RESUMO
BACKGROUND: Acute promyelocytic leukemia is a cytogenetically well defined entity. Nevertheless, some features observed at diagnosis are related to a worse outcome of the patients. METHODS: In a prospective study, we analyzed peripheral (PB) leukocyte count, immunophenotype, methylation status of CDKN2B, CDKN2A and TP73; FLT3 and NPM1 mutations besides nuclear chromatin texture characteristics of the leukemic cells. We also examined the relation of these features with patient's outcome. RESULTS: Among 19 cases, 4 had a microgranular morphology, 7 presented PB leukocytes >10x109/l, 2 had FLT3-ITD and 3 had FLT3-TKD (all three presenting a methylated CDKN2B). NPM1 mutation was not observed. PB leukocyte count showed an inverse relation with standard deviation of gray levels, contrast, cluster prominence, and chromatin fractal dimension (FD). Cases with FLT3-ITD presented a microgranular morphology, PB leukocytosis and expression of HLA-DR, CD34 and CD11b. Concerning nuclear chromatin texture variables, these cases had a lower entropy, contrast, cluster prominence and FD, but higher local homogeneity, and R245, in keeping with more homogeneously distributed chromatin. In the univariate Cox analysis, a higher leukocyte count, FLT3-ITD mutation, microgranular morphology, methylation of CDKN2B, besides a higher local homogeneity of nuclear chromatin, a lower chromatin entropy and FD were associated to a worse outcome. All these features lost significance when the cases were stratified for FLT3-ITD mutation. Methylation status of CDNK2A and TP73 showed no relation to patient's survival. CONCLUSION: in APL, patients with FLT3-ITD mutation show different clinical characteristics and have blasts with a more homogeneous chromatin texture. Texture analysis demonstrated that FLTD-ITD was accompanied not only by different cytoplasmic features, but also by a change in chromatin structure in routine cytologic preparations. Yet we were not able to detect chromatin changes by nuclear texture analysis of patients with the FTLD-TKD or methylation of specific genes.
Assuntos
Biomarcadores Tumorais/genética , Montagem e Desmontagem da Cromatina , Cromatina/ultraestrutura , Metilação de DNA , Leucemia Promielocítica Aguda/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Feminino , Fractais , Humanos , Imunofenotipagem , Leucemia Promielocítica Aguda/imunologia , Leucemia Promielocítica Aguda/mortalidade , Leucemia Promielocítica Aguda/patologia , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Nucleofosmina , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
La leucemia promielocítica (LPM) subtipo M3 representa del 5-15 por ciento en la clasificación FAB de las leucemias mieloides agudas (LMA). Está asociada con características genéticas únicas que incluyen la translocación recíproca t(15;17)(q22;q12). El mecanismo por el que ocurre la t(15;17) no se conoce. Las leucemias de estirpe mieloide expresan diversos antígenos de diferenciación tumoral como son la proteinasa 3 (PR 3) y la mieloperoxidasa (MPO) que se encuentran sobreexpresados en el promielocito. Se plantea que participan en la maduración y en la regulación de la división celular. Existe poca información acerca de la respuesta inmune de pacientes con LPM dirigida contra las células tumorales. En nuestro trabajo se detectó la presencia de anticuerpos contra los antígenos de diferenciación tumoral PR3 y MPO en diferentes fases del tratamiento de la enfermedad, mediante inmunofluorescencia indirecta. Los anticuerpos anti PR3 y anti MPO se detectaron en aquellos pacientes sin tratar y en fase de inducción, no así en la consolidación y mantenimiento, de ahí su posible utilidad como marcadores de diferenciación celular.
ABSTRACT Promyelocytic leukemia (PML) subtype M3 represents the 5-15 percent in the FAB classification of acute myeloid leukemias (AML). It is associated with the unique genetic features including the reciprocal t-translocation (15;17) (q22;q12). The mechanism of t is unknown. The myeloid leukemias express different tumoral differentiation antigens such as the proteinase 3 (PR 3) and myeloperoxidase (MPO) which are over-expressed in promyelocyte. It is involved in maturation and regulation of cell division. There is scarce information on the immune response of patients with PLM against tumor cells. In our paper we detected presence of antibodies to RP3 and MPO tumor differentiation antigens in different phases of disease treatment by indirect immunofluorescence. Anti-MPO and anti-PR3 antibodies were detected in those patients without treatment and in induction phase but not in the consolidation and maintenance, thus its potential usefulness as cellular differentiation markers.
Assuntos
Humanos , Masculino , Feminino , Antígenos de Diferenciação , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/imunologia , Técnica Indireta de Fluorescência para AnticorpoRESUMO
La leucemia promielocítica (LPM) está asociada con características genéticas únicas que incluyen la translocación recíproca t(15;17)(q22;q12) con la formación del complejo PML-RARa, el cual representa más del 95 por ciento de las proteínas de fusión en la LPM y resulta una diana potencial de la respuesta inmune. Los tratamientos actuales conocidos por terapias de diferenciación combinan el ácido trans retinoico (ATRA) y las antraciclinas, así como el trióxido de arsénico; resultan en una sobrevida a largo plazo y la cura potencial aproximadamente entre el 70 y 80 por ciento de los pacientes. En estudios realizados en ratones con LPM inmunizados con una vacuna de ADN elaborada mediante la fusión del oncogen PML-RARa al fragmento C de la toxina tetánica como adyuvante, conjuntamente con ATRA, se produjeron títulos elevados de anticuerpos. Se logró una sobrevida significativa, entre 120-300 días, comparada con la de ratones a los que se administró placebo. En nuestro trabajo se detectó la presencia de anticuerpos contra la proteína de fusión PML-RARa en diferentes fases del tratamiento, lo que indica la capacidad de respuesta de estos pacientes frente al antígeno tumoral. Los anticuerpos anti PML-RARa se detectaron en aquellos pacientes sin tratar y en fase de inducción, no así en la consolidación y mantenimiento, de ahí su posible utilidad como marcador de diferenciación celular a partir de la presencia o ausencia de la proteína de fusión en la célula leucémica.
Promyelocytic leukemia (PML) is associated with unique genetic features including the t reciprocal translocation (15; 17) (q22; q22) with PML-RARa, which accounted for more than 95 percent of fusion protein in PML and is a potential target of immune response. The current treatments known as differentiation therapies combine the trans-retinoic acid (TRA) and the anthracyclines, as well as the trioxide arsenic (As(2)0(3)); become in a long-term survival and the potential cure in about the 70 to 80 percent of patients. In studies conducted in LPM-mice immunized with a DNA vaccine made the PML-RARa oncogene fusion to C fragment of tetanus toxin as coadjuvant together with TRA were antibodies high titers. It was possible to achieve a significant survival among 120-130 days compared with that of placebo-mice. In our paper it was detected the presence of antibodies to PML-RARa fusion protein in different phases of treatment indicating the response to these patients to tumor antibody. The anti-PML-RARa were detected in those non-treated patients and in induction fusion, but not in consolidation and maintenance, thus its potential usefulness as cellular differentiation marker from presence or absence of fusion protein un leukemic cell.
Assuntos
Humanos , Anticorpos , Leucemia Promielocítica Aguda/imunologia , Proteínas de Fusão OncogênicaRESUMO
Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor alpha (RARalpha) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARalpha fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARalpha develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARalpha TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 +/- 16.68, 10.83 +/- 8.11, 7.4 +/- 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARalpha TM present a specific immunophenotype.
Assuntos
Antígenos CD/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Promielocítica Aguda/imunologia , Proteínas de Fusão Oncogênica/imunologia , Animais , Antígenos CD/genética , Medula Óssea/imunologia , Medula Óssea/patologia , Catepsina G , Catepsinas , Citometria de Fluxo , Genótipo , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/genética , Camundongos , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Serina Endopeptidases , Baço/imunologia , Baço/patologiaRESUMO
Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46 percent, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.
Assuntos
Animais , Camundongos , Antígenos CD/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Promielocítica Aguda/imunologia , Proteínas de Fusão Oncogênica/imunologia , Antígenos CD/genética , Medula Óssea/imunologia , Medula Óssea/patologia , Catepsinas , Citometria de Fluxo , Genótipo , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/genética , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Serina Endopeptidases , Baço/imunologia , Baço/patologiaRESUMO
Acute promyelocytic leukemia (APL) is a subtype acute myeloid leukemia in which leukemic promyelocytes predominate in the bone marrow (BM). Rapid diagnosis is critical for treatment decision since all-trans-retinoic acid must be administrated promptly. The microgranular variant may be of difficult diagnosis, as it may be confused with other diseases on morphological grounds. The purpose of this study was to determine if the microgranular variant has the same antigenic profile as the classical hypergranular type. The immunophenotype of leukemic cells from the bone marrow of 50 patients, with the PML-RARalpha gene rearrangement confirmed by RT-PCR, was determined by flow cytometry using a large panel of 22 monoclonal antibodies and a polyclonal anti-TdT antibody. Thirty-four cases were classified as classical APL and 16 as microgranular APL. The immunophenotypic profile of the two subtypes was indistinguishable concerning the presence or absence of these antigens, including the absence of reactivity for the HLA-DR antigen. The simultaneous immunophenotypic combination of a unique major cell population, heterogeneous intensity of expression of CD13, and the typical pattern of CD15/CD34 expression were similarly present in the hypergranular and microgranular subtypes. Homogeneous expression of CD33 was observed in 76% of the classical APL cases and in 100% of the microgranular cases. Additionally, we have studied two cases of PLZF-RARalpha APL that also displayed the same immunophenotype described for classical APL. Thus, the immunophenotypic profile highly characteristic of the PML-RARalpha gene rearrangement was also observed in microgranular and PLZF-RARalpha variants of APL.
Assuntos
Antígenos CD/biossíntese , Rearranjo Gênico , Leucemia Promielocítica Aguda/diagnóstico , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Adolescente , Adulto , Idoso , Antígenos CD34/biossíntese , Medula Óssea/imunologia , Medula Óssea/metabolismo , Antígenos CD13/biossíntese , Criança , Análise Citogenética , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Antígenos CD15/biossíntese , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We describe an 8 year old boy who had received chemotherapy for an acute promyelocytic leukemia and developed a secondary leukemia 27 months after the diagnosis of this first malignancy. Blasts cells were positive for cytoplasmic markers CD22, CD3 and myeloperoxidase. Cell surface T and myeloid-associated markers were also detected. Cytogenetic study disclosed monosomy 7. The patient achieved complete remission, but relapsed 15 months later with identical immunophenotypic and cytogenetic findings. Three-lineage commitment is proved by the expression of specific criteria for myeloid, and lymphoid T and B typing. A multipotent immature progenitor must be the target of leukemogenic agents. The prognosis is obviously ominous.
Assuntos
Moléculas de Adesão Celular , Cromossomos Humanos Par 7 , Lectinas , Leucemia Promielocítica Aguda/genética , Leucemia/genética , Monossomia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos B/análise , Biomarcadores Tumorais , Complexo CD3/análise , Criança , Humanos , Imunofenotipagem , Cariotipagem , Leucemia/imunologia , Leucemia/patologia , Leucemia Promielocítica Aguda/imunologia , Masculino , Peroxidase/análise , Recidiva , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Se efectuó la determinación de inmunocomplejos circulantes (ICC) así como la medición del sistema complemento por la vía clásica, vía alterna, actividad del factor B y la cuantificación del tercer (C3) y cuarto (C4) componentes de complemento en 30 pacientes con leucemia promielocítica (LPM) al diagnóstico, 22 de los cuales presentaron coagulación intravascular diseminada (CID). Se demostró la existencia de niveles elevados de ICC en los enfermos con CID y una disminución significativa de la actividad de la vía clásica, los componentes C3 y C4 en los enfermos con CID, al compararlos con el grupo de pacientes que no presentaba el trastorno de la hemostasia y los controles normales, lo que sugiere la posible participación de estos parámetros en el fenómeno de la CID en estos enfermos
Assuntos
Humanos , Adulto , Coagulação Intravascular Disseminada/imunologia , Complexo Antígeno-Anticorpo/análise , Proteínas do Sistema Complemento , Leucemia Promielocítica Aguda/imunologiaRESUMO
Six cases of microgranular variant acute promyelocytic leukemia (M3v) were studied by use of a multiparameter approach including morphology, cytochemistry, flow cytochemistry, flow cytometry, cytogenetics, and gene rearrangement. Three of six cases demonstrated both myeloid and monocytoid associated surface markers by flow cytometry. One of six cases had strong alpha-naphthyl-butyrate esterase (alpha-NBE) activity in addition to myeloperoxidase activity. There was no correlation between percentage of positive monocytoid surface markers and intensity of cytoplasmic alpha-NBE activity. Four of six cases also had a T-cell-associated surface antigen. Further studies indicated that the T-cell markers appeared to be on the promyelocytes and that the T-B receptor gene was not rearranged. Similarly, cytogenetics studies indicated only one clonal abnormality t(15q+; 17q-). Whether these cases represent true "lineage infidelity" remains to be answered. Future important studies are needed on normal hematopoietic progenitor cells at early stages of development and childhood to study lineage-specific characteristics and to determine whether co-expression normally exists during early development.