RESUMO
The single-arm, phase 2 ENESTfreedom trial assessed the potential for treatment-free remission (TFR; i.e., the ability to maintain a molecular response after stopping therapy) following frontline nilotinib treatment. Patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase with MR4.5 (BCR-ABL1⩽0.0032% on the International Scale (BCR-ABL1IS)) and ⩾2 years of frontline nilotinib therapy were enrolled. Patients with sustained deep molecular response during the 1-year nilotinib consolidation phase were eligible to stop treatment and enter the TFR phase. Patients with loss of major molecular response (MMR; BCR-ABL1IS⩽0.1%) during the TFR phase reinitiated nilotinib. In total, 215 patients entered the consolidation phase, of whom 190 entered the TFR phase. The median duration of nilotinib before stopping treatment was 43.5 months. At 48 weeks after stopping nilotinib, 98 patients (51.6%; 95% confidence interval, 44.2-58.9%) remained in MMR or better (primary end point). Of the 86 patients who restarted nilotinib in the treatment reinitiation phase after loss of MMR, 98.8% and 88.4%, respectively, regained MMR and MR4.5 by the data cutoff date. Consistent with prior reports of imatinib-treated patients, musculoskeletal pain-related events were reported in 24.7% of patients in the TFR phase (consolidation phase, 16.3%).
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/psicologia , Masculino , Pessoa de Meia-Idade , Pirimidinas/efeitos adversos , Qualidade de VidaRESUMO
In the phase 3 Evaluating Nilotinib Efficacy and Safety in Clinical Trials-Newly Diagnosed Patients (ENESTnd) study, nilotinib resulted in earlier and higher response rates and a lower risk of progression to accelerated phase/blast crisis (AP/BC) than imatinib in patients with newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP). Here, patients' long-term outcomes in ENESTnd are evaluated after a minimum follow-up of 5 years. By 5 years, more than half of all patients in each nilotinib arm (300 mg twice daily, 54%; 400 mg twice daily, 52%) achieved a molecular response 4.5 (MR(4.5); BCR-ABL⩽0.0032% on the International Scale) compared with 31% of patients in the imatinib arm. A benefit of nilotinib was observed across all Sokal risk groups. Overall, safety results remained consistent with those from previous reports. Numerically more cardiovascular events (CVEs) occurred in patients receiving nilotinib vs imatinib, and elevations in blood cholesterol and glucose levels were also more frequent with nilotinib. In contrast to the high mortality rate associated with CML progression, few deaths in any arm were associated with CVEs, infections or pulmonary diseases. These long-term results support the positive benefit-risk profile of frontline nilotinib 300 mg twice daily in patients with CML-CP.
Assuntos
Mesilato de Imatinib/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Pirimidinas/administração & dosagem , Glicemia/metabolismo , Colesterol/sangue , Seguimentos , Humanos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mieloide de Fase Crônica/sangue , Leucemia Mieloide de Fase Crônica/mortalidade , Pirimidinas/farmacologia , Medição de Risco , Resultado do TratamentoRESUMO
PURPOSE: L19-TNF is an armed antibody that selectively targets human TNF to extra domain B-fibronectin on tumour blood vessels. We performed a phase I/II first-in-man trial with L19-TNF monotherapy in metastatic solid cancer patients to study safety and signs of clinical activity. METHODS: Six cohorts of patients were treated with increasing (1.3-13 µg/kg) doses of intravenous L19-TNF on day 1, 3, and 5 of repeated 3-weekly cycles, and 12 colorectal cancer patients were treated at 13 µg/kg. PK, antibody formation, changes in lymphocyte subsets, 5-HIAA plasma levels as well as safety and clinical activity were analysed. RESULTS: Thirty-four patients received at least one L19-TNF dose. The serum half-life of L19-TNF at 13 µg/kg was 33.6 min, and maximum peak serum concentration was 73.14 µg/L. Mild chills, nausea and vomiting but no haemato- or unexpected toxicity were observed. Grade 3 lumbar pain in bone metastasis was the only dose-limiting toxicity found in one patient. Objective tumour responses were not detected. Transient stable disease occurred in 19 of 31 evaluable patients. CONCLUSIONS: Intravenous L19-TNF on day 1, 3, and 5 of a 3-weekly schedule was safe up to 13 µg/kg, but did not result in objective tumour responses. The maximally tolerated dose (MTD) was not reached, allowing for further dose escalation of L19-TNF possibly in combination with chemotherapy.
Assuntos
Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Adulto , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Estudos de Coortes , Citocinas/administração & dosagem , Citocinas/química , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Neoplasias/patologiaRESUMO
The transcription factor Wilms' tumour gene 1 (WT1) is important as a prognostic marker as well as in the detection and monitoring of minimal residual disease in leukaemia and myelodysplastic syndromes. Evidence has accumulated over the past decade to show that WT1 is a key molecule for tumour proliferation in a large number of human neoplasms most prominent in acute leukaemias, making it a suitable target for therapeutic strategies. Based on animal results, showing safety and efficacy of immunization with WT1 peptides and protein, early clinical trials in leukaemia have recently been initiated. The First International Conference on WT1 in Human Neoplasia was held in Berlin, March 11--12, 2004. This report reviews the current knowledge on the role of WT1 in tumour promotion and as a diagnostic and therapeutic target, and summarizes the data presented and discussed in this meeting.
Assuntos
Neoplasias/etiologia , Proteínas WT1/fisiologia , Animais , Genes do Tumor de Wilms , Humanos , Imunoterapia , Leucemia/diagnóstico , Leucemia/etiologia , Leucemia/terapia , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
High-dose chemotherapy with subsequent autologous stem cell transplantation is believed to be of therapeutic benefit in patients with acute myeloid leukemia (AML), especially when no allogeneic bone marrow donor is available. One of the main risks is contamination of the stem cell preparations with leukemic blasts, which may account for a higher relapse rate compared to allogeneic bone marrow transplantation. Since overexpression of WT1 is common in leukemic blasts, we investigated, whether PBSCs from AML patients express WT1 at a higher level as compared to patients with solid cancers. PBSCs of seven patients with AML and of five patients with solid cancers were investigated for WT1 expression. Total WT1 copy count was determined in a standardized quantitative real time RT-PCR. WT1 expression was found in all AML PBSCs with an average copy number of 49.99 +/- 61.09. In solid cancers WT1 expression was statistically significantly lower with a copy number of 3.51 +/- 1.92. In AML patients with sustained complete remission we found a nearly significantly lower WT1 expression than in patients who relapsed within the first year after stem cell transplantation. Our data show a higher WT1 expression in PBSCs of AML patients compared to patients with solid cancers. This finding might indicate a contamination with leukemic blasts. Quantification of WT1 in PBSCs might therefore be useful to estimate the risk of relapse after autologous stem cell transplantation in AML patients.
Assuntos
Genes do Tumor de Wilms/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/terapia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco/normas , Doença Aguda , Adolescente , Adulto , Biomarcadores Tumorais/análise , Feminino , Humanos , Separação Imunomagnética , Leucaférese/métodos , Leucaférese/normas , Leucemia Mieloide/sangue , Leucemia Mieloide/diagnóstico , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , Recidiva , Indução de Remissão , Transplante AutólogoRESUMO
Hirudin, the anticoagulatory polypeptide of the leech Hirudo medicinalis, strongly inhibits thrombus formation by specifically interacting with thrombin. For diagnostic purposes, hirudin should be superior to other anticlotting compounds because it only minimally alters the mineral, protein, and cellular blood constituents. To test this hypothesis, hirudinized and routinely processed venous blood from 80 healthy volunteers and patients was subjected to a variety of automated blood tests. A strong correlation was found between the results of automated complete blood counts obtained from K(2)-ethylenediaminetetraacetic acid (EDTA) anticoagulated and hirudinized blood (1000 antithrombin units [ATU] hirudin/ml). In addition, clinical chemistry and serological infection parameters (asparlat amintransferase [ASAT], lactate dehydrogenase [LDH], sodium, and so on, and antibodies against hepatitis B and C and human immunodeficiency virus [HIV]1/2, respectively) correlated well when measured in serum as compared with hirudinized plasma. Contrary to single clotting factors, global coagulation parameters (activated partial thromboplastin time [aPTT], prothrombin time [PT]) could not be measured in hirudinized blood. Recombinant hirudin neither interfered with immunophenotyping of mononuclear cells using FACScan analysis, nor did it alter the detection of Wilms' tumor gene expression by RT-PCR technology even at high doses (5000 ATU hirudin). Thus, a hirudin-containing blood sampling tube can be designed as a universal blood sampling tube (UBT) for testing the majority of diagnostic blood parameters.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Fibrinolíticos/farmacologia , Hirudinas/farmacologia , Anticorpos Antivirais/sangue , Sangue/efeitos dos fármacos , Contagem de Células Sanguíneas/normas , Análise Química do Sangue/normas , Testes de Coagulação Sanguínea/normas , Coleta de Amostras Sanguíneas/normas , Estado Terminal , DNA de Neoplasias/análise , Ácido Edético/farmacologia , Fibrinolíticos/normas , Genes do Tumor de Wilms/fisiologia , Hirudinas/normas , Humanos , Imunofenotipagem/normas , Testes Sorológicos/normasRESUMO
BACKGROUND: Patients with advanced multiple myeloma (stage III or progressive myeloma) received the CAD protocol every three weeks: cyclophosphamide 200 mg/m2 i.v./orally days 1-4, adriamycin 30 mg/m2 i.v. on day 1 and dexamethasone 40 mg p.o. days 1-4. PATIENTS AND METHODS: Forty-six patients with a median age of sixty years (range 34-84 years) were enrolled. According to Durie-Salmon 44 patients were in stage III, 2 in stage II; 6 patients had renal insufficiency (stage B). Twenty-three patients were pre-treated at least with melphalane/prednisone. RESULTS: Remission rates were as follows: complete remission 4%, partial remission 70%, minimal change 11%, no change 11%, progressive disease 4%. After an observation time of 14 months the median progression free interval for 33 patients not treated with subsequent high-dose chemotherapy with stem-cell support was more than 14 months. Overall, treatment was well tolerated. After 209 cycles given febrile neutropenia occurred in 11% of cycles including one fatal outcome. Neutropenia or thrombocytopenia grade 3-4 WHO was recorded in 18% and 6% of the cycles, respectively. CONCLUSIONS: This study shows that CAD is an effective regimen with an overall remission rate of 74%. The CAD protocol should be further evaluated in prospective trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ciclofosfamida/administração & dosagem , Dexametasona/administração & dosagem , Progressão da Doença , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Neutropenia/induzido quimicamente , Trombocitopenia/induzido quimicamente , Resultado do TratamentoRESUMO
PURPOSE: Two identical, concurrent, parallel, multicenter, randomized, double-blind, double-dummy trials were conducted to compare the efficacy and safety of zoledronic acid and pamidronate for treating hypercalcemia of malignancy (HCM). PATIENTS AND METHODS: Patients with moderate to severe HCM (corrected serum calcium [CSC] > or = 3.00 mmol/L [12.0 mg/dL]) were treated with a single dose of zoledronic acid (4 or 8 mg) via 5-minute infusion or pamidronate (90 mg) via 2-hour infusion. A protocol-specified pooled analysis of the two parallel trials was performed. Clinical end points included rate of complete response by day 10, response duration, and time to relapse. RESULTS: Two hundred eighty-seven patients were randomized and evaluated for safety; 275 were evaluated for efficacy. Both doses of zoledronic acid were superior to pamidronate in the treatment of HCM. The complete response rates by day 10 were 88.4% (P = .002), 86.7% (P = .015), and 69.7% for zoledronic acid 4 mg and 8 mg and pamidronate 90 mg, respectively. Normalization of CSC occurred by day 4 in approximately 50% of patients treated with zoledronic acid and in only 33.3% of the pamidronate-treated patients. The median duration of complete response favored zoledronic acid 4 and 8 mg over pamidronate 90 mg with response durations of 32, 43, and 18 days, respectively. CONCLUSION: Zoledronic acid is superior to pamidronate; 4 mg is the dose recommended for initial treatment of HCM and 8 mg for relapsed or refractory hypercalcemia.
Assuntos
Difosfonatos/uso terapêutico , Hipercalcemia/tratamento farmacológico , Imidazóis/uso terapêutico , Adulto , Idoso , Feminino , Humanos , Hipercalcemia/etiologia , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Pamidronato , Ensaios Clínicos Controlados Aleatórios como Assunto , Ácido ZoledrônicoRESUMO
We present the clinical course of a 56-year-old female patient with a primary tracheal leiomyosarcoma. The diagnostic approach and pathological classification of this seldom described tumour remains extremely difficult. We discuss the symptoms as well as the diagnostic and therapeutic procedures, including multimodal chemotherapy with organ-preserving surgery leading to complete remission.
Assuntos
Leiomiossarcoma/patologia , Neoplasias da Traqueia/patologia , Quimioterapia Adjuvante , Feminino , Humanos , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/cirurgia , Pessoa de Meia-Idade , Neoplasias da Traqueia/tratamento farmacológico , Neoplasias da Traqueia/cirurgiaRESUMO
The Wilms' tumor gene (WT1) encodes a transcriptional regulator involved in growth and differentiation of various tissue types. A continuous over-expression of WT1 was found in leukemic blasts, thus suggesting an oncogenic function. Solid cancer entities have also been described as expressing WT1. We systematically analyzed WT1 expression in small-cell and non-small-cell lung cancer, colon cancer and glioblastoma patients and in the respective tumor cell lines. Using reverse transcription/polymerase chain reaction, we found WT1 expression in glioblastoma (5 of 8), lung (5 of 11), and colon cancer (5 of 15) cell lines. While WT1 was expressed in only 1 of 12 lung cancer and 1 of 5 glioblastoma specimens, it was not detected in colon cancer or macroscopically tumor-free colon and lung tissue. In addition, HT29 colon cancer cells showed a loss of WT1 expression when grown to confluence or induced to differentiate by sodium butyrate. From this evidence, testing for WT1 expression is not clinically relevant for colon cancer, lung cancer, or glioblastoma patients. WT1 expression in cancer cell lines can probably be attributed to optimized in vitro growth conditions.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/biossíntese , Glioblastoma/metabolismo , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição/biossíntese , Neoplasias do Sistema Nervoso Central , Células HL-60 , Células HT29 , Humanos , Células K562 , Células Tumorais Cultivadas , Proteínas WT1RESUMO
Continuous Wilms' tumor gene (WT1) expression is a typical feature of leukemic blasts in AML, ALL, and blast crisis CML patients. It is easily detectable by a variety of RT-PCR protocols, which differ mainly in their sensitivity. The nuclear WT1 protein can be found in blasts of approximately 50-60% of acute leukemia patients at diagnosis. Conversely, WT1 is only transiently expressed in normal hemopoiesis. Early CD34+ hemopoietic progenitors express WT1, whereas no WT1 mRNA transcripts can be found in mature blood cells and differentiation-induced committed CD34- progenitors. As a powerful complementary diagnostic tool, testing for WT1 expression can be helpful to discriminate between eosinophilic leukemia (EoL) patients and patients with idiopathic hypereosinophilic syndromes. Conflicting data about the usefulness of testing for WT1 expression to monitor minimal residual disease (MRD) in treated leukemia patients will be discussed. Finally, research strategies to circumvent shortcomings in detecting leukemia-associated WT1 expression will be outlined.
Assuntos
Proteínas de Ligação a DNA/análise , Síndrome Hipereosinofílica/diagnóstico , Leucemia/genética , Fatores de Transcrição/análise , Doença Aguda , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/genética , Diagnóstico Diferencial , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/diagnóstico , Neoplasia Residual , Proteínas Nucleares/análise , Prognóstico , Controle de Qualidade , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Proteínas WT1RESUMO
Systemic mycosis caused by Cryptococcus neoformans frequently becomes life threatening in patients with cellular immunodeficiencies. In contrast to AIDS patients, there are only a few reports of concurrent systemic cryptococcosis in patients with Hodgkin's disease (HD). Only two of 75 (2.7%) patients with HD who were consecutively admitted to our hospital in the past decade developed Cryptococcus neoformans infection. Both had stage IVB (Ann Arbor) HD with bone marrow involvement and absolute lymphopenia (< 1/nl). We have reviewed the literature and analyzed the data of 54 cases with concurrent cryptococcosis and HD. Presence of HD for > or = 12 months, stage IV disease, absolute lymphopenia (< 1/nl), and extensive pretreatment were the most common features among these patients and must be regarded as predisposing for acquiring a cryptococcal infection. In our patients antimycotic therapy was successful using liposomal amphotericin B (lipAmB) simultaneously with cytotoxic therapy for HD. Drug level measurements performed in one patient revealed a higher level of amphotericin B in CSF when the liposomal formulation was administered as compared with the level in CSF after administration of conventional amphotericin B. To our knowledge, this is the first report on antimycotic treatment of cryptococcosis with lipAmB in patients with HD. Regarding the favorable therapeutic index of lipAmB as compared with conventional amphotericin B, the drug should be considered as a less toxic and perhaps more effective alternative in the therapy of acute cryptococcosis, especially when cytotoxic treatment is administered simultaneously.
Assuntos
Criptococose/etiologia , Doença de Hodgkin/complicações , Infecções Oportunistas/microbiologia , Adulto , Anfotericina B/líquido cefalorraquidiano , Anfotericina B/uso terapêutico , Antifúngicos/líquido cefalorraquidiano , Antifúngicos/uso terapêutico , Criptococose/líquido cefalorraquidiano , Criptococose/tratamento farmacológico , Criptococose/imunologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/imunologia , Doença de Hodgkin/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/líquido cefalorraquidiano , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/imunologiaRESUMO
In patients presenting with immature eosinophilic precursors it is notoriously difficult to distinguish acute eosinophilic leukaemia (EoL) from the benign idiopathic hypereosinophilic syndrome (HES), based on morphological, cytochemical and immunophenotyping criteria, alone. Cytogenetic analysis or fluorescence in situ hybridization (FISH) can help in discriminating between these rare haematological disorders, but often treatment decisions cannot wait for the results of these time-consuming techniques. Recently, we and others found Wilms' tumour (WT1) gene expression to be increased in virtually all patients with acute leukaemias, whereas normal haemopoietic progenitors express the WT1 gene at much lower levels or not at all. To determine whether detection of WT1 gene expression is useful to distinguish EoL from HES patients, we analysed, by RT-PCR, bone marrow or blood mononuclear cells from EoL (n=3), HES (n=3) and reactive eosinophilia patients (n = 4) for WT1 gene expression. Using our WT1-RT-PCR protocol, we found WT1 gene expression to be restricted to EoL patients. By detecting WT1 mRNA transcripts in the cerebrospinal fluid using RT-PCR, we were also able to diagnose isolated CNS-relapsed leukaemia, initially confused with bacterial meningitis, in an EoL patient. In conclusion, we show that WT1-RT-PCR is a powerful complementary diagnostic tool to distinguish acute eosinophilic leukaemia from the hypereosinophilic syndromes. This observation needs confirmation in a larger series of EoL and HES patients.
Assuntos
Genes do Tumor de Wilms , Síndrome Hipereosinofílica/diagnóstico , Leucemia Eosinofílica Aguda/diagnóstico , Adulto , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Diagnóstico Diferencial , Feminino , Expressão Gênica , Humanos , Síndrome Hipereosinofílica/genética , Leucemia Eosinofílica Aguda/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseAssuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Genes do Tumor de Wilms , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/biossíntese , Antígenos CD , Antígenos CD34 , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Desenvolvimento Embrionário e Fetal , Sangue Fetal , Feto , Idade Gestacional , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas WT1 , Dedos de ZincoRESUMO
The WT1 gene encodes a transcriptional regulator which during embryogenesis is involved in growth control and differentiation of diverse tissues. It is also expressed in few human malignancies, including acute leukemia. We tested 3 different monoclonal antibodies (MAbs H2, H7, HCl7) and the polyvalent serum WTC-19 for WT1 protein detection in mononuclear cell (MNC) preparations of 104 newly diagnosed acute leukemia patients. Using RT-PCR, these MNC preparations were also analyzed for WT1 gene expression. MAbs H2, H7 and HCl7 and the polyclonal WTC-19 exhibited nuclear immunoreactivity in 63 of 99, 28 of 56, 38 of 60 and 22 of 43 WT1 gene-expressing leukemia samples, respectively. With these antibodies, no WT1 immunoreactivity was found in MNCs from blood of healthy volunteers, from CD34+ progenitor cell-enriched leukapheresis products of patients conditioned for peripheral stem cell harvest or from reactive bone marrow. Contrary to WTC-19, all MAbs reacted highly specifically with the WT1 protein (0.71 vs. 1.0). The WT1 protein was heterogeneously detected in leukemia blast preparations by all antibodies, irrespective of cell morphology. Very few HL60 cells and blasts from newly diagnosed leukemia patients interspersed among normal blood MNCs (50 blasts among 5 x 10(5) MNCs) were easy to identify by indirect immunofluorescence using MAbs H2 and HCl7. Taken together, MAbs H2 and HCl7 were superior to MAb H7 and the polyvalent WTC-19 in detecting the WT1 nuclear protein.
Assuntos
Anticorpos Monoclonais , Proteínas de Ligação a DNA/análise , Leucemia Mieloide Aguda , Proteínas de Neoplasias/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras , Fatores de Transcrição/análise , Especificidade de Anticorpos , Proteínas de Ligação a DNA/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Proteínas de Neoplasias/imunologia , Fatores de Transcrição/imunologia , Células Tumorais Cultivadas/química , Proteínas WT1RESUMO
The wt1 gene is located on chromosome 11p13 and encodes a zinc finger motif-containing transcription factor involved in regulation of growth and differentiation. Its expression was shown during embryonic development in various tissues as well as in a few human malignancies including acute leukemias. Using RT-PCR, we found wt1 gene expression in blast cells of the majority of 150 acute leukemia patients. Particularly, the wt1 transcript was detected in 12 of 14 (86%) pre-pre-B-ALL patients, in 33 of 41 (80%) cALL patients, in 23 of 31 (74%) T-ALL patients, and in 53 of 57 (93%) AML patients. Additionally, mononuclear cells from CML patients expressed the wt1 gene only when diagnosed with blast crisis. In contrast to acute human leukemias, mononuclear cells from reactive bone marrow (n = 4), and peripheral blood of healthy volunteers (n = 20), as well as normal peripheral CD34+ hematopoietic progenitors (n = 6) did not express the wt1 gene at detectable levels. Using the anti-WT1 MoAb 6F-H2 in an immunofluorescence assay on single cell level, we found the translated WT1 protein only in nuclei of leukemia blast cells but not in nuclei of normal CD34+ hematopoietic progenitor cells. Blast cells of 12 of 20 leukemia patients (60%) all tested positive for the wt1 gene expression by RT-PCR displayed a strong nuclear immunofluorescence. Its expression in the majority of human acute leukemias but not in normal mononuclear blood cells and normal CD34+ hematopoietic progenitors qualifies the wt1 gene transcript as a 'pan-acute leukemic' marker probably useful in monitoring minimal residual disease after chemotherapy and in detecting leukemic blast cells in purged or unpurged hematopoietic stem cell preparations intended to be used for autologous bone marrow transplantation.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Expressão Gênica , Genes do Tumor de Wilms , Leucemia/genética , Fatores de Transcrição/biossíntese , Adulto , Sequência de Bases , Crise Blástica , Northern Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Primers do DNA , Proteínas de Ligação a DNA/análise , Imunofluorescência , Humanos , Leucemia/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/genética , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Fatores de Transcrição/análise , Transcrição Gênica , Células Tumorais Cultivadas , Proteínas WT1 , Dedos de ZincoRESUMO
We report expression of the wt1 (Wilms' tumor) gene by cultured human melanoma cells. Using RNA polymerase chain reaction analysis, wt1 transcripts were detected in 7 of 9 melanoma cell lines but not in 5 normal melanocyte strains. In Northern blot analysis, steady-state wt1 mRNA levels were found in 2 of 4 melanoma lines but not in normal melanocytes. Sequence analysis of the wt1 cDNA expressed by melanoma cell line WM 902-B revealed the presence of 4 previously published splice variants but no evidence for mutations in the coding region. Previous work has shown that WT1 modulates transcription after binding to the early growth response (EGR)-1 sites present in the platelet-derived growth factor (PDGF)-A chain promoter; the PDGF-A chain gene is known to be expressed by various melanoma cell lines. Based on these findings, we studied the relationship of wt1 and PDGF-A chain gene expression in melanoma cell lines. Co-expression of the wt1 and the PDGF-A chain genes was observed in 2 melanoma cell lines with mutated p53 but not in 2 melanoma cell lines with wild-type p53; this result is consistent with a previous report showing that, in the context of absent or mutated p53, WT1 acts as a transcriptional activator, whereas in the presence of wild-type p53 it acts as a repressor.
Assuntos
Expressão Gênica , Genes do Tumor de Wilms , Melanócitos/metabolismo , Melanoma/metabolismo , Sequência de Bases , Northern Blotting , DNA Complementar/química , Genes p53 , Humanos , Melanoma/genética , Dados de Sequência Molecular , Mutação , Fator de Crescimento Derivado de Plaquetas/genética , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo.
Assuntos
Nevo/patologia , Neoplasias Cutâneas/patologia , Sequência de Bases , Cálcio/análise , Divisão Celular , Meios de Cultura Livres de Soro/química , Fator 2 de Crescimento de Fibroblastos/análise , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Hormônios Estimuladores de Melanócitos/farmacologia , Melanoma/etiologia , Dados de Sequência Molecular , Nevo/química , Nevo/congênito , RNA Mensageiro/análise , Neoplasias Cutâneas/química , Neoplasias Cutâneas/congênito , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
In a panel of metastatic melanoma cell lines we found steady-state mRNA transcripts for multiple growth factors including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-A, PDGF-B, transforming growth factor (TGF)- beta 1, TGF- alpha, melanoma growth-stimulating activity (MGSA), interleukin (IL)-1 alpha, and IL-1 beta but not insulin-like growth factor (IGF)-1 or IGF-2. Expression of growth factor genes was constitutive because prior to RNA extraction melanoma cells were maintained in a chemically defined culture medium free of exogenous growth factors. Each of four cell lines had an individual pattern of expression of either two, four, five, or seven growth factors; however, all cell lines shared expression of the bFGF gene. Two strains of normal melanocytes expressed TGF- beta 1 but not bFGF, PDGF, TGF- alpha , or MGSA mRNA at detectable levels. We tested growth-modulatory effects of the growth factors most frequently expressed by melanoma cells (bFGF, TGF- alpha, TGF- beta, PDGF). None of these stimulated melanoma cell growth consistently, whereas exogenous, acid-activated TGF- beta inhibited melanoma growth at concentrations greater than 10 ng/ml, suggesting that bioactive TGF- beta may represent a physiologic growth inhibitor. Neither neutralizing antisera to PDGF or TGF- alpha nor a monoclonal antibody to the epidermal growth factor (EGF)-receptor inhibited melanoma cell growth. Our results indicate that multiple growth factors are expressed simultaneously and constitutively by melanoma cells but not normal melanocytes in culture. Expression of bFGF is a common feature underscoring the significance of bFGF as an autocrine factor for melanoma cells as described earlier. Secreted PDGF and TGF- alpha are apparently not involved in or not essential for autocrine growth stimulation of melanoma cells.