Acute Megakaryocytic Leukemia Research Articles

Acute Megakaryocytic Leukaemia (AMKL)-like Disease in a Cynomolgus Monkey ( Macaca fascicularis)

A 5-year-old male cynomolgus monkey (Macaca fascicularis) with a clinical history of bleeding tendency, severe anaemia, thrombocytopenia and elevated serum concentration of liver-related enzymes was examined post mortem. Ecchymotic haemorrhages were present on the left eyelid and forehead. The liver, kidney and spleen were markedly enlarged and the kidneys had capsular petechiae. Microscopically, numerous atypical cells resembling myeloid cells were observed in the bone marrow, and myelofibrosis was present. Atypical cells were also present in the blood vessels of the liver, kidney, spleen, lymph nodes, lung, heart, bladder, adrenal gland and brain. Some neoplastic cells had oval or pleomorphic macronuclei and others were multinucleated. Immunohistochemically, the majority of the neoplastic cells had granular cytoplasmic expression of the megakaryocyte-associated antigens Von Willebrand Factor and CD61-IIIa, but were negative for myeloperoxidase. A diagnosis of acute megakaryocytic leukaemia (AMKL)-like disease was made. This would appear to be the first report of AMKL-like disease in non-human primates. This monkey was infected with simian retrovirus type D and it is possible that this viral infection was associated with the development of neoplasia.

Unbalanced chromosome 1 abnormalities leading to partial trisomy 1q in four infants with Down syndrome and acute megakaryocytic leukemia

BackgroundChildren with Down syndrome (DS) have an increased risk of childhood acute leukemia, especially acute megakaryoblastic leukemia (AMKL) also called acute myeloid leukemia (AML) type M7. Here four yet unreported infants with such malignancies are reported.ResultsAn unbalanced translocation involving chromosome 1 was identified by GTG banding in all cases. These were characterized in more detail by molecular cytogenetic approaches. Additional molecular analysis revealed in three of the four cases mutations in exon 2 of the GATA binding protein 1 (globin transcription factor 1), located in Xp11.23.ConclusionOur results corroborate that abnormalities of chromosome 1 are common in DS-associated AMKL. Whether this chromosomal region contains gene(s) involved in hematopoietic malignant transformation remains to be determined.

Long-term Complete Remission in a 71-year-old Patient with AML-M7 after Low-dose Cytarabine Induction and Intermediate-dose Cytarabine Consolidation Treatment

The authors describe the case of a 71-year-old patient with acute megakaryocytic leukemia (AML-M7) who was successfully treated with low-dose cytarabine induction followed by intermediate-dose cytarabine consolidation therapy. The patient presented with infection and rapidly increasing blood blasts. The diagnosis was consistent with AML-M7 with a normal karyotype. Peripheral blood blasts decreased rapidly upon low-dose cytarabine administration, and the patient achieved complete remission after two courses of low-dose cytarabine (10 bid for 12 days). Consolidation therapy with intermediate-dose cytarabine (1.0 bid on day 1, 3 and 5) was then instituted without serious complication. He remained in complete remission at the time of writing 47 month after diagnosis. In spite of multiple poor prognostic factors, this patient showed excellent treatment outcome through low-dose cytarabine induction and intermediate-dose cytarabine consolidation. It needs to be validated whether acute leukemia with a megakaryocytic morphology is exceptionally sensitive to cytarabine.

Ectopic Expression of ERG and FLI1 Enforce a Megakaryocyte Phenotype Independent of GATA-1

Specific genes that contribute to Transient Myeloproliferative Disorder (TMD) and DS Acute Megakaryocytic Leukemia (AMKL) remain largely undefined. To date, only the X-linked transcription factor GATA1, which is mutated in these disorders, has been implicated in contributing to DS hematopoietic malignancies. We recently reported that overexpression of either of the chromosome 21 ETS family members ERG or ETS2 led to a dramatic increase in megakaryopoeisis from wild-type or Gata1-knockdown fetal liver hematopoietic progenitors. Furthermore, we demonstrated that expression of ETS2 or ERG caused a significant increase in megakaryocyte colony formation and led to immortalization of Gata1-knockdown fetal liver hematopoietic progenitors in vitro. In new studies to define the mechanisms by which these ETS proteins promote megakaryopoiesis, we discovered that the ETS family member FLI1, which shares significant homology with ERG, significantly increased CD41 and CD42 expression and the degree of polyploidization of megakaryocytes in vitro in a manner that was nearly identical to that of ectopic ERG. Furthermore, FLI1 also immortalized Gata1-knockdown fetal liver progenitors. To characterize the effects of ectopic ETS family members on transcription, we performed genome wide expression profiling using RNA isolated from fourth generation immortalized Gata1-knockdown cells expressing ETS2, ERG, FLI1 or GFP alone. Principal component analysis showed that the ERG and FLI1 gene expression patterns clustered tightly with one another and were well separated from both the MIGR1 and ETS2 signatures. Unsupervised hierarchical clustering further revealed that 726 genes were differentially expressed in FLI1, ERG, ETS2 immortalized cells compared to MIGR1 control cells. Of these, genes associated with the erythroid lineage, including Klf1, Lmo2, glycophorin A, Egr1, Ank1 and Eraf were strongly repressed. These findings are consistent with recent publications showing that FLI1 promotes the development of megakaryocytes at the expense of red blood cells and suggest that ERG and ETS2 mimic FLI1 in our assays. Notably, we did not observe significant increases in the expression of Hox genes or Meis1, genes whose expression is often associated with transformed hematopoietic cells. However, we did detect increased expression of Bmi1, which has an essential role in regulating the proliferative capacity of both normal and malignant hematopoietic progenitors, as well as increased expression of Jak2 and Stat5, two genes whose activation is associated with myeloproliferative disease and megakaryocytic transformation, in ETS-family transduced cells. Next, to determine whether ETS-family expression affected the immunophenotype of Gata1-knockdown fetal liver progenitors, immortalized cells were assessed for cell surface expression of c-kit and CD41. ERG and FLI1 expressing colonies included a substantial proportion of CD41+/c-kit- cells, indicative of megakaryocytic maturation. Furthermore, colonies formed from progenitors transduced with ERG were comprised of greater than 90% CD41+ cells, confirming that nearly all of the cells within the immortalized colonies are of the megakaryocyte lineage. Finally, because our gene array data showed increased Jak2 and Stat5 mRNA levels in ETS-family transduced cells, and activating mutations in JAK3 are associated with AMKL, we assessed whether JAK/STAT signaling was altered in the immortalized cells. Using flow cytometry, we found that cells within the ERG and FLI1 cultures consistently showed significant increases in phospho-STAT3 staining in comparison to the MIGR1 control. The increases in STAT3 phosphorylation suggest that ERG and FLI1 promote immortalization of Gata1-knockdown fetal liver progenitors in part by activation of the JAK/STAT pathway. Together our data support the hypothesis that an increased dosage of ETS factors, including ETS2 and ERG, contribute to a pro-megakaryocytic phenotype and to the leukemogenic process in DS TMD and AMKL.

Biclonal (CD34+ CD42b+) Myeloblastic and Megakaryoblastic Transformation of Chronic Myeloproliferative/Myelodysplastic Disorders

Despite the widespread use of molecular and genetic assays (i.e. JAK2V617F, FLT-3, bcr-abl, PML-RARalpha) for diagnoses of myeloid neoplasms, some disease entities remain difficult to define. For instance, distinguishing between blastic phase of bcr-abl negative chronic myeloproliferative disease, unclassifiable (CMPD-U), transformed myelodysplastic/myeloproliferative disease-unclassifiable (MDS/MPD-U), acute megakaryocytic leukemia (AML FAB-M7), and idiopathic myelofibrosis (IMF) per the 2001 WHO classification system remains challenging. Common to all of these diagnoses are marrow dysplasia, presence of immature myelocytic and megakaryocytic cells, and reticulin myelofibrosis. Here we describe our institute experience with 11 patients (pts) who presented between 2004–2008 with acute myeloid neoplasms characterized by prominent myeloblastic and megakaryoblastic cell populations with myelofibrosis. Morphological diagnoses were: MDS-refractory anemia with excess blasts (n=1), blastic phase of MPD/MDS with myelofibrosis (n=5), atypical or mixed acute myelocytic and megakaryoblastic leukemia (n=4), and acute megakaryoblastic leukemia (n=1). Median age at diagnosis was 69 years (range 20–89). Six pts were male (55%) and five female. The majority presented with pancytopenia with a median white blood cell count of 2.55 × 109/l (range<0.01–237.5), median hemoglobin of 9.6gm/dl (range 5.6–10.6), and median platelet count of 57 × 109/l (range 14–296). The median percentage of peripheral blood blasts was 7% (range 0–52%) with a median of 28.5% (range 18.5–73%) blasts in the bone marrow. Organomegaly was present in 4/11 pts (1 hepatomegaly, 3 splenomegaly) at diagnosis. Only two pts reported a known antecedent MDS diagnosis (7 and 9 months prior to AML). One pt had relapsed disease with a prior diagnosis of AML FAB M1. Bcr-abl was negative in all cases; FLT-3 TKD/ITD was negative in all 9/9 cases tested (2 not available). 10/11 marrows underwent correlative cytogenetic tests showing normal karyotype in 2 pts and complex in 4 pts. Four pts had chromosome 7 aberrations (−7/del (7), t(7;17), der (7). One AML had inv(3)(q21q26). Dual immunohistochemical staining for CD34 and CD42b expression confirmed the presence of separate abnormal myeloblastic (CD34+) and megakaryoblastic cells (CD42b+) in all samples with a minority of cells expressing both antigens. All patients underwent therapy for AML/MDS: 8 received cytarabine/anthracyclines/etoposide (ADE) or Hidac-based induction regimens, 1 received subcutaneous cytarabine, 1 received Zarnestra therapy, and 1 Vidaza only. 7/11 pts were refractory to or died during chemotherapy. Three patients achieved complete remission, and two underwent allogeneic stem cell transplantation with overall survival (OS) of 18 and 8.5 months. One relapsed prior to autologous transplant. Median OS for 10 pts was 6.2months (range 0.5–18.5 months) with one pt lost to follow up.Conclusions: Acute bcr-abl negative myeloid disorders displaying biclonal CD34+ myeloblastic and CD42b+ megakaryoblastic cell populations with myelofibrosis most likely represent leukemic evolution from prior chronic (undiagnosed) MPD or MDS/MPD. Dual immunohistochemical staining for CD34/CD42b is invaluable in distinguishing between the two immature blastic populations with coexpression of both markers on some cells suggestive of a common leukemia stem cell origin. Clinical outcomes of these patients are poor, and allogeneic stem cell transplantation remains the best long-term treatment option if remission can be achieved.

Vascular Branching Morphogenesis of the Placenta Requires Expression of Ott1(Rbm15) in the Trophoblast Compartment.

OTT1(RBM15) is a transcriptional activator/repressor known to be a fusion partner in t(1;22) infant acute megakaryocytic leukemia. Conditional deletion of Ott1 in adult mice resulted in expansions of the megakaryocytic, myeloid and pluripotent Lin−Sca-1+c-Kit+ populations and a block in pre B lymphoid development, suggesting a global regulatory role in hematopoiesis. Germline deletion of Ott1 is homozygous lethal after E9.5, producing developmentally delayed embryos with open notochords. Further investigation into the cause of embryonic death revealed a placental defect characterized by arrested vascular branching morphogenesis of the invading fetal vessels, leading to embryonic demise through inadequate oxygenation. Histopathology and in situ hybridization of Ott1-deficient placentas identified a dramatic reduction in the spongiotrophoblast and syncytiotrophoblast layers. Expression of Vegf, a critical element of placental vasculogenesis, was still observed, thus excluding the requirement for Ott1 in its induction. By using Sox2-cre with a conditional allele of Ott1, complete excision of Ott1 was achieved within the embryo yet the placental trophoblast compartment was left undeleted. These embryos were now able to form normal placentas with intact vascular branching morphogenesis and survive to term. This result demonstrates that Ott1-mediated vascular branching morphogenesis is regulated by the trophoblast compartment rather than the invading fetal vasculature. Additionally, the Sox2-cre-deleted pups manifested cardiac ventricular septal defects and hyposplenia and succumbed during the perinatal period. Hox, Notch and hypoxia-related pathways are important for both cardiac and splenic development, however downstream target gene expression was not perturbed. In summary, Ott1-dependent pathways, in addition to their implicit involvement in a congenital leukemia, are essential for vascular branching morphogenesis in the placenta and organogenesis of the mesoderm-derived heart, spleen and blood. Mutation of OTT1 or alterations in its regulation may therefore underlie human disorders such as placental insufficiency, asplenia or congenital cardiac malformations.

Ott1 (Rbm15) Is Required for the Large to Small Pre B-Cell Transition

The OTT1 gene is fused with the MAL gene in t(1;22) infant-associated acute megakaryocytic leukemia generating a chimeric protein, OTT1-MAL. OTT1 is a transcriptional activator/repressor related to the spen/SHARP/Mint family. Mint was shown to regulate follicular versus marginal zone B-cell development in the spleen; however, Ott1's physiologic role in B cell development is not fully understood. Recent data utilizing conditional deletion of a targeted Ott1 allele in mice delineated multiple regulatory roles for Ott1 in myeloid and lymphoid differentiation. Previous work by our laboratory showed that loss of Ott1 in addition to causing a myeloid and megakaryocytic expansion, results in a block in B development prior to the B220+CD43-IgM+ stage. We have characterized Ott1-deficient B-cell progenitors to identify stage- and process-specific requirements for Ott1 in pre B development. Ott1-deleted bone marrow and fetal liver could not generate pre B colonies in methylcellulose, however, we were able to establish IL-7-dependent pro B cell lines in vitro and observed no significant differences in proliferation, apoptosis or the ability to form v-Abl-transformed cell lines. Activated Ras or overexpression of Bcl2 failed to rescue pre B colony formation. In vivo, Ott1 null fetal liver pre B-cells expressed Ig heavy chain but failed to express the B-cell receptor (BCR) on their surface even though kappa rearrangement was detectable in vitro. In comparison to wildtype cells, B220+CD43-IgH+ Ott1 null cells were larger in size, had lower levels of IL-7R, but proliferated at higher levels and with an associated increase in apoptosis. Moreover, these cells had normal pre-BCR proximal signaling as judged by phospho-Blnk and phospho-Erk, but increased phosphorylation of S6 after IL7 and Ig stimulation. Ott1 null large pre B-cells had normal expression of Myc, but higher levels of expression of Cyclin D1. Taken together, these data indicate that loss of Ott1 results in enhanced proliferation and apoptosis in the pre B-cell compartment causing a developmental block at the large to small pre B-cell transition. Differentiation blocks at the pro and pre B stage through mutations in B cell regulatory genes, such as PAX5, BTK and BLNK, have been recently demonstrated in acute lymphocytic leukemias. It is plausible that mutations in OTT1, given its position in the tightly regulated process of B cell development, may likewise contribute to pre B-leukemogenesis.

OTT-MAL Is a Deregulated Activator of Serum Response Factor-Dependent Gene Expression

The OTT-MAL/RBM15-MKL1 fusion protein is the result of the recurrent translocation t(1;22) in acute megakaryocytic leukemia in infants. How it contributes to the malignancy is unknown. The 3' fusion partner, MAL/MKL1/MRTF-A, is a transcriptional coactivator of serum response factor (SRF). MAL plays a key role in regulated gene expression depending on Rho family GTPases and G-actin. Here we demonstrate that OTT-MAL is a constitutive activator of SRF and target gene expression. This requires the SRF-binding motif and the MAL-derived transactivation domain. OTT-MAL localizes to the nucleus and is not regulated by upstream signaling. OTT-MAL deregulation reflects its independence from control by G-actin, which fails to interact with OTT-MAL in coimmunoprecipitation experiments. Regulation cannot be restored by reintroduction of the entire MAL N terminus into the fusion protein. OTT-MAL also caused a delayed induction of the MAL-independent, ternary complex factor-dependent target genes c-fos and egr-1 and the mitogen-activated protein kinase/Erk pathway. With testing in heterologous tissue culture systems, however, we observed considerable antiproliferative effects of OTT-MAL. Our data suggest that the deregulated activation of MAL-dependent and -independent promoters results in tissue-specific functions of OTT-MAL.

Megakaryocytic blast crisis as a presenting manifestation of chronic myeloid leukemia

Acute megakaryocytic leukemia is a rare form of acute myelogenous leukemia and may occur either de novo or by transformation of a preexisting myelodysplastic or myeloproliferative process including blast crisis of chronic myeloid leukemia (CML). Megakaryocytic blast crisis as the presenting manifestation of CML is extremely rare. We describe such a patient with no prior hematologic disease who presented with acute megakaryoblastic leukemia and extramedullary involvement, in whom the leukemic cells carried the BCR-ABL1 translocation as part of a complex karyotype. Using targeted sequential fluorescence in situ hybridization (T-FISH) technique, we detected two copies of BCR-ABL1 fusion gene in the leukemic blasts while the neutrophils carried a single copy of BCR-ABL1 fusion gene, thereby proving the origin of the megakaryoblastic leukemia from a previously undiagnosed CML clone. Blast crisis as a presenting manifestation of CML is rare and detecting clonal evolution of acute leukemia by specialized cytogenetic techniques may have important diagnostic and therapeutic implications.

Tetrasomy 21 as a sole acquired abnormality without GATA1 gene mutation in pediatric acute megakaryoblastic leukemia: A case report and review of the literature

We report a case of pediatric acute megakaryocytic leukemia (AMKL) showing 48,XX,+21,+21 as a sole acquired cytogenetic abnormality without the mutation of GATA1 gene. A physical examination showed a phenotypically normal female. Bone marrow findings showed diffuse infiltration of leukemic blasts having scanty cytoplasm with budding blebs and prominent nucleoli, which were negative for myeloperoxide (MPO) stain, Sudan black B stain and periodic acid–Schiff stain. Immunophenotyping of leukemic cells revealed positive expression of CD34, CD13, CD33, CD117, CD41, CD61, CD7 and negative expression of TdT, anti-MPO, CD64, CD56, CD2, CD3, CD5, CD10, CD19, CD20 and CD22. A fluorescence in situ hybridization analysis showed four distinct AML1 signals in 284 of 300 interphase nuclei. The entire six exons of the GATA1 gene (7757 bp) were directly sequenced. We could not find any mutations, including known polymorphisms, which are known to be involved in transient myeloproliferative disorder and acute megakaryocytic leukemia of Down syndrome. After achieving complete remission, the tetrasomy 21 disappeared.

Platelet Factor-4 Is an Indicator of Blood Count Recovery in Acute Myeloid Leukemia Patients in Complete Remission

To investigate whether serum biomarkers can be used to indicate the responsiveness of acute myeloid leukemia to remission induction chemotherapy, we performed MALDI-TOF protein profile analysis of patient sera. The resulting spectra revealed a protein (or peptide) peak at m/z 7764 that varied in intensity; its intensity was much higher in samples from patients in complete remission than in those from patients with resistant disease or in samples taken prior to treatment (at the time of diagnosis). Using fractionation, trypsin digestion, MS/MS, and protein molecular weight analyses, we identified the m/z 7764 protein as platelet factor-4 (PF4). This identification was confirmed by a magnetic bead-based MALDI immunoassay. Statistical comparison of PF4 levels and platelet counts in patient sera revealed a significant positive correlation between the two variables. This study demonstrates that PF4 protein levels are a good indicator for the recovery of blood count in the complete remission of acute myeloid leukemia. The linear positive correlation curve indicates that blood count recovery of platelets to >100,000/mm(3) is equivalent to a serum PF4 recovery level of >2.492 microg/ml.

GATA1Mutation in Transient Myeloproliferative Disorder of Down Syndrome

Children with Down syndrome (DS) have a higher risk of developing leukemia than do healthy children, and they especially have a higher risk for developing transient myeloproliferative disorder (TMD) or acute megakaryocytic leukemia (AMKL). In recent studies, it has been reported that most of these patients have acquired mutation of the GATA1 gene, which encodes the erythroid/megakaryocytic transcription factor GATA1. GATA1 mutations have not been found in AMKL patients who did not have DS and other hematologic malignancies in DS. Most of the GATA1 mutations in DS-TMD/AMKL are nonsense mutations that are mainly located in exon 2. We observed a nonsense mutation in exon 2 of GATA1 [c.189_190delCA (Tyr63X)] in one case of DS-TMD. The GATA1 mutation has been thought to be an early event in the leukemogenesis of DS-TMD/AMKL and it could be used as a stable molecular marker to assess the treatment response or to monitor for the recurrence of DS-TMD/AMKL.

The role of the proto-oncogene ETS2 in acute megakaryocytic leukemia biology and therapy

Acute myeloid leukemia (AML) in Down syndrome (DS) children has several unique features including a predominance of the acute megakaryocytic leukemia (AMkL) phenotype, higher event-free survivals compared to non-DS children using cytosine arabinoside (ara-C)/anthracycline-based protocols and a uniform presence of somatic mutations in the X-linked transcription factor gene, GATA1. Several chromosome 21-localized transcription factor oncogenes including ETS2 may contribute to the unique features of DS AMkL. ETS2 transcripts measured by real-time RT-PCR were 1.8- and 4.1-fold, respectively, higher in DS and non-DS megakaryoblasts than those in non-DS myeloblasts. In a doxycycline-inducible erythroleukemia cell line, K562pTet-on/ETS2, induction of ETS2 resulted in an erythroid to megakaryocytic phenotypic switch independent of GATA1 levels. Microarray analysis of doxycycline-induced and doxycycline-uninduced cells revealed an upregulation by ETS2 of cytokines (for example, interleukin 1 and CSF2) and transcription factors (for example, TAL1), which are key regulators of megakaryocytic differentiation. In the K562pTet-on/ETS2 cells, ETS2 induction conferred differences in sensitivities to ara-C and daunorubicin, depending on GATA1 levels. These results suggest that ETS2 expression is linked to the biology of AMkL in both DS and non-DS children, and that ETS2 acts by regulating expression of hematopoietic lineage and transcription factor genes involved in erythropoiesis and megakaryopoiesis, and in chemotherapy sensitivities.

Mutations of GATA1, FLT3, MLL-partial tandem duplication, NRAS, and RUNX1 genes are not found in a 7-year-old Down syndrome patient with acute myeloid leukemia (FAB-M2) having a good prognosis

The prognosis of leukemia developed in Down syndrome (DS) patients has improved markedly. Most DS leukemia occurs before 3 years of age and is classified as acute megakaryocytic leukemia (AMKL). Mutations in the GATA1 gene have been found in almost all DS patients with AMKL. In contrast, it has been shown that occurrence of DS acute myeloid leukemia (DS-AML) after 3 years of age may indicate a higher risk for a poor prognosis, but its frequency is very low. Age is one of the significant prognostic indicators in DS-AML. The prognostic factor of gene alterations has not been reported in older DS-AML patients. We here describe the case of a 7-year-old DS boy with AML-M2, who had no history of transient abnormal myelopoiesis or any clinical poor prognostic factors, such as high white blood cell counts or extramedullary infiltration. We molecularly analyzed the GATA1, FLT3, MLL-partial tandem duplication, NRAS, and RUNX1 (previously AML1) genes and did not detect any alterations. The patient has lived for more than 5 years after treatment on the AML99-Down protocol in Japan. This suggests that a patient lacking these genes alterations might belong to a subgroup of older DS-AML patients with good prognosis. Accumulation of more data on older pediatric DS-AML patients is needed.

Análise morfológica, imunofenotípica e molecular na identificação da leucemia megacariocítica aguda (LMA-M7)

Acute megakaryocytic leukemia (AML-M7) is a rare subtype of acute myeloid leukemia (AML), which has recently been incorporated in the FAB (French-American-British) classification. It represents from 3 to 5% of AML cases, is frequently associated to myelofibrosis and is a subtype with poor prognosis. The diagnosis of AML-M7 is initially based on the morphologic characteristics of leukemic cells. Bone marrow aspirates or biopsy shows a population of pleomorphic and basophilic cells which may present cytoplasmatic blebs. Morphologic and cytochemical criteria are not enough for a correct diagnosis. It is necessary to differentiate acute megakaryocytic leukemia with other subtypes of acute myeloid leukemia mainly in cases in which blast cells are undifferentiated, as are the cases of AML-M0, AML-M1 and of acute lymphoid leukemia, subtypes L1 and L2. The use of immunopherotypic techniques is essential for a differential diagnosis showing a population of leukemia cells with the absence of most surface lymphoid and myeloid markers, yet relevant for the antigen of the megakaryocytic expression: CD41a (complex glycoproteic IIb/IIIa), CD42b (glycoprotein Ib) and/or CD61 (glycoprotein IIIa), there by providing the for correct classification in 98% of the cases. Chromosomal abnormalities identified by cytogenetic techniques and molecular analysis are important for determining the prognosis and definition of the therapheutic regime.

Massive periosteal reaction a presenting feature of acute megakaryocytic leukemia

Acute megakaryoblastic leukemia (AML M7) is a biologically heterogeneous form of acute myeloid leukemia accounting for 14.6% of cases. In many instances in the past, AML M7 has been classified as undifferentiated leukemia, myelodysplasia, myelofibrosis or some other disease because of its complex clinical presentation or the difficulty of obtaining and interpreting bone marrow samples. However, with currently available morphological, cytochemical, cytogenetic and immunophenotypic methods, AML M7 can now be reliably diagnosed. Although the radiographic spectrum of bony changes in leukemia have been well characterized, skeletal X-ray abnormalities in the setting of AML M7 in pediatric patients have been described in few reports that were associated with bone marrow fibrosis. Here we report on a 14-month-old girl who presented with a massive periosteal reaction of the extremities and clavicles associated with myelofibrosis, a presenting feature of AML M7. The bone changes were very unusual in this case.

Lineage Shift of a Megakaryocytic-Erythroid Leukemia Cell Line by Adhesion to Fibronectin.

Acute megakaryocytic leukemia (AMgL) occurs in about 1% of AMLs and has poor prognosis in adults. Research of AMgL has been hampered by this rarity. To overcome this obstacle, the established immortal AMgL cell lines are useful materials for the study of megakaryocytic malignancies. A few cell lines with the characteristics of megakaryocytes have been established, but half of the cell lines are derived from blastic crisis of CMLs. In normal hematopoiesis, erythroblasts and megakaryocytes have a common precursor. And erythroid and megakaryocytic leukemias frequently share their phenotypes simultaneously. However, the mechanisms of lineage shift between the two remain unknown. We recently established an AMgL cell line named JAS-R. Megakaryocytic-erythroid JAS-R cells showed the phenotypic change by adhesion to culture dishes. Adherent JAS-R cells (named JAS-RAD) show megakaryocytic phenotype concomitant with erythropoietin secretion and MDR1 expression, while suspension cells (JAS-REN) show erythroid characters. Because the adhesion requires bivalent cation and is inhibited by RGDS tetrapeptide, it is presumably mediated by integrins. As for ligands, fibronectin was found to be the most powerful, using dishes coated with collagens (Type I and type IV), laminin, fibronectin and poly-D-lysine. Next we studied the phenotypic changes of JAS-REN cells along with the adhesion to fibronectin. JAS-REN cells were seeded on fibronectin-coated dishes and cultured for 48 hours. Then adherent and floating cells were harvested, separately. Cells were also treated with phorbol ester (TPA). Flow cytometry analysis disclosed that adherent cells had megakaryocytic phenotype with increased CD41a and CD61, and with decreased CD235a. The gene expression profile revealed that FLI1, RUNX1 and GFI1 were increased in fibronectin-adherent cells while GATA1, FOG1 and NFE2 were not changed. These characteristics of fibronectin-adherent cells resembled JAS-RAD. Similar results were observed in TPA-treated cells. Among these transcription factors, the induction of FLI1 was prominent. The promoter activity of FLI1 studied by the luciferase assay was higher in JAS-RAD than JAS-REN. These data suggest the substantial change of AMgL blast cells depending on the growth milieu in vivo. These changes might influence the result of chemotherapy

Targeting Pim Kinases in Hematological Malignancies.

The three Pim kinases represent a small subfamily of serine/threonine kinases known to be involved in a number of signaling pathways as downstream effectors and potent inhibitors of apoptosis. Unlike most other kinases, Pim kinases lack a regulatory domain which means they are controlled largely at the transcriptional level and that the mRNA expression levels of Pim kinases in cells correlate with their activity. While normal expression of Pim-1 kinase is seen in cells of hematopoietic origin, examination of gene expression of Pim-1 in different malignancies using cDNA microarray analysis suggests that Pim-1 is overexpressed in a large percentage of ALL, AML, CML, DLBCL, Prostatic Adenocarcinoma, Bladder, and Oral cancers. Pim-2 is also largely expressed in ALL, AML, Squamous Cell Lung and Adenocarcinoma of the Lung while Pim-3 is restricted to Melanoma, Pancreatic Adenocarcinoma, Gastric cancers. This implicates Pim-1 and Pim-2 in the onset and progression in several of hematological malignancies and therefore they represent interesting potential targets for drug development. To evaluate the potential of Pim-1 and -2 as a drug targets we have identified and synthesized a series of Pim kinase inhibitors using our proprietary CLIMB™ drug discovery process. Through the use of CLIMB™, the published Pim-1 kinase crystal structure was used to build several models that were then used to predict potential small molecule inhibitors of Pim-1 and Pim-2 from a large virtual library. A subset of leads, based on calculated binding energies as well as additional physical chemical properties, were screened using a number of in silico physicochemical and ADMET prediction algorithms to determine which compounds were most likely to be successful in a biological context. Lead candidates were initially screened using biochemical enzyme-based and cell-based assays. Cell-based activity was determined in HEL (acute megakaryocytic leukemia), K562 (chronic myeloid leukemia), MO7e (myeloid leukemia), and other human leukemia and lymphoma cell lines. From several lead candidates a series of analogs were produced with improved inhibitory activity and pharmacokinetic characteristics. In the Pim-1 and Pim-2 in vitro kinase assay and in the cell-based assay a number of leads exhibited inhibitory activity with IC50 concentrations in the low nanomolar range. Here we present the details of the biochemical and cell-based assay results as well as the activity in tumor xenograft models of our Pim kinase inhibitors.

Reciprocal Regulation of DSCR1 (Down Syndrome Critical Region 1) Expression and NFAT (Nuclear Factor of Activated T Cells) Activation in Megakaryocytes.

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Distinct Clones are Associated with the Development of Transient Myeloproliferative Disorder and Acute Megakaryocytic Leukemia in a Patient with Down Syndrome

Children with Down syndrome (DS) have an approximately 20-fold higher incidence of leukemia than unaffected children, and most leukemia cases with DS present as acute megakaryocytic leukemia (AMKL). At least 10% of neonates with DS develop transient myeloproliferative disorder (TMD), and 20% to 30% of patients with TMD develop AMKL. Mutations in the GATA1 gene are identified not only in AMKL patients but also in TMD patients; however, sequential analysis of GATA1 is not often performed in the same patients. We describe a child with DS who developed TMD followed by AMKL and have identified different mutations in the GATA1 gene during the course of TMD and AMKL. Distinct clones were associated with the development of TMD and AMKL in this patient.