Identification of Normal and Abnormal Human Megakaryocytes Based on Acid Fast Metachromasia after Staining with Basic Black MSP

Megakaryocytes from normal persons and from patients with immune thrombocytopenic purpura, myelodysplastic disorders, Hypersplenism, and essential thrombocythemia displayed vivid magenta metachromatic staining of the cytoplasm when stained with basic black MSP followed by brief exposure to dilute hydrochloric acid. Under the same conditions, other hematopoietic cells were completely decolorized. Acid fast metachromasia of megakaryocytes facilitates their identification, particularly in cases of small and atypical megakaryocytes found in disease states.     

Differential expression of circular RNAs in bone marrow-derived exosomes from essential thrombocythemia patients

Circular RNAs (circRNA) are closely associated with the pathogenesis of various hematological diseases. However, little is known about the potential functions of circRNAs in essential thrombocythemia (ET) development. The circRNA profile alterations in the bone marrow of ET patients were mainly investigated in this study. The sizes of exosomes derived from human bone marrow tissues were validated by the nanoparticle tracking analysis (NTA) method. CD63 and TSG101 expressions in exosomes were analyzed by western blot analysis. The profiles and differential expression of circRNAs in bone-derived exosomes were characterized by high-throughput sequencing. Herein, circular structures and expression of circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction, respectively. The circRNA-miRNA-mRNA networks were predicted using the Cytoscape software. And we detected the effect of circ_0014614 on the transformation of K562 cells into megakaryocytes. Exosomes derived from the bone marrow of ET patients and healthy volunteers showed a diameter between 70 and 140 nm and expressed high CD63 and TSG101. Meanwhile, the circRNA profiles were significantly altered in bone marrow–derived exosomes from ET patients, among which circDAP3, circASXL1, and circRUNX1 were significantly downregulated in ET patients, thus conferring a new insight into the role of circRNAs in the pathogenesis of ET. Besides this, circRNA-encoding genes and miRNA-mRNA networks targeted by this three circRNA were involved in various biological processes and signaling pathways. And circ_0014614 could inhibit K562 cells' differentiation into megakaryocytes. The predictions of the potential function of these three differentially expressed circRNAs along with their interaction with specific miRNAs could provide a basis for circRNA-based ET diagnosis and treatment.     

Abnormalities of platelet aggregation in chronic myeloproliferative disorders

A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnormalities may be due to deficiency of platelet granules, arahidonic acid metabolism defects or platelet membrane glycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76 patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro by measuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. These results were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We found complex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent, decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almost absent platelet response to all agonists while in some patients a normal platelet aggregation was noted. The correlation between these results and template bleeding time, thrombotic or hemorrhagic events and the type of diseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigating platelet aggregation may be useful not only for the assesment of the hemostatic balance in chronic myeloproliferative disorders but also for a better insight into cell abnormalities occuring in these pathologic conditions.     

HRAS mutation positive multiple myeloma in the type 2 CALR mutation positive essential thrombocythemia: A case report

Out of BCR-ABL negative myeloproliferative neoplasm (MPNPh⁻) patients, 3%–14% display a concomitant monoclonal gammopathy of unknown significance (MGUS). In most cases, the diagnosis of plasma cell dyscrasia is either synchronous with that of MPNPh⁻ or occurs later on. We present a 50-year-old patient with type 2 CALR Lys385Asnfs*47 mutation positive essential thrombocythemia (ET) who developed symptomatic multiple myeloma (MM) 13 years after the diagnosis of ET during PEG-INF2α treatment. The NGS study performed at the time of the MM diagnosis revealed the HRAS Val14Gly/c.41T〉G mutation and the wild type CALR, JAK2 and MPL gene sequence. In the presented case, the complete molecular remission of ET was achieved after 16 months of PEG-INF2α treatment. The origin of MM cells in MPNPh⁻ patients remains unknown. Published data suggests that type 2 CALRins5 up-regulate the ATF6 chaperone targets in hematopoietic cells and activate the inositol-requiring enzyme 1α-X-box-binding protein 1 pathway of the unfolded protein response (UPR) system to drive malignancy. It cannot be excluded that endoplasmic reticulum stress induced by the increased ATF6 resulted in an abnormal redox homeostasis and proteostasis, which are factors linked to MM. The presented case history and the proposed mechanism of mutant CALR interaction with UPR and/or ATF6 should initiate the discussion about the possible impact of the mutant CALR protein on the function and genomic stability of different types of myeloid cells, including progenitor cells.     

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- ( e.g. FIP1L1-PDGFRA) or inter-chromosomal ( e.g. ETV6-PDGFRB ) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera ( JAK2 V617F and other JAK2 mutations), essential thrombocythemia ( JAK2 V617F and MPL5 15 mutations), primary myelofibrosis ( JAK2 V617F and MPL515 mutations), systemic mastocytosis ( KIT D816V and other KIT mutations) and stem cell leukaemia/lymphoma ( ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets.     

Relationship between chromosomal changes complexity and disease aggressiveness in myeloid and lymphoid disorders

In this paper are presented four cases, with unusual chromosomal abnormalities, identified at the first presentation, among over 100 patients with myeloid and lymphoid acute and chronic leukemias cytogenetically investigated. The complexity and nature of cytogenetic abnormalities was in direct relationship with the disease evolution. The first case, a 22 years old man with acute lymphoblastic leukemia type L3, exhibited many structural changes in bone marrow cells with diploid number of chromosomes: del(3)(q26); del (5)(p13); t(8;14) (q24;q32); del(9)(p11q11);inv(15)(p12qter). The second case, a 62 years old woman, diagnosed as poorly differentiated acute leukemia, refractory to treatment, showed hiperdiploidy (48–54 chromosomes) and 3–4 markers derived from chromosomes 5 and 12. The third case, a young man of 27 years old, diagnosed as acute myeloid leukemia, apart of Philadelphia chromosome, presented trisomy 16, both in diploid and aneuploid cells. None of these three patients did respond to any medical therapy. Their rapid death was a powerful proof of the correlation between the complexity of genome changes and disease aggressiveness. In the fourth case, a constitutional translocation t(3;5)(q26.3;q21) identified in a 72 years old woman with essential thrombocythemia, appeared not to be involved in the etiology of the disease. In this case, the treatment with hydroxyurea was successful and the disease evolution was favourable. In conclusion, we appreciate that in the three cases of myeloid and lymphoid leukemias it was a direct relationship between the complexity of genomic changes and the aggressiveness of the disease.