesão da Célula Epitelial , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Humanos , Queratinas/genética , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Paclitaxel/administração & dosagem , Valor Preditivo dos Testes , Presenilina-2 , Prognóstico , RNA Mensageiro , RNA Neoplásico , Receptor ErbB-2/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medição de Risco , Fatores de Risco , Análise de Sobrevida , Tiotepa/administração & dosagem , Resultado do Tratamento
14.
Blood ; 106(5): 1851-6, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15870173

RESUMO

Human erythrocyte R-type pyruvate kinase (RPK) deficiency is an autosomal recessive disorder produced by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Survival of patients with severe RPK deficiency has been associated with compensatory expression in red blood cells (RBCs) of M2PK, an isoenzyme showing wide tissue distribution. We describe a novel homozygous null mutation of the PKLR gene found in a girl with a prenatal diagnosis of PK deficiency. The mutant PK gene revealed an 11-nucleotide (nt) duplication at exon 8, causing frameshift of the PKLR transcript, predicting a truncated protein inferred to have no catalytic activity. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected no M2PK expression in the peripheral blood red cell fraction. The expression of mutant RPK mRNA in the RBCs was almost 6 times higher than that detected in a control patient with hereditary spherocytosis. This molecular phenotypic analysis of the null mutation in the PKLR gene provides evidence for a lack of M2PK in the mature RBCs of this patient and suggests that normal red cell functions and survival are achieved through a population of young erythroid cells released into the circulation in response to anemia.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Mutação de Sentido Incorreto , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Criança , Análise Mutacional de DNA , Éxons , Saúde da Família , Feminino , Humanos , Masculino , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espanha/epidemiologia
15.
Stem Cells ; 23(3): 324-34, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15749927

RESUMO

Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about 10 x 10(8) and only 25% of these units reach the target cell dose of 2 x 10(7)/kg in UCBT patients weighing 50-70 kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 +/- 50.1 ml and 1.26 +/- 0.52 x 10(9) NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2%-100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over 0.8 x 10(9) NCs, resulting in a 36% cell increment (1.46 +/- 0.52 x 10(9) NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of 2 x 10(7)/kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sangue Fetal/citologia , Adulto , Antígenos CD/análise , Contagem de Células Sanguíneas , Coleta de Amostras Sanguíneas/instrumentação , DNA/sangue , DNA/genética , Células Precursoras Eritroides/citologia , Feminino , Sangue Fetal/metabolismo , Citometria de Fluxo , Células Precursoras de Granulócitos/citologia , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Leucócitos Mononucleares/citologia , Perfusão , Placenta , Reação em Cadeia da Polimerase , Gravidez , Cordão Umbilical
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