RESUMEN
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome characterized by monocytic proliferation in the presence of dysplastic bone marrow changes, inflammatory symptoms, and propensity for transformation to acute myeloid leukemia (AML), with a poor prognosis and limited treatment options. Unlike the α and ß isoforms, the phosphatidylinositol-3-kinase (PI3K)-δ signaling protein is predominantly expressed by hematopoietic cells and therefore has garnered interest as a potential target for the treatment of lymphomas and leukemias. We revealed a pattern of increased PIK3CD:PIK3CA ratio in monocytic M5 AML patients and cell lines, and this ratio correlated with responsiveness to pharmacological PI3K-δ inhibition in vitro. Because CMML is a disease defined by monocytic clonal proliferation, we tested the PI3K-δ inhibitor umbralisib as a single agent and in combination with the JAK1/2 inhibitor ruxolitinib, in CMML. Our ex vivo experiments with primary CMML patient samples revealed synergistic inhibition of viability and clonogenicity with this combination. Phospho-specific flow cytometry revealed that dual inhibition had the unique ability to decrease STAT5, ERK, AKT, and S6 phosphorylation simultaneously, which offers a mechanistic hypothesis for the enhanced efficacy of the combination treatment. These preclinical data indicate promising activity by co-inhibition of PI3K-δ and JAK1/2 and support the use of ruxolitinibâ¯+â¯umbralisib combination therapy in CMML under active clinical investigation.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Pirazoles/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Sinergismo Farmacológico , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Terapia Molecular Dirigida , Nitrilos , PirimidinasRESUMEN
Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.
Asunto(s)
Biomarcadores de Tumor/análisis , Citometría de Flujo/métodos , Leucemia Mielomonocítica Crónica/diagnóstico , Síndromes Mielodisplásicos/diagnóstico , Neutrófilos/enzimología , Peroxidasa/metabolismo , Anciano , Estudios de Casos y Controles , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Masculino , Síndromes Mielodisplásicos/enzimología , Pronóstico , Estudios Prospectivos , Curva ROC , Estudios RetrospectivosRESUMEN
Kidney failure is common in haematologic malignancies. However, the nephrotoxic effect of lysozyme is seldom recognized. We present a 78-year-old male with chronic myelomonocytic leukaemia who developed progressive kidney failure due to increased production of lysozyme.
Asunto(s)
Leucemia Mielomonocítica Crónica/enzimología , Muramidasa/metabolismo , Insuficiencia Renal/enzimología , Anciano , Progresión de la Enfermedad , Humanos , Leucemia Mielomonocítica Crónica/complicaciones , Hígado/enzimología , Masculino , Insuficiencia Renal/etiologíaRESUMEN
A subset of patients with chronic myelomonocytic leukemia (CMML) presents with significance myelofibrosis. In myelodysplastic syndromes, significant myelofibrosis has been associated with adverse outcomes and p53 dysregulation. However, in CMML the clinical and molecular correlates of significant myelofibrosis at presentation remain poorly understood. From a cohort of 651 CMML patients, we identified retrospectively 20 (3.1%) cases with moderate to severe reticulin fibrosis (CMML-F) detected at diagnosis, and we compared them to CMML patients without fibrosis (n=631) seen during the same period. Patients with CMML-F had a median age of 69.8 years (range, 24.8 to 91.2 y) and most (13; 65%) were men. Patients with CMML-F differed significantly from other CMML patients across the following parameters: white blood count, absolute monocyte count, serum lactate dehydrogenase level, splenomegaly, and bone marrow blast percentage. Notably, the frequency of JAK2 p.V617F mutation was higher in CMML-F patients compared with other CMML patients (P<0.001). Most CMML-F patients (12/20; 60%) had myeloproliferative CMML. Dysregulation of p53 was uncommon in CMML-F. CMML-F patients tended to have a shorter median overall survival compared with other CMML patients (P=0.079). Multivariate analysis using the Cox proportional hazards model showed an independent association between CMML-F and overall survival (P=0.047). In summary, unlike typical CMML, CMML-F is commonly associated with JAK2 p.V617F. The high frequency of myeloproliferative features and JAK2 p.V617F mutation, and the low frequency of p53 dysregulation, suggest that fibrosis in the context of CMML has a different pathogenesis from that previously reported in myelodysplastic syndrome.
Asunto(s)
Biomarcadores de Tumor/genética , Proliferación Celular , Janus Quinasa 2/genética , Leucemia Mielomonocítica Crónica/genética , Mutación , Mielofibrosis Primaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Femenino , Predisposición Genética a la Enfermedad , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/patología , Leucemia Mielomonocítica Crónica/terapia , Masculino , Persona de Mediana Edad , Tasa de Mutación , Fenotipo , Mielofibrosis Primaria/enzimología , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/terapia , Pronóstico , Reticulina/análisis , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/genética , Adulto JovenRESUMEN
Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.
Asunto(s)
Quinasas Janus/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/enzimología , Animales , Proliferación Celular/efectos de los fármacos , Genes ras , Humanos , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/enzimología , Leucemia Mielomonocítica Juvenil/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Mutantes , Trastornos Mieloproliferativos/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal/efectos de los fármacosRESUMEN
The frequency of RAS mutations in chronic myelomonocytic leukemia (CMML) suggests that activation of the MAPK pathway is important in CMML pathogenesis. Accordingly, we hypothesized that mutations in other members of the MAPK pathway might be overrepresented in RAS(wt) CMML. We performed targeted next generation sequencing analysis on 70 CMML patients with known RAS mutation status. The study group included 37 men and 33 women with a median age of 67.8 years (range, 28-86 years). Forty patients were RAS(wt) and 30 were RAS(mut) ; the latter included KRAS = 17; NRAS = 12; KRAS + NRAS = 1. Five patients (7.1% of total group; 12.5% of RAS(wt) group) with RAS(wt) had kinase domain BRAF mutations. The BRAF mutations were of missense type and involved exon 11 in one patient and exon 15 in four patients. All BRAF(mut) patients had CMML-1 with low-risk cytogenetic findings. Two (40%) of the five patients with BRAF(mut) patients transformed to acute myeloid leukemia during follow-up. In summary, we demonstrate that a subset of patients with RAS(wt) CMML harbors BRAF kinase domain mutations that are potentially capable of activating the MAPK signaling pathway.
Asunto(s)
Leucemia Mielomonocítica Crónica/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Sistema de Señalización de MAP Quinasas , Masculino , Persona de Mediana Edad , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that has been implicated in various aspects of adaptive and innate immune responses. Nevertheless, the role of CYLD in the function of specific types of immune cells remains elusive. In this report we have used conditional gene targeting in mice to address the role of the deubiquitinating activity of CYLD in the myelomonocytic lineage. Truncation of the deubiquitinating domain of CYLD specifically in myelomonocytic cells impaired the development of lethal LPS-induced endotoxic shock and the accumulation of thioglycollate-elicited peritoneal macrophages. Our data establish CYLD as a regulator of monocyte-macrophage activation in response to inflammatory stimuli and identify it as a potential target for therapeutic intervention in relevant inflammatory disorders in humans.
Asunto(s)
Cisteína Endopeptidasas , Inflamación/prevención & control , Leucemia Mielomonocítica Crónica/enzimología , Ubiquitinación , Animales , Enzima Desubiquitinante CYLD , Inmunidad , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos Peritoneales , Ratones , Monocitos , Choque Séptico/prevención & controlAsunto(s)
Análisis Mutacional de ADN/métodos , Leucemia Mielomonocítica Crónica/enzimología , Proteínas Tirosina Quinasas/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielomonocítica Crónica/etiología , Leucemia Mielomonocítica Crónica/genética , ProteómicaRESUMEN
The recently approved drugs 5-azacitidine (5AC) and 5-aza-2'-deoxyazacytidine (DAC) are in wide clinical use for the treatment of myelodysplastic syndrome (MDS) of all types and chronic myelomonocytic leukemia (CMML). These agents were developed based upon an understanding of the importance of epigenetic changes in malignancy, and they have been evaluated in randomized clinical trials, which demonstrate response rates between 20% and 40% in patients for whom no previous standard of care was available. As understanding of the epigenetic changes characteristic of the malignant phenotype improves, we are able to target other regulators of chromatin conformation that contribute to aberrant gene transcription and dysregulated cell growth. The histone deacetylase (HDAC) inhibitors belong to one class of therapeutics developed using this paradigm. Although responses using HDAC inhibitors alone in MDS have been modest, robust preclinical data drive clinical trials in which they are utilized in combination with DNA methyltransferase (DNMT) inhibitors. Combination therapy offers the possibility of hematologic improvement and remission to myelodysplastic patients with previously untreatable disease.
Asunto(s)
Metilasas de Modificación del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Azacitidina/análogos & derivados , Azacitidina/uso terapéutico , Ensayos Clínicos como Asunto , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Metilación de ADN , Decitabina , Quimioterapia Combinada , Histona Desacetilasas/metabolismo , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Síndromes Mielodisplásicos/enzimologíaRESUMEN
Extramedullary hematopoiesis (EMH) in the spleen is a characteristic feature of the chronic myeloproliferative disorders (CMPDs) and various other neoplastic or reactive myeloid conditions. However, the origin of these hematopoietic precursor cells and the molecular mechanisms underlying their development in the spleen is uncertain. The V617F mutation in the Janus Kinase 2 gene (JAK2(V617F)) was recently shown to be frequently and preferentially present in the peripheral blood and bone marrow cells of CMPD patients, and the resulting dysregulation of its downstream targets is important to CMPD pathogenesis. To determine the occurrence and potential role of JAK2(V617F) in splenic EMH cells, we studied splenectomy specimens from 47 patients with significant EMH. JAK2(V617F) was detected by real-time PCR melting curve analysis in 22 specimens, including 11/17 chronic idiopathic myelofibrosis, 7/7 polycythemia vera, 1/1 essential thrombocythemia, 1/3 CMPD unclassifiable, 1/5 chronic myelomonocytic leukemia, 0/5 chronic myelogenous leukemia, 1/3 myelodysplastic syndrome and 0/6 acute myeloblastic leukemia cases, whereas only the JAK2 wild-type allele was detected in the other 25. Nineteen of 20 cases with adequate bone marrow samples available for molecular examination demonstrated concordant JAK2 genotypes. Laser-capture microdissection was then used to enrich the EMH and non-EMH splenic cell fractions, confirming that the mutant alleles specifically originated from the EMH cells. Furthermore, megakaryocytes in the JAK2(V617F)-positive splenectomy specimens expressed higher levels of Bcl-xL, an antiapoptotic protein and downstream target of the JAK2/STAT5 pathway. Thus, JAK2(V617F) is frequently present in splenic EMH cells associated with CMPD, but it is rarely identified in splenic EMH cells associated with other myeloid disorders. Our results indicate that the precursor cells leading to extramedullary hematopoietic expansion in CMPD most likely originate from the transformed bone marrow clone. Also, dysregulation of downstream pathways such as Bcl-xL may be important to CMPD disease pathogenesis in the spleen.
Asunto(s)
Hematopoyesis Extramedular/genética , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/genética , Células de la Médula Ósea/química , Células de la Médula Ósea/enzimología , Enfermedad Crónica , Humanos , Inmunohistoquímica , Janus Quinasa 2/metabolismo , Rayos Láser , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Crónica/sangre , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Megacariocitos/química , Megacariocitos/enzimología , Microdisección/métodos , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/genética , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/metabolismo , Policitemia Vera/sangre , Policitemia Vera/enzimología , Policitemia Vera/genética , Reacción en Cadena de la Polimerasa , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/enzimología , Mielofibrosis Primaria/genética , Estudios Retrospectivos , Bazo/química , Bazo/enzimología , Trombocitemia Esencial/sangre , Trombocitemia Esencial/enzimología , Trombocitemia Esencial/genética , Estados Unidos , Proteína bcl-X/análisisRESUMEN
Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.
Asunto(s)
Alelos , Leucemia Linfoide/genética , Leucemia Mieloide/genética , Mutación Puntual , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Anciano , Sustitución de Aminoácidos , Estudios de Casos y Controles , Activación Enzimática/genética , Femenino , Humanos , Janus Quinasa 2 , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfoide/enzimología , Leucemia Mieloide/enzimología , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genéticaAsunto(s)
Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Mutación/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Preescolar , Humanos , Janus Quinasa 2 , Leucemia Mielomonocítica Crónica/terapia , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Carbonic anhydrases (CAs) are key enzymes that regulate acid-base homeostasis in both normal and pathological conditions. Recent studies have shown that they are functionally involved in the growth and invasion of cancer cells. However, there are only a few publications on CAs in hematological malignancies. EXPERIMENTAL DESIGN: Here we investigated the presence of CA isozymes in six malignant hematopoietic cell lines and malignant blast cells of bone marrow samples collected from patients with acute myeloid leukemia, acute lymphoblastic leukemia, or chronic myelomonocytic leukemia. RESULTS: Because three of the malignant hematopoietic cell lines expressed CA II, we also set out to examine its expression in a series of bone marrow samples. Positive reactions were found in 16 of 26 cases (62%) of acute myeloid leukemia, 11 of 15 cases (73%) of acute lymphoblastic leukemia, and 1 of 2 cases (50%) of chronic myelomonocytic leukemia. CONCLUSIONS: The results indicate that CA II expression is not restricted to only one cell lineage but may result from a genetic aberration that occurs in both myeloid and lymphatic lineages or in their progenitor cell. Because CA II is expressed in most patients with leukemic blast cells, CA inhibitors may prove to be of value as an adjunct to chemotherapy for such cancers.
Asunto(s)
Anhidrasa Carbónica II/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mielomonocítica Aguda/enzimología , Leucemia Mielomonocítica Crónica/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Enfermedad Aguda , Anemia Refractaria con Exceso de Blastos/metabolismo , Antígenos CD34/metabolismo , Médula Ósea/metabolismo , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Células Tumorales CultivadasRESUMEN
We report on the fusion of the monocytic leukemia zinc finger protein (MOZ) gene to the adenoviral E1A-associated protein p300 (p300) gene in acute monocytic leukemia M5 associated with a t(8;22)(p11;q13) translocation. We studied two patients with double-color fluorescence in situ hybridization (FISH) using the yeast artificial chromosome 176C9 and the bacterial artificial chromosome clone H59D10 specific to the MOZ and p300 genes, respectively. Both probes were split in the patients' chromosome metaphase cells, and the two derivative chromosomes were each labeled with both probes. We showed by Southern blot the rearrangement of the MOZ gene, and cloned the fusion transcripts in one patient carrying the t(8;22) by reverse transcription-polymerase chain reaction using MOZ- and p300-specific primers. Both fusion transcripts were expressed. This result defines a novel reciprocal translocation involving two acetyltransferases, MOZ and p300, resulting in an abnormal transcriptional co-activator that could play a critical role in leukemogenesis.
Asunto(s)
Acetiltransferasas/genética , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 8/genética , Leucemia Monocítica Aguda/genética , Proteínas Nucleares/genética , Transactivadores/genética , Translocación Genética/genética , Acetiltransferasas/aislamiento & purificación , Secuencia de Aminoácidos , Proteína p300 Asociada a E1A , Reordenamiento Génico , Histona Acetiltransferasas , Humanos , Hibridación Fluorescente in Situ , Leucemia Monocítica Aguda/enzimología , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Datos de Secuencia Molecular , Proteínas Nucleares/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transactivadores/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
We analyzed the relative expression of Hematopoietic cell phosphatase (HCP) in mononuclear cells (MNC) of peripheral blood (PB), bone marrow (BM) and spleen of patients with juvenile myelomonocytic leukemia (JMML) and normal donors. Two regions of HCP with alternative exon skipping of exon 6 or exon 12 are described. There was no difference in the expression of the amplified HCP cDNA regions in MNC of JMML patients compared to normal donors. The two forms of exon skipping were present in unstimulated MNC of JMML patients or normal donors. In contrast, phytohemagglutinin (PHA) stimulated MNC of normal donors, Epstein-Barr Virus (EBV) transformed B-cells of JMML patients, BFU-E and CFU-GM derived colonies of JMML patients, and the cell lines K562 and HEL did not or only barely express these two forms of exon skipping. These results may indicate that alternative HCP exon skipping may be associated with the proliferative state of the cell.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mielomonocítica Crónica/enzimología , Proteínas de Neoplasias/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Células de la Médula Ósea/enzimología , División Celular , Línea Celular Transformada , Transformación Celular Viral , Niño , Preescolar , ADN Complementario/genética , Inducción Enzimática , Exones/genética , Femenino , Genes ras , Herpesvirus Humano 4 , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular , Células K562/patología , Leucemia Mielomonocítica Crónica/genética , Leucocitos Mononucleares/enzimología , Activación de Linfocitos , Masculino , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/enzimología , Fitohemaglutininas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Bazo/enzimología , Transcripción Genética , Dominios Homologos srcRESUMEN
We used peripheral blood (PB) and bone marrow (BM) smears in the development of two methods based on cytomorphology and esterase cytochemistry in combination with fluorescence in situ hybridization (FISH). The first method involves photodocumentation of May-Grünewald-Giemsa (MGG)-stained cells, followed by destaining in methanol-acetic acid, fixation in paraformaldehyde, and digestion with protease and RNAse before FISH using alpha-satellite probes that specify chromosomes X, 7, 8, and 17. On average, two hybridization signals were seen in 94.5% of disomic BM cells. The hybridization sensitivity was found to vary, however, both among morphologically defined hematopoietic cell lineages and among differentation levels within a lineage. In the second method, an esterase staining technique was followed by the same treatment as for MGG-stained cells. The esterases and FISH signals could be simultaneously visualized and the method was found suitable for rapid screening of in situ signals in cytochemically defined granulocytes and lymphocytes but not in monocytes. The combined methods proved very useful in elucidating the clinical significance of chromosomal abnormalities seen in two cases of leukemia.
Asunto(s)
Células Sanguíneas/patología , Médula Ósea/patología , Cromosomas Humanos , Esterasas/análisis , Histocitoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Células Sanguíneas/enzimología , Médula Ósea/enzimología , Ciclo Celular/genética , Humanos , Interfase/genética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/patología , Coloración y EtiquetadoRESUMEN
The aim of this study was to evaluate the activity of topotecan in patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). Forty-seven patients with a diagnosis of MDS (n = 22) or CMML (n = 25) were treated. The median age was 66 years. Chromosomal abnormalities were present in 70% and thrombocytopenia less than 50 x 10(3)/microL in 51%. Evaluation of outcome and of differences among subgroups was performed according to standard methods; the criteria for response were those used for acute leukemia. Topotecan was administered as 2 mg/ m2 by continuous infusion over 24 hours daily for 5 days (10 mg/m2 per course) every 3 to 4 weeks until remission, then once every month for a maximum of 12 courses. Thirteen patients (28%) achieved a complete response (CR) and six (13%) had hematologic improvement. A CR was achieved in six of 22 patients with MDS (27%) and in seven of 25 with CMML (28%). All eight patients who presented with cytogenetic abnormalities (five chromosome 5 or 7 abnormalities) who achieved CR were cytogenetically normal in CR. Characteristics for which there was evidence of association with a higher response rate were lack of prior chemotherapy, less than 10% marrow monocytes, and absence of RAS oncogene mutations. In contrast, CR rates were similar in patients with or without abnormal karyotypes. Mucositis occurred in 64% of patients (severe in 19%) and diarrhea in 32% (severe in 13%). Febrile episodes occurred in 85% of patients and documented infections in 47%. With a median follow-up duration of 8 months, the 12-month survival rate was 38%, median survival time 10.5 months, and median remission duration 7.5 months. We conclude that topotecan has significant activity in MDS and CMML, with acceptable side effects. Future studies will investigate topotecan combined with topoisomerase II reactive agents, cytarabine, or hypomethylating agents (azacytidine and decitabine).
Asunto(s)
Antineoplásicos/uso terapéutico , Camptotecina/análogos & derivados , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Topoisomerasa I , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Camptotecina/efectos adversos , Camptotecina/farmacología , Camptotecina/uso terapéutico , Diarrea/inducido químicamente , Femenino , Fiebre/inducido químicamente , Estudios de Seguimiento , Genes ras , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/mortalidad , Tablas de Vida , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Inducción de Remisión , Estomatitis/inducido químicamente , Análisis de Supervivencia , Tasa de Supervivencia , Topotecan , Resultado del TratamientoAsunto(s)
Transformación Celular Neoplásica/metabolismo , Leucemia Mielomonocítica Crónica/genética , Proteína Oncogénica p21(ras)/fisiología , Factores de Transcripción/fisiología , Translocación Genética/fisiología , Humanos , Leucemia Mielomonocítica Crónica/enzimología , Modelos Biológicos , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiología , Transducción de SeñalRESUMEN
When purified control neutrophils were primed with GM-CSF, a significant increase in FMLP-induced MPO release was observed (mean +/- S.E.M., 3.4 +/- 0.8 mU/10(7) unprimed cells compared to 6.5 +/- 1.1 mU/10(7) primed cells, p < 0.001). This MPO release was greatly augmented by Cytochalasin B (Cy B), but after the addition of Cy B the priming effects of GM-CSF became less obvious. Exposure to GM-CSF without FMLP did not enhance MPO release. Within whole blood, FMLP produced negligible MPO release, but priming with GM-CSF prior to FMLP always resulted in a significant increase in MPO release. Myelodysplastic neutrophils released similar amounts of MPO in response to FMLP, compared with control cells (3.4 +/- 0.8 mU/10(7) control cells compared to 2.7 +/- 0.3 mU/10(7) MDS cells, p > 0.05). Priming with GM-CSF produced an increase in FMLP-stimulated MPO release comparable with control cells. In terms of total MPO content, although some MDS patients exhibited low levels, as a group there was no significant difference from controls (169 +/- 21 mU/10(7) control cells compared with 157 +/- 19 mU/10(7) MDS cells). These findings suggest that MPO activity is not a universal defect in MDS and cannot account for the defects in respiratory burst activity in these neutrophils.