Pan myeloid antigen-negative pediatric acute megakaryoblastic leukemia.

Acute megakaryoblastic leukemia (AMKL) is a relatively common type of acute myeloid leukemia in children. We describe two unusual cases of AMKL that by flow cytometry (FC) lacked expression of any commonly evaluated myeloid antigens. One case presented as a periorbital myeloid sarcoma and clinically was thought to be a solid tumor. In both cases, the leukemic blasts were variably positive for the megakaryocytic marker CD61. Cytogenetics confirmed the presence of the t(1;22) in one case. Cytogenetics and inclusion of megakaryocytic markers in FC panels when evaluating pediatric specimens is critical for appropriate diagnosis for myeloid antigen negative AMKL.

Spontaneous remission of childhood acute marrow fibrosis and megakaryoblastic leukemia.

Spontaneous remission in 2 children with myelofibrosis, one with megakaryocytic acute myeloblastic leukemia and t(1;22) (with recurrence later) and one with Down syndrome and GATA1 mutation (permanent), are described. One had sepsis and was treated with antibiotics and blood products, whereas the other received only blood products. Remission was spontaneous, without chemotherapy treatment. Possible explanations for these outcomes include immunologic response to sepsis by a leukemia-specific T-cell response or the release of various cytokines, such as tumor necrosis factor and interleukin-2, during infections. Natural killer and cytotoxic T cells transfused with blood products might have also triggered an immune response.

Acute megakaryoblastic leukaemia (AMKL) in children: a comparison of AMKL with and without Down syndrome.

To characterize childhood acute megakaryoblastic leukaemia (AMKL), we reviewed 45 children with AMKL diagnosed between 1986 and 2005 at Nagoya University Hospital and Japanese Red Cross Nagoya First Hospital. Twenty-four patients (53%) had AMKL associated with Down syndrome (DS-AMKL) and 21 (47%) had non-DS-AMKL. The median age of the DS-AMKL patients was 21 months (range, 8-38 months) and that of non-DS-AMKL patients was 15 months (range, 2-185 months). The morphology of blast cells was categorized into three groups according to the stage of megakaryocyte maturation. The blast cells were more immature in DS-AMKL than in non-DS-AMKL in terms of morphology and immunophenotyping. Cytogenetic abnormalities of leukaemic cells were classified into seven categories: normal karyotype including constitutional trisomy 21 in DS-AMKL; numerical abnormalities only; t(1;22)(p13;q13); 3q21q26 abnormalities; t(16;21)(p11;q22); -5/del(5q) and/or -7/del(7q); and other structural changes. The outcome of children with either DS-AMKL or non-DS-AMKL is excellent. The 10-year overall survival estimate was 79% [95% confidence interval (CI): 54-90] for DS-AMKL and 76% (95% CI: 58-91) for non-DS-AMKL (P = 0.81) with a median follow-up of 78 months (range, 20-243 months). Our study shows the diverse heterogeneity of childhood AMKL and the need for subclassification according to cytogenetic and morphological features.

Cytoplasmic CD3 expression in infant acute megakaryoblastic leukemia: A new ambiguous lineage subtype?

Several conventions have been established in order to define and characterize Mixed Phenotype Acute Leukemia (MPAL). However, megakaryocytic markers have not been included in the definition of MPAL neither in the European Group for the Immunological Characterization of Leukemias (EGIL) proposal nor in any of the WHO Classification of Tumors issues. We report four pediatric acute leukemia (AL) cases (prevalence: 0.18%) with megakaryoblasts co-expressing the T-specific antigen CD3 (cytoplasmic), together with a very homogeneous antigen profile of immature cells and other lymphoid traits. In one case, the presence of epsilon CD3 mRNA was confirmed as well on sorted CD34+ blasts. All four cases were infants, and two of them disclosed trisomy 21 in the blast population (not constitutional) without being children with Down Syndrome. They were homogeneously treated with AML schemes, achieving all four CR. However, 3 patients relapsed early. Only one patient is alive and remain disease-free, with a long follow-up. Even though cyCD3 was the only T cell marker expressed, its specificity entails the consideration of these cases as a new subtype of MPAL Megakaryoblastic/T, keeping this in mind when designing diagnostic panels. Detection and report of these cases are necessary so as to further characterize them in order to define the most appropriate treatment.

A case of acute megakaryoblastic leukaemia (FAB M7), a rare type of acute myeloid leukemia (AML), in a teenager.

Acute Megakaryoblastic Leukaemia (AML, M7) is a rare type of acute myeloid leukemia (AML) evolving from primitive megakaryoblasts. It accounted for 1.2% of newly diagnosed AML according to Eastern Cooperative Oncology Group (ECOG) trials between 1984 and 1997. Patients may present with a broad variety of symptoms including low-grade fever, easy bruising, and life-threatening conditions. We report a rare case of AML, M7 in a 19-year-old lady who presented with weakness and fatigue. She was diagnosed as a case of AML, M7 on the basis of peripheral blood finding, bone marrow examination report, radiological findings and immunophenotyping.

CD34+, CD41+ acute megakaryoblastic leukemia in a dog.

A clinically normal, 5-year-old intact female German Shepherd dog was presented to the local veterinarian to be spayed. Results of a preoperative CBC included mild nonregenerative anemia, severe thrombocytopenia, and 17% unclassified cells. On cytologic examination of aspirates from the dog's enlarged spleen and peripheral lymph nodes, a population of primitive round cells that occasionally resembled megakaryocytes was observed. A bone marrow aspirate specimen was markedly hypercellular with approximately 65% of marrow cells comprising a homogeneous population of immature hematopoietic cells similar to those found in the spleen, lymph nodes, and peripheral blood. Using immunocytochemical stains with canine-specific antibodies, all neoplastic cells strongly expressed cytoplasmic CD41 and 20-70% of the neoplastic cells expressed CD34 weakly to moderately. Rare (<0.5%) neoplastic cells weakly expressed vWF. The cells were negative for all other markers. Based on these results and the morphology of the neoplastic cells, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. In spite of treatment, results of a CBC performed 1 week later indicated progressive anemia and thrombocytopenia, and the dog was euthanized. To our knowledge, this report documents the first case of canine AMegL diagnosed with both anti-canine CD34 and CD41 antibodies.

Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis.

A megakaryoblastic cell line, termed T-33, was established from the peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis. T-33 cells have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for over 2 years. Giemsa-banded karyotypes were female hyperdiploid with a modal chromosomal number of 51, all cells including Philadelphia chromosome. The cells showed strong positivity for periodic acid-Schiff and alpha-naphthyl acetate esterase, and weak for alpha-naphthyl butyrate esterase, but were negative for myeloperoxidase. Flow cytometric analysis of cell surface markers showed the existence of HLA-DR, MY-7, MY-9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes. Most of the cells fixed with acetone were positive for Factor VIII, platelet glycoprotein IIb-IIIa, IIIa (Yukb), and Ib, but negative for glycophorin A and hemoglobin. Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% after the treatment with 12-O-tetradecanoylphorbol-13-acetate. The cells contained small dense granules negative for platelet peroxidase, their number increasing threefold after 12-O-tetradecanoylphorbol-13-acetate treatment. Such treated cells frequently showed a complex of the demarcation membrane in the cytoplasm. T-33 responded thrombin to exhibit calcium influx. This cell line may be useful for the study of the early stage of megakaryocytic differentiation in human megakaryopoiesis.

Acute megakaryoblastic leukemia: establishment of a new cell line (MKPL-1) in vitro and in vivo.

A megakaryoblastic cell line (MKPL-1) was newly established from the bone marrow of an adult patient with acute megakaryoblastic leukemia. This cell line grew in single cell suspension with a doubling time of 30 h and consisted of large primitive blasts with persistent development of giant cells carrying multilobed nuclei. MKPL-1 cells were positive for platelet GPIIb/IIIa (CD41) and GPIIIa (CD61), and expressed OKM5 (CD36), MY7 (CD13), and MY9 (CD33) antigens in the absence of erythroid and lymphoid markers. The cytochemical and morphologic characteristics of MKPL-1 were also consistent with those of megakaryoblasts. The cells did not, however, express ultrastructural platelet peroxidase which is considered to be another marker of the megakaryocytic lineage. Cytogenetic analysis of MKPL-1 revealed a model chromosome number of 92 with abnormal chromosomes including those found in the patient's bone marrow cells. Furthermore, MKPL-1 cells were serially transplanted into nude mice for nine passages with production of lethal tumors and leukemic manifestation. Thus, our megakaryoblastic cell line which can be maintained both in vitro and in vivo would be useful for further studies of the biology of megakaryopoiesis and megakaryoblastic leukemia.

Different expression of CD36 antigen molecule on the surface of megakaryocyte lineage leukemias and megakaryocyte leukemia cell lines MEG-01 and HEL.

Peripheral blood leukemic cells from four patients with peroxidase negative acute leukemia, which expressed neither myeloid nor lymphoid cell surface antigens, were analyzed by using monoclonal antibodies (MoAb) capable of recognizing megakaryocyte-platelet-related antigens. Leukemic cells from one case reacted with 5F1 MoAb, whereas cells from all the tested cases reacted with OKM5 MoAb, which belongs to the same CD group as 5F1 (CD36). Also, culture cells from megakaryoblastic leukemia cell line, MEG-01, and human erythroleukemia cell line, HEL, showed a different pattern of expression for the CD36 antigen molecule detected by 5F1 and OKM5 MoAb, individually. Furthermore, we have demonstrated that the epitopes recognized by 5F1 and OKM5 MoAb appear on the same CD36 molecule on the surface of HEL cells by means of the two-color analysis using FACS-IV. On the basis of our experiments, we conclude that, CD36 molecule, a receptor for TSP, is synthesized and expressed in at least two ways, inside the cells and on the surface of megakaryocyte lineage leukemias and megakaryocytic leukemia cell lines MEG-01 and HEL. This is strongly suggestive that thrombospondin (TSP)-mediated adhesion represents an alternative pathway for cytoadherence, and that CD36 expression on various kinds of cells may lack some essential modifications or components necessary for the TSP receptor activity.

CD34+ megakaryoblastic leukaemic cells are CD38-, but CD61+ and glycophorin A+: improved criteria for diagnosis of AML-M7?

The aim of this flow cytometry study in acute megakaryoblastic leukaemia (AML-M7) was to describe the membrane phenotype of CD34+ progenitor subsets and compare these with the phenotypes expressed by other AML FAB types. Following conventional histopathological diagnosis mononuclear cells from bone marrow and blood were examined in seven patients with AML-M7 and compared with results from 26 sequential patients with AML-M0 to AML-M6. The CD34+ subsets in AML-M7 patients differed from that of patients with AML-M0 to AML-M6 as the CD34+ CD61+ and the CD34+ Glycophorin A+ subsets were median 31% and 20%, respectively, compared to 4% and 2% in the AML-M0 to AML-M6 (P = 0.0005). Only 1% of the CD34+ progenitors were CD34+ CD38+ in AML-M7 compared to 72% in other AML subtypes (P < 0.000). These findings suggest that the CD34+ cell compartment in AML-M7 consists of early lineage-specific progenitors. In conclusion, flow cytometry analysis of CD34+ subsets may improve the diagnostic safety in AML-M7 and consequently the prognostic significance of immunophenotyping in acute leukaemia.

Cell surface c-kit receptors in human leukemia cell lines and pediatric leukemia: selective preservation of c-kit expression on megakaryoblastic cell lines during adaptation to in vitro culture.

We produced a monoclonal antibody MTK1 which recognized c-kit protein. Using MTK1, 31 leukemia cell lines and 76 leukemia blasts from pediatric patients were analyzed for expression of the c-kit receptor by flow cytometry. The c-kit receptor was detectable on four of four cell lines assigned to the megakaryo/erythromegakaryoblastic lineage and on one of seven cell lines of myeloid lineage. C-kit expression was not seen on any of 20 cell lines of erythroid and lymphoid lineages. Furthermore, c-kit was expressed on 16 of 24 nonlymphoid blasts without platelet surface antigens (67%) and on six of eight non-lymphoid blasts with platelet surface antigens (75%), but was not detectable on 44 lymphoid blasts from pediatric leukemia patients. In these cases CD34 was expressed on 26 of 32 myeloid blasts (81%) and on 27 of 44 lymphoid blasts (61%). The findings indicate a dominant expression of the c-kit receptor on established cell lines assigned to the megakaryo/erythromegakaryoblastic lineage, though a high percentage of leukemic myeloblasts also expressed the c-kit receptor on their surface.

Characterization of two novel sublines established from a human megakaryoblastic leukemia cell line transfected with p210(BCR-ABL).

Disease progression in chronic myelogenous leukemia (CML) is usually accompanied by chromosomal abnormalities such as an additional Ph chromosome, trisomies of chromosome 8 or 19, or i(17) in addition to the standard translocation t(9;22) (q34;q11). However, detailed studies of the various steps involved during this evolution are difficult to perform, thereby making the study of cell lines that contain the transposed genes BCR-ABL, especially those of human origin, an important focus. In this analysis we investigated the human megakaryoblastic cell line MO7e and its subline transfected with BCR-ABL, MO7e/p210. Initial studies demonstrated that the phenotype of the MO7e line was consistent with a megakaryocytic lineage as originally described and was growth factor dependent in liquid culture. The MO7e/p210 subline, however, was growth factor independent and could be further separated into two distinct sublines based on expression of glycophorin A using the monoclonal antibody R10. The subline R10 negative (R10-) was similar to the parent line MO7e but R10 positive (R10+) cells had a distinct erythroid phenotype. In addition, the R10- and R10+ sublines demonstrated strikingly different colony morphology when cultured in semisolid medium. Furthermore, R10+ cells had additional chromosomal abnormalities not detected in the R10- population. These results demonstrate that the insertion of the BCR-ABL in this human leukemia cell line resulted in two distinct subpopulations of cells, each now growth factor independent, but one with a phenotype and karyotype identical to the parent cell line and the other with a different phenotype and additional chromosomal abnormalities. These two subpopulations derived from the MO7e/p210 transfected cell line may prove useful in further understanding the multistep events that occur in the progression of this disease.

Flow cytometric analysis of peroxidase negative acute leukemias by monoclonal antibodies--II. Acute megakaryoblastic and acute pro-megakaryocytic leukemias.

In this report, we have described three cases of acute megakaryoblastic leukemia (AMKL) which were demonstrated by the presence of megakaryocyte-platelet-related cell-surface antigens detected by utilizing flow cytometry and monoclonal antibodies in addition to both PPO activity which was shown by ultrastructural cytochemistry and emergence of differentiation antigens while culturing these leukemic cells. The blasts of one case possessed both platelet GpIb and GpIIb/IIIa cell-surface antigens detected by 5F1 (CD36), AN51 (CDw42), and J15, P2 and HPL2 (CDw41), respectively, whereas the remaining two cases almost completely lacked Gp1b cell-surface antigen. Hence, the former was diagnosed as immature (pro) megakaryocytic leukemia and the latter as AMKL from the viewpoint of immunophenotypic analysis as discussed in this article.

Myeloperoxidase-positive acute megakaryoblastic leukemia.

Acute megakaryoblastic leukemia (FABM7) is an unusual but well recognized form of acute myelogenous leukemia in which the bone marrow blast cells are phenotypically recognized by the demonstration of cytoplasmic platelet peroxidase or surface staining for the IIb/IIIa platelet-specific glycoprotein. Herein, the authors report a case of acute megakaryoblastic leukemia that satisfies the accepted French-American-British criteria and in which the blast cells also exhibit evidence of myeloid differentiation, including surface MY7 (CD13) by flow cytometry and immunocytochemical positivity for myeloperoxidase. These findings suggest that megakaryoblasts may be closely related to myelomonoblasts, that they have the potential to partially differentiate along multiple phenotypic lines, and that aberrant phenotypes can occur that do not correspond to known stages of normal maturation. The authors illustrate the difficulty in classification of these aberrant phenotypes by standard cytochemical and morphologic criteria.

Acute megakaryoblastic leukemia simulating carcinoma.

Acute megakaryoblastic leukemia has emerged as an important subset of early childhood leukemia. It often presents a diagnostic dilemma because of its many morphologic manifestations and propensity to mimic metastatic carcinoma. An abdominal mass was identified by sonographic and computed tomographic scans in a 10-month-old girl, who had anemia and thrombocytopenia. An open biopsy of the 3-cm, peripancreatic mass showed cohesive nests and sheets of tumor cells with focal spindling and desmoplasia. Although the diagnosis of acute megakaryoblastic leukemia was established from a bone marrow aspirate using immunocytochemical techniques and karyotype analysis, a coexistent abdominal epithelial malignant neoplasm could not be excluded entirely by light microscopic examination alone. The megakaryoblastic nature of the abdominal tumor was established by immunocytochemical stains for glycoprotein IIIa on paraffin-embedded tissue.

Dysmegakaryopoiesis resembling acute megakaryoblastic leukemia in treated acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by trilineage dysplasia, including atypical megakaryocytes. Acute megakaryoblastic leukemia (FAB M7) is particularly associated with atypical megakaryocytic hyperplasia (AMH). Fifteen patients with nonmegakaryoblastic AML developed AMH after therapy, comprising 12.6% of cases of AML diagnosed from 1986 to 1989. Platelet counts were normal in nine patients and decreased in six. Blasts comprised less than 5% of cells in 40% of the biopsies, ranged from 5-15% in 53%, and comprised more than 30% of cells in 7%. Numerous small hypo- and hyperlobated megakaryocytes were seen in all specimens, often occurring in clusters, and were more easily seen in sections than in smears. Subsequent biopsies in 13 patients showed a remission marrow in seven, increased blasts in three, and AML in three; none showed AMH. AMH resembling acute megakaryoblastic leukemia may be seen transiently after treatment of AML.

Cytochemical profile of megakaryoblastic leukaemia: a study with cytochemical methods, monoclonal antibodies, and ultrastructural cytochemistry.

A cytochemical study using: Sudan black B; alpha-naphthyl acetate (ANAE) staining; estimation of alpha-naphthyl butyrate (ANBE) esterase activity; acid phosphatase activity; and 5' nucleotidase activity was carried out in 15 cases of megakaryoblastic leukaemia. These included cases of M7 acute myeloid leukaemia and blast crises of chronic granulocytic leukaemia. The megakaryoblastic nature of the blasts was first established using two monoclonal antibodies against platelet glycoproteins, and by estimating the platelet/peroxidase reaction at ultrastructural level. Our findings suggest that megakaryoblasts have a typical cytochemical profile comprising positive ANAE staining and acid phosphatase activity with a predominant localisation in the Golgi zone and negative or weak ANBE activity. A similar positive cytochemical pattern was also found in five cases of erythroleukaemia (M6). The specificity of the 5'nucleotidase activity for megakaryoblasts was not confirmed. In most cases of megakaryoblastic leukaemia there was no 5'nucleotidase activity only two cases showed positive reactions--reactions were positive in several cases of myeloblastic and lymphoblastic leukaemia. We suggest that cytochemical methods may be useful in diagnosing M6 and M7 acute leukaemia because less than 40% of leukaemic cells react with specific monoclonal antibodies.

Expression of CD56/NCAM on hematopoietic malignant cells. A useful marker for acute monocytic and megakaryocytic leukemias.

We investigated the expression of CD56 (a neural cell adhesion molecule, NCAM) and CD57 in various hematopoietic and non-hematopoietic malignant cells, using Leu-19 and Leu-7 monoclonal antibodies. Although both molecules are commonly defined as a natural killer cell marker, we found that CD56 was highly expressed on blasts from patients with acute monocytic (4/6) and megakaryocytic (3/3) leukemias. In the latter, FACS two-color analysis revealed that leukemic megakaryoblasts simultaneously expressed CD56 and platelet-related antigens. Among leukemic cell lines, one myelocytic, three monocytic, and two megakaryocytic lines were positive for CD56. On the other hand, except for one large granular lymphocytic leukemia and one multiple myeloma cell line, none of the lymphoid leukemia cell lines or lymphoblasts from patients with acute lymphocytic leukemia (ALL) (0/15), non-Hodgkin's lymphoma (NHL) (0/2), and central nervous system (CNS) leukemia (0/2) reacted with Leu-19 antibody for CD56. The expression of CD56 in leukemia cells was not significantly affected by 12-O-tetradecanoylphorbol-13-acetate (TPA). By contrast, all hematopoietic materials were negative for CD57, while non-hematopoietic neuroblastoma cell lines expressed this molecule (4/5) as well as CD56 (5/5). Cytogenetically, the NCAM gene is located at chromosome 11q23, and chromosome breaks were often observed at this location in various leukemias. Blasts from all five acute non-lymphocytic leukemia (ANLL) patients and cell lines with 11q23-proximal chromosomal breaks were positive, while those from one ALL patient with an 11q23 abnormality were negative for CD56, necessitating further studies to clarify the link between the 11q23 abnormality and CD56 expression.

Acute megakaryoblastic leukemia.

In 1985 acute megakaryoblastic leukemia was included in the FAB classification system of hematological neoplasias with the designation of AML M7. It occurs in all age groups with two peaks in distribution. The one is in adults and the other in children 1 to 3 years of age especially in those with Down's syndrome. The diagnosis of AML M7 requires more than 30% of the nucleated bone marrow cells being megakaryoblasts. The more common types of AML MO-M6 have to be excluded by morphological and cytochemical analysis whereas immunology is needed to exclude ALL. The megakaryocytic nature of the leukemia has to be proven by ultrastructural demonstration of platelet peroxidase or by immunological demonstration of CD61, CD42, CD41 on the surface of the leukemic blasts. Megakaryocytic/megakaryoblastic leukemias show a wide morphologic spectrum. In some instance small cells dominate, clearly showing megakaryocytic differentiation with scant amounts of cytoplasm and with nuclei showing dense chromatin. On the other hand, there are cases with larger cells resembling ALL-L2 blasts with moderate amounts of rather basophilic cytoplasm which in some instances contain azurophilic granules. Cytoplasmic blebs and protrusions are the most prominent feature of many cases. The nuclei of these cells are round with more finely reticulated chromatin and with prominent nucleoli. The megakaryoblastic nature of these cells can be suggested by morphology. However, according to our experience there are cases of c-ALL with the very same morphologic picture. Consequently, immunologic phenotyping of these cases is necessary in any instance. Cytochemistry is of limited diagnostic value in megakaryoblastic leukemias. Usually it is used to exclude the more common types of leukemia.