ABSTRACT
Abstract The present study evaluated 56 patients diagnosed with Chronic Lymphocytic Leukemia (CLL) and a control group of 44 clinically healthy subjects with no previous history of leukemia. Genetic expressions of AKT and microRNAs were evaluated by quantitative PCR (qPCR). A significant increase in AKT gene expression in patients when compared to controls was observed (p = 0.017). When the patients were stratified according to Binet subgroups, a significant difference was observed between the subgroups, with this protein kinase appearing more expressed in the B+C subgroup (p = 0.013). Regarding miRNA expression, miR-let-7b and miR-26a were reduced in CLL patients, when compared to controls. However, no significant differences were observed in these microRNA expressions between the Binet subgroups (A versus B+C). By contrast, miR-21 to miR-27a oncogenes showed no expression difference between CLL patients and controls. AKT protein kinase is involved in the signaling cascade that occurs with BCR receptor activation, leading to increased lymphocyte survival and protection against the induction of cell death in CLL. Thus, increased AKT protein kinase expression and the reduction of miR-let-7b and miR-26a, both tumor suppressors, may explain increased lymphocyte survival in CLL patients and may be promising markers for the prognostic evaluation of this disease.
Subject(s)
Humans , Male , Female , Protein Kinases , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Patients , Gene Expression/genetics , Apoptosis , MicroRNAs/pharmacology , Healthy VolunteersABSTRACT
Chronic lymphocytic leukemia (CLL) is a blood cancer characterized by the accumulation of clonal B-lymphocytes. This study evaluated the mRNA gene expression of miR-15a, miR-16- 1, ZAP-70, and Ang-2 by qPCR, as well as the plasma levels of Bcl-2 by Elisa immunoassay, in CLL patients and healthy controls. Significant differences were observed when comparing patients and controls regarding miR-15a (p < 0.001), miR-16-1 (p < 0.001) mRNA, Ang-2 gene expression, and Bcl-2 plasma levels (p < 0.001). When stratified by risk, differences were maintained with a significantly reduced expression in high-risk patients. A positive correlation was observed between miR-15a and platelets (R2 = 0.340; p = 0.009) as well as between Bcl-2 and leukocytes (R2 = 0.310; p = 0.019). Conversely, negative correlations were observed between ZAP-70 and platelets (R2 = - 0.334; p = 0.011), between miR-15a and lymphocytes (R2 = - 0.376; p = 0.004), as well as between miR-16-and lymphocytes (R2 = - 0.515; p = 0.00004). The data suggest that a reduction in miR-15a and miR-16-1 expressions, in addition to an overexpression of Bcl-2, are associated with the reduction in apoptosis and, consequently, to a longer survival of lymphocytes, thus contributing to lymphocyte accumulation and aggravation of the disease. By contrast, Ang-2 expression was significantly higher in A than in B + C Binet groups. This context leads to the speculation that this biomarker should be investigated in more robust studies within populations with a still relevantly indolent form of the disease in an attempt to identify those patients with a greater potential for an aggravation of the disease
Subject(s)
Humans , Male , Female , Biomarkers/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , ZAP-70 Protein-Tyrosine Kinase/analysis , Patients , Enzyme-Linked Immunosorbent Assay/instrumentation , Gene Expression , ApoptosisABSTRACT
Introduction: The laboratory diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), disease that is categorized by reduced synthesis of glycosylphosphatidylinositol (GPI) anchor, is based on the detection of blood cells deficient in GPI-anchored proteins by flow cytometry. PNH clones have been detected in patients with aplastic anaemia (AA) and myelodysplastic syndrome (MDS), with therapeutic implications. Objectives: To validate a sensitive assay for detection of GPI-anchored protein-deficient cells, by flow cytometry, and to analyze the clone frequency in AA and MDS patients. Methods: Samples from 20 AA patients, 30 MDS patients and 20 adult volunteers (control group) were analyzed using monoclonal antibodies to CD16, CD24, CD55 and CD59 (neutrophils); CD14 and CD55 (monocytes); CD55 and CD59 (erythrocytes); besides fluorescent aerolysin reagent (FLAER) (neutrophils and monocytes) and lineage markers. The proportions of PNH cells detected in neutrophils and monocytes, using different reagent combinations, were compared by analysis of variance (ANOVA) and Pearson's correlation. Results: PNH cells were detected in five (25%) AA patients, and the proportions of PNH cells varied from 0.14% to 94.84% of the analyzed events. PNH cells were not detected in the MDS patients. However, by the analysis of these samples, it was possible to identify the technical challenges caused by the presence of immature and dysplastic circulating cells. FLAER showed clear distinction of GPI-deficient cells. Conclusion: Multiparameter flow cytometry analysis offers high sensitivity and accuracy in the detection of subclinical PNH clones. FLAER shows excellent performance in detection of PNH neutrophils and monocytes...
Introdução: O diagnóstico laboratorial da hemoglobinúria paroxística noturna (HPN), doença caracterizada por deficiência de síntese da molécula de ancoragem glicosilfosfatidilinositol (GPI), baseia-se na detecção de células sanguíneas deficientes em proteínas ancoradas ao GPI, por citometria de fluxo. Clones de células com fenótipo HPN podem ser detectados em pacientes com anemia aplásica (AA) e síndrome mielodisplásica (SMD), com implicações terapêuticas. Objetivos: Validar técnica sensível para detecção de células HPN, por citometria de fluxo, e avaliar a frequência dos clones em pacientes com AA e SMD. Métodos: Amostras de 20 pacientes com AA, 30 pacientes com SMD e 20 voluntários (controles) foram analisadas, utilizando anticorpos monoclonais anti-CD16, CD24, CD55 e CD59 (neutrófilos); CD14 e CD55 (monócitos); e CD55 e CD59 (hemácias); além do reagente de aerolisina fluorescente (FLAER) (neutrófilos e monócitos) e marcadores de linhagem celular. A comparação do tamanho dos clones HPN detectados em neutrófilos e monócitos, pelas diferentes combinações de reagentes, foi realizada por análise de variância (ANOVA) e correlação de Pearson. Resultados: Em cinco (25%) pacientes com AA foram identificadas células HPN, em proporções entre 0,14% e 94,84% dos eventos analisados. O clone não foi detectado nos pacientes com SMD. Contudo, a análise dessas amostras permitiu evidenciar as dificuldades técnicas secundárias à presença de células imaturas e displásicas circulantes. O reagente FLAER propiciou separação precisa das células alteradas. Conclusão: A análise multiparamétrica por citometria de fluxo apresenta sensibilidade...