Morphological and cytogenetic studies have demonstrated there is a diverse heterogeneity for acute megakaryoblastic leukemia (AMKL). In children, there are 2 major AMKL subgroups, with the disease developing in patients with and without Down syndrome (DS-AMKL and non-DS-AMKL, respectively). Somatic mutations of the GATA1 gene are found in children with DS-AMKL. Recently, activating mutations of the JAK3 gene have been found in both DS-AMKL and non-DS-AMKL patients. Furthermore, JAK2V617F plays a critical role in the pathogenesis of myeloproliferative disorders that are found in adults with non-DS-AMKL. To examine the biological characteristics of the DS-AMKL and non-DS-AMKL blast cells, we compared the morphology, immunophenotypes, gene alterations (GATA1, JAK2, JAK3) and the expression of the hematopoietic transcription factors in blast cells derived from 29 DS-AMKL and 21 non-DS-AMKL children. AMKL was defined by the presence of blast cells that expressed at least one platelet-associated antigen (CD36, CD41, CD42, or CD61). Written informed consent for banking and molecular analysis of the blast cells was obtained from the parents of each of the patients. Blast cell morphology was categorized as follows: type 1, completely undifferentiated blasts; type 2, intermediately differentiated blasts with cytoplasmic blebs; type 3, blasts with dysmegakaryocytopoiesis; and type 4, blasts with deep blue cytoplasm. The respective distributions of the morphology of the blasts in DS-AMKL and non-DS-AMKL patients were as follows: type 1 (41%, 39%), type 2 (4%, 39%), type 3 (11%, 22%), and type 4 (44%, 0%). Type 4 blasts were only seen in DS-AMKL. Atypical expression of lymphoid-associated antigen CD7 was detected in 90% and 53% of the immunophenotyped-patients with DS-AMKL and non-DS-AMKL, respectively (p = 0.003). Glycophorin A was only detected on the blasts of 34% of the patients with DS-AMKL (p = 0.012). Interestingly, 78% of the type 4 blasts were positive for glycophorin A. The GATA1, JAK2 and JAK3 mutations were analyzed in bone marrow or peripheral blood samples. High-molecular weight DNA was extracted from the samples using standard methods. We amplified the genomic DNA corresponding to exon2 of GATA1, exon14 of JAK2, and all 23 exons of the JAK3 gene, respectively, with the amplified products sequenced directly on a DNA sequencer (310; Applied Biosystems, Foster City, CA) using a BigDye terminator cycle sequencing kit (Applied Biosystems). The frequencies of the gene mutations in DS-AMKL and non-DS-AMKL were distributed as follows: GATA1 (100%, 8%), JAK2V617F (6%, 0%) and JAK3 (12%, 8%). To define the differentiation stage based on the molecular level, we quantified the expression of the transcription factors related to the megakaryo-erythroid cell lineages. The expression of the transcription factors (GATA1, GATA2, FOG-1, NF-E2, SCL, PU.1, C/EBPa, c-mpl, Epo-R, CD41b, bglobin) was analyzed by real time RT-PCR in blasts that were sorted by flow cytometry. Total RNA was extracted from the purified blasts. Synthesis of cDNA was performed using a Thermoscript RT-PCR system (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. Real-time RT-PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Branchburg, NJ). Normalized gene expression levels are given as the ratio between the mean value for the target gene and that for the GAPDH gene in each sample. Median bglobin transcript levels in DS-AMKL blasts were significantly higher than those seen for the non-DS-AMKL blasts (p = 0.006). Particularly, median bglobin and GATA1 transcript levels in the type 4 blasts, which were only found in DS-AMKL, were significantly higher than those found in the blasts of other types (p = 0.033 and p = 0.021, respectively). Median GATA1, NF-E2 and Epo-R transcript levels in type 3 blasts, which differentiated into mature megakaryocytes, were significantly lower than those found in the blasts of other types (p = 0.030, p = 0.036, and p = 0.028, respectively). In conclusion, blasts of DS-AMKL differentiated not only into the megakaryocyte but also the erythroid lineage because of the overexpression of glycophorin A and bglobin under the GATA1 gene mutations. Considering the totally different gene alteration patterns and the levels of transcription factors that were observed, DS-AMKL needs to be classified into a category that differs from non-DS-AMKL.

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