Megakaryoblastic Differentiation Research Articles

Improved identification of clinically relevant Acute Leukemia subtypes using standardized EuroFlow panels versus non-standardized approach.

Rare acute leukemia (AL) components or subtypes such as blastic plasmacytoid dendritic cell neoplasm (BPDCN) or early T-cell precursor acute Lymphoblastic Leukemia (ETP-ALL) can be difficult to detect by routine flow cytometry due to their immunophenotypes overlapping with other poorly differentiated AL. We hypothesized that using standardized EuroFlow™ Consortium approach could better diagnose such entities among cases that previously classified as acute myeloid leukemia (AML)-M0, AML with minimal differentiation, AML with myelodysplasia-related changes without further lineage differentiation, and AL of ambiguous lineage. In order to confirm this hypothesis and assess whether these AL subtypes such as BPDCN and ETP-ALL had previously gone undetected, we reanalyzed 49 banked cryopreserved sample cases using standardized EuroFlow™ Consortium panels. We also performed target sequencing to capture the mutational commonalities between these AL subtypes. Reanalysis led to revised or refined diagnoses for 23 cases (47%). Of these, five diagnoses were modified, uncovering 3 ETP-ALL and 2 typical BPDCN cases. In 12 AML cases, a variable proportion of immature plasmacytoid dendritic cell and/or monocytic component was newly identified. In one AML case, we have identified a megakaryoblastic differentiation. Finally, in five acute lymphoblastic leukemia (ALL) cases, we were able to more precisely determine the maturation stage. The application of standardized EuroFlow flow cytometry immunophenotyping improves the diagnostic accuracy of ALs and could impact treatment decisions.

Unusual CD34 positivity in acute myeloid leukaemia with myelodysplasia-related changes with megakaryoblastic differentiation.

Acute megakaryoblastic leukaemia is a rare entity with distinct immunophenotype profile and cytogenetic lesions. Herein, we report a case of acute myeloid leukaemia with myelodysplastic‐related changes with megakaryoblastic differentiation in absence of recurrent genomic lesions such as t(1;22), inv(3) and t(3;3).One of the peculiarities of this case is the positivity of CD34+ within the abnormal megakaryoblasts; CD61 immunohistochemistry highlights the heavily infiltration of bone marrow from abnormal megakaryoblasts.

Pathologic Features of Down Syndrome Myelodysplastic Syndrome and Acute Myeloid Leukemia: A Report From the Children's Oncology Group Protocol AAML0431.

Detailed diagnostic features of acute myeloid leukemia in Down syndrome are lacking, leading to potential misdiagnoses as standard acute myeloid leukemia occurring in patients with Down syndrome. To evaluate diagnostic features of acute myeloid leukemia and myelodysplastic syndrome in patients with Down syndrome. Diagnostic bone marrow samples from 163 patients enrolled in the Children's Oncology Group study AAML0431 were evaluated by using central morphologic review and institutional immunophenotyping. Results were compared to overall survival, event-free survival, GATA1 mutation status, cytogenetics, and minimal residual disease results. Sixty myelodysplastic syndrome and 103 acute myeloid leukemia samples were reviewed. Both had distinctive features compared to those of patients without Down syndrome. They showed megakaryocytic and erythroid but little myeloid dysplasia, and marked megakaryocytic hyperplasia with unusual megakaryocyte morphology. In acute myeloid leukemia cases, megakaryoblastic differentiation of blasts was most common (54 of 103, 52%); other cases showed erythroblastic (11 of 103, 11%), mixed erythroid/megakaryoblastic (20 of 103, 19%), or no differentiation (10 of 103, 10%). Myelodysplastic syndrome and acute myeloid leukemia cases had similar event-free survival and overall survival. Leukemic subgroups showed interesting, but not statistically significant, trends for survival and minimal residual disease. Cases with institutional diagnoses of French American British M1-5 morphology showed typical features of Down syndrome disease, with survival approaching that of other cases. Myelodysplastic syndrome and acute myeloid leukemia in Down syndrome display features that allow discrimination from standard cases of disease. These distinctions are important for treatment decisions, and for understanding disease pathogenesis. We propose specific diagnostic criteria for Down syndrome-related subtypes of acute myeloid leukemia and myelodysplastic syndrome.

Lysine-Specific Demethylase 1A as a Promising Target in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy characterized by the accumulation of incompletely differentiated progenitor cells (blasts) in the bone marrow and blood, and by suppression of normal hematopoiesis. It has recently become apparent that the AML genome is characterized by recurrent mutations and dysregulations in epigenetic regulators. These mutations frequently occur before the onset of full blown leukemia, at the pre-leukemic phase, and persist in residual disease that remains after therapeutic intervention, thus suggesting that targeting the AML epigenome may help to eradicate minimal residual disease and prevent relapse. Within the AML epigenome, lysine-specific demethylase 1 A (LSD1) is a histone demethylase that is found frequently overexpressed, albeit not mutated, in AML. LSD1 is a required constituent of critical transcription repressor complexes like CoREST and nucleosome remodeling and deacetylase (NuRD), and abrogation of LSD1 expression results in impaired self-renewal and proliferation, and increased differentiation and apoptosis in AML models and primary cells, particularly in AMLs with MLL- and AML1-rearrangements, or erythroid and megakaryoblastic differentiation block. On this basis, a number of LSD1 inhibitors have been developed in the past decade, and few of them are currently being tested in clinical trials for patients with AML, along with other malignancies. To date, the most promising application of this therapeutic strategy appears to be combination therapy of LSD1 inhibitors with all-trans retinoic acid (ATRA) to reactivate myeloid differentiation in cells that are not spontaneously susceptible to ATRA treatment. In this review, we provide an overview of LSD1 function in normal hematopoiesis and leukemia, and of the current clinical application of LSD1 inhibitors for the treatment of patients with AML.

Myeloid sarcoma with megakaryoblastic differentiation presenting as a breast mass

Myeloid sarcoma is an extramedullary tumor that consists of myeloblasts or immature myeloid cells. The neoplasm can occur in any part of the body, including the bone, periosteum, lymph nodes, skin, and soft tissue and they may occur de novo or in association with acute myeloid leukemia, myeloproliferative neoplasms and myelodysplastic syndromes. Most cases display a myelomonocytic or pure monoblastic morphology. Tumors with megakaryoblastic differentiation are extremely uncommon and may occur in association with transformation of a myeloproliferative disorder. Myeloid sarcoma presenting as a breast mass is very rare and diagnostically challenging. We report a case of myeloid sarcoma with megakaryoblastic differentiation in the breast of a patient with history of essential thrombocythemia. The mass was composed of undifferentiated pleomorphic malignant cells in trabecular cords and nests with many scattered giant malignant cells and brisk abnormal mitoses. On immunohistochemistry, the neoplastic cells were positive for CD61, CD31, CD34, Factor VIII and CD43 which confirmed the diagnosis. To our knowledge, this is the first report of myeloid sarcoma with megakaryoblastic morphology occurring in the breast. Here we discuss the clinicopathologic features of this rare entity and the challenges involved in making this difficult diagnosis.

Basophilia and megakaryoblastic differentiation in a case of acute myeloid leukemia: An unusual morphological combination

Basophilia is commonly associated with chronic myelogenous leukemia, notably in the accelerated phase or during blast crisis. It is also associated with other myeloproliferative neoplasms. However, its association with acute leukemia is very rare and is described in association with acute basophilic leukemia and few acute myeloid leukemias (AMLs) with recurrent genetic abnormalities such as t(6;9)(p23;q34). Herein, we describe the morphological features and discuss the differential diagnosis of a case of AML with the blasts showing previously unreported unusual combination of megakaryoblastic and basophilic differentiation along with peripheral blood and bone marrow basophilia.

Myeloid sarcoma with megakaryoblastic differentiation mimicking a sellar tumor

Myeloid sarcoma (MS) is a localized extra-medullary tumor mass of immature myeloid cells, arising de novo or related to acute myeloid leukemia, of which it can be a forerunner, a coinciding or late event. Less commonly, MS represents an acute blastic transformation of myelodysplastic syndromes or myeloproliferative neoplasms. This rare condition commonly consists of a proliferation of more or less immature cells with a myeloid immunophenotype, very exceptional cases showing a megakaryoblastic or erythroid differentiation. The most common localization of MS is the skin, lymph node, soft tissues and bones, but CNS involvement is exceedingly rare, with no cases reported in the sellar region. We report a 54-year-old man, affected by myeloproliferative neoplasm, JAK2 V617F-positive of 13 years duration, who acutely presented with a third cranial nerve palsy; neuroradiology documented a space-occupying lesion at the level of the sellar, upper clival and right parasellar regions, that was sub-totally removed with a trans-sphenoidal approach. The histological examination documented a proliferation of large, blastic cells, frequently multinucleated; a diagnosis of MS with megakaryoblastic differentiation, arising in a background of chronic idiopathic myelofibrosis, was suggested by immunohistochemistry, owing to CD42b, CD45, CD61 and LAT (linker for activation of T cells) positivity. In addition, homozygous JAK2 V617F mutation was detected from the myeloid sarcoma specimen. A few weeks after surgery, an acute blastic leukemic transformation occurred and, despite chemotherapy, the patient died 2 months after surgery. To the best of our knowledge, this is the first MS case with megakaryoblastic differentiation arising within the CNS.

Cyto-morphological features of extramedullary acute megakaryoblastic leukemia on fine needle aspiration and cerebrospinal fluid cytology: A case report

Extramedullary deposits may be the presenting feature of acute myeloid leukemia. An early and accurate diagnosis on cytology will aid in correct patient management. This is especially true for patients with acute megakaryoblastic leukemia (AML M7), where bone marrow aspiration may yield only a dry tap. While cytomorphological features of myeloid sarcoma of other types are well recognized due to its rarity, there are only two case reports discussing the morphological details of megakaryoblastic differentiation on aspiration cytology. We present the case of a 25-year-old patient with extramedullary involvement of lymph node and cerebrospinal fluid by AML M7, describing in detail, the morphological features on aspiration as well as exfoliative cytology.

Retroviral mediated expression of the human myeloid nuclear antigen in a null cell line upregulates Dlk1 expression.

The human myeloid nuclear differentiation antigen (MNDA) is a hematopoietic cell specific nuclear protein. MNDA and other related gene products interact with and alter the activity of a large number of proteins involved in regulating specific gene transcription. MNDA and related genes exhibit expression characteristics, which suggest functions unique to specific lineages of cells, in addition to mediating the effects of interferons. Cells of the human K562 myeloid line do not express MNDA and are relatively immature compared to lines that express MNDA (HL-60, U937, and THP1). The hypothesis that MNDA influences the expression of specific genes was tested by creating MNDA expressing K562 cells using stable retroviral mediated gene transfer followed by evaluation of transcription profiles. Two macroarrays containing a total of 2,350 cDNAs of known genes showed a specific up-regulation of Dlk1 expression in MNDA expressing K562 cell clones. Real time quantitative RT-PCR analysis confirmed an average of over 3- and 7-fold upregulation of Dlk1 in two clones of MNDA expressing K562 cells. The effects on Dlk1 were also confirmed by Northern blotting. Dlk1 is essential for normal hematopoiesis and abnormal expression is a proposed marker of myelodysplastic syndrome. Additional screening of transcription profiles after induced erythroid and megakaryoblastic differentiation showed no additional gene transcripts altered by the presence of MNDA. These results indicate that MNDA alters expression of a gene essential for normal hematopoiesis.

Regulation of c-myc mRNA Decay in Vitro by a Phorbol Ester-inducible, Ribosome-associated Component in Differentiating Megakaryoblasts

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of c-myc expression, because its constitutive expression blocks differentiation. TPA-induced differentiation of K562 cells causes rapid down-regulation of c-myc expression, due in part to an mRNA decay rate that is 4-fold faster compared with dividing cells. A cell-free mRNA decay system reconstitutes TPA-induced destabilization of c-myc mRNA, but it requires at least two components for reconstitution. One component fractionates to the post-ribosomal supernatant from either untreated or treated cells. This component is sensitive to cycloheximide and micrococcal nuclease. The other component is polysome-associated and is induced or activated by TPA. Although in dividing cells c-myc mRNA decays via a sequential pathway involving removal of the poly(A) tract followed by degradation of the mRNA body, TPA activates a deadenylation-independent pathway. The cell-free mRNA decay system reconstitutes this alternate decay pathway as well.

Human megakaryoblastic proliferation and differentiation events observed by phase-contrast cinematography.

Microcinematographic documentation of mitoses, amitoses, endomitoses, or cytoplasmic fusion shortly after completion of mitoses was done in bone marrow specimens of patients with quantitative platelet disorders and controls. In patients with platelet disorders, most mitoses with cell duplication occurred in large promegakaryocytes after 4-fold nuclear and cytoplasmic enhancement. Normal specimens showed polyploidization happening in small megakaryoblasts, while mitoses with cell duplication were seen only after cultivation in freeze-thawed sera of patients with platelet disorders. Frequently, lymphocytes were observed to contact megakaryoblastic cells undergoing mitoses, amitoses and endomitoses and to enter the cytoplasm of megakaryocytes (emperipolesis), leaving it again after several hours.

Translocation t(15;17) in acute myelogenous leukemia with atypical megakaryoblastic features: Diagnostic, clinical, and therapeutic implications

We describe the first reported case of acute myelogenous leukemia with characteristics of megakaryoblastic differentiation and the t(15;17) chromosomal translocation, which has been associated with promyelocytic leukemia. The diagnostic, clinical, and therapeutic implications are discussed.

Granulocytic sarcoma of megakaryoblastic differentiation complicating chronic idiopathic myelofibrosis

We report the development of soft tissue granulocytic sarcoma with megakaryoblastic differentiation in a patient with chronic idiopathic myelofibrosis, which has hitherto been undescribed. Although an artifactual signet ring appearance of the blasts was found in the formalin-fixed tissue and caused diagnostic problems, the histological appearance on B-5 fixed material and immunophenotyping confirmed the diagnosis. Cytogenetic analysis showed 47,XY, + der(1)del(1) (?p13) at initial presentation and 47,XY, del(1)(?p13), +der(1)del(1) (?p13) at the time of soft tissue blastic transformation, indicating that partial trisomy 1 might be of pathogenetic significance.

Immunological definition of acute minimally differentiated myeloid leukemia (MO) and acute undifferentiated leukemia (AUL).

Immunophenotyping has become an important tool in the diagnosis of acute leukemia for several reasons. Indeed the use of a standardized panel of monoclonal antibodies (MoAb) to B and T cells, and myeloid cells, as well as non lineage restricted antigens, permits allocation of more than 98% of acute leukemia to their respective lineage. In ALL, immunophenotyping has established a basis for precise and biologically oriented classification of the disease which may be of prognostic importance. In AML immunological markers are particularly important for identification of acute leukemia with minimal myeloid, erythroblastic or megakaryoblastic differentiation. Immunological markers also allow the identification of acute leukemias with minimal or aberrant marker expression, acute biphenotypic leukemia in which single cells coexpress different lineage associated markers and acute bilineage leukemia where there are two separate blast cell populations (usually lymphoid and myeloid). There is sometimes confusion in the literature about the definition of acute unclassifiable and acute undifferentiated leukemia. This is mainly due to misinterpretation of phenotypic data or to the lack of relevant lineage specific markers in these studies, especially for the detection of cytoplasmic antigens. Indeed, it is important to stress that in hematopoietic precursors, antigens detected by monoclonal antibodies first appear in the cytoplasm during early differentiation and are only expressed on the membrane later. This has been demonstrated not only for the T lineage (Cy CD3), the B lineage (CyCD22) but also for the myeloid lineage (CyCD13).(ABSTRACT TRUNCATED AT 250 WORDS)

2 Immunophenotypic classification of acute lymphoblastic leukaemia

Recent advances in immunophenotyping of acute leukaemias have led to important insights into normal haematopoietic differentiation as well as into the cellular diversity and origins of leukaemic blasts, especially in acute lymphoblastic leukaemia (ALL). These advances include the production and standardization of monoclonal antibodies (mAbs) to a variety of lymphoidand myeloid-lineage-associated antigens, the characterization of reactivity patterns of these reagents with leukaemic blasts, the establishment of an internationally accepted cluster of differentiation (CD) nomenclature and, finally, technical innovations in the evaluation of antigen expression on individual leukaemic cells. Immunophenotyping has become an important element in the modern diagnosis of acute leukaemia for several reasons. First, use of a standardized panel of mAbs to B-cell, T-cell and myeloid as well as non-lineage-restricted antigens permits allocation of more than 98% of acute leukaemias to their respective lineage (Janossy et at, 1989; Campana et al, 1990a). Secondly, in ALL, immunophenotyping has established a solid basis for precise and biologically oriented classification of the disease (Foon and Todd, 1986; Greaves, 1986); and, in acute myeloid leukaemia (AML), immunologic markers are particularly important for identifying acute leukaemia with minimal myeloid or megakaryoblastic differentiation (Bennett et al, 1985; Bennett et al, 1991; Buccheri et al, 1992). Thirdly, based on recent observations that leukaemic blasts frequently disclose aberrant or asynchronous antigen expression compared with normal haematopoietic cell differentiation, leukaemia-associated phenotypic features have been used to detect minimal residual disease (MRD) in both ALL and AML (reviewed in Campana et al, 1991a; van Dongen et al, •992). Finally, immunophenotyping alone or in conjunction with more recently developed cytogenetic and molecular-biological techniques has identified biologically and clinically distinct subsets within the major diagnostic groups and has been essential for

False-positive flow cytometric platelet glycoprotein IIb/IIIa expression in myeloid leukemias secondary to platelet adherence to blasts

Because neither morphologic nor routine cytochemical features are pathognomonic, the diagnosis of acute megakaryoblastic leukemia (AML- M7) is difficult, requiring either specialized ultrastructural or immunologic techniques. Because the ultrastructural techniques are cumbersome, immunologic assays for expression of several platelet- specific antigens, such as CD41a (platelet glycoprotein IIb/IIIa), have become the primary method used to detect megakaryoblastic differentiation. In our flow cytometric analysis of the immunophenotypes of over 1,000 cases of AML from patients registered to Southwest Oncology Group (SWOG) Treatment Protocols, we found that 38% of cases demonstrated CD41a reactivity. Because this frequency of CD41a expression by flow cytometry greatly exceeded the number of morphologically defined cases of AML-M7, we postulated that the reaction may be caused by platelets adherent to leukemic blasts. To investigate this hypothesis, we performed a side-by-side comparison of flow cytometric and cytospin immunofluorescence studies on 37 cases of adult de novo AML that demonstrated a wide range of CD41a expression by flow cytometric analyses. We found that the expression of CD41a detected by flow cytometric techniques was secondary to adherent platelets or platelet fragments in 85% of cases. Many of these cases also expressed the lineage-specific carbohydrate, LNF III (CD15), which may mediate platelet adhesion to mature monocytes and neutrophils. Only 15% of the CD41a flow cytometrically positive cases demonstrated true diffuse membrane and cytoplasmic positivity on cytospin slides indicative of megakaryoblastic differentiation. Cytospin immunofluorescence for CD41a should be performed on all cases of suspected AML-M7. If only flow cytometric techniques are used, adherent platelets may result in the erroneous diagnosis of this AML subtype.

False-Positive Flow Cytometric Platelet Glycoprotein IIb/IIIa Expression in Myeloid Leukemias Secondary to Platelet Adherence to Blasts

Because neither morphologic nor routine cytochemical features are pathognomonic, the diagnosis of acute megakaryoblastic leukemia (AML-M7) is difficult, requiring either specialized ultrastructural or immunologic techniques. Because the ultrastructural techniques are cumbersome, immunologic assays for expression of several platelet-specific antigens, such as CD41a (platelet glycoprotein IIb/IIIa), have become the primary method used to detect megakaryoblastic differentiation. In our flow cytometric analysis of the immunophenotypes of over 1,000 cases of AML from patients registered to Southwest Oncology Group (SWOG) Treatment Protocols, we found that 38% of cases demonstrated CD41a reactivity. Because this frequency of CD41a expression by flow cytometry greatly exceeded the number of morphologically defined cases of AML-M7, we postulated that the reaction may be caused by platelets adherent to leukemic blasts. To investigate this hypothesis, we performed a side-by-side comparison of flow cytometric and cytospin immunofluorescence studies on 37 cases of adult de novo AML that demonstrated a wide range of CD41a expression by flow cytometric analyses. We found that the expression of CD41a detected by flow cytometric techniques was secondary to adherent platelets or platelet fragments in 85% of cases. Many of these cases also expressed the lineage-specific carbohydrate, LNF III (CD15), which may mediate platelet adhesion to mature monocytes and neutrophils. Only 15% of the CD41a flow cytometrically positive cases demonstrated true diffuse membrane and cytoplasmic positivity on cytospin slides indicative of megakaryoblastic differentiation. Cytospin immunofluorescence for CD41a should be performed on all cases of suspected AML-M7. If only flow cytometric techniques are used, adherent platelets may result in the erroneous diagnosis of this AML subtype.

The dyad symmetry element is the molecular target for c-fos induction and inhibition during K 562 differentiation along mutually exclusive lineages

The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.

The Dyad Symmetry Element Is the Molecular Target for c-fos Induction and Inhibition During K 562 Differentiation Along Mutually Exclusive Lineages

Abstract The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13- acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.

Increased EPA/TIMP and Jun/Fos characterize TPA-induced megakaryoblastic differentiation of K562 cells

Increased EPA/TIMP and Jun/Fos characterize TPA-induced megakaryoblastic differentiation of K562 cells