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In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: β-catenin, PPAR-γ and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of β-catenin immunoreactive cells was significantly higher in PV then in ET (p < 0.01) or PMF group of patients (p < 0.01) and also in ET versus PMF group of patients (p < 0.01). Erythroid lineage showed absent β-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-γ was localized mostly in megakaryocytes and the highest number of PPAR-γ immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed β-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-γ immunopositive megakaryocytes characterized by abnormal maturation.

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF-1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF-1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34 + cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34 + cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation-related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Introduction Philadelphia-negative myeloproliferative neoplasms (Ph− MPN) are characterized by overproduction of one or more blood cell lines. Methods We studied the proliferative characteristics of 91 patients with de novo Ph− MPN. Colony-forming cells ( CFC) and endogenous colonies ( EC), from bone marrow ( BM) and/or peripheral blood ( PB), were analyzed by colony assay based on methylcellulose. The level of circulating CD34+ cells was determined by flow cytometry. Results The total number of PB CFC in primary myelofibrosis ( PMF) was increased compared to the control sample ( P < 0.01) and essential thrombocythemia ( ET) ( P < 0.05). The highest number of BM and PB EC was observed in polycythemia vera ( PV) ( P < 0.01). Increased levels of CD34+ cells characterized early-prefibrotic (57%) and advanced-fibrotic PMF (90%) as compared to PV (34%) and ET (32%) ( P < 0.01). In the whole Ph− MPN group, the total number of PB CFC ( P < 0.01), PB EC ( P < 0.05), and CD34+ cells ( P < 0.01) correlated with the degree of BM fibrosis. Higher levels of circulating CD34+ cells in PMF correlated with the total number of PB EC ( P < 0.05) and degree of BM fibrosis ( P < 0.01). Conclusions Exploration of the PB proliferative characteristics of Ph− MPN on diagnosis may be helpful in revealing early-prefibrotic PMF. Monitoring the levels of circulating CD34+ cells may provide a sensitive indicator of fibrotic evolution in PV and PMF. [ABSTRACT FROM AUTHOR]