Increase in Frequency of Myeloid-Derived Suppressor Cells in the Bone Marrow of Myeloproliferative Neoplasm: Potential Implications in Myelofibrosis.

The Philadelphia-negative myeloproliferative neoplasms (MPNs), defined as clonal disorders of the hematopoietic stem cells, are characterized by the proliferation of mature myeloid cells in the bone marrow and a chronic inflammatory status impacting the initiation, progression, and symptomatology of the malignancies. There are three main entities defined as essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), and genetically classified by JAK2V617F, CALR, or MPL mutations. In MPNs, due to the overproduction of inflammatory cytokines by the neoplastic cells and non-transformed immune cells, chronic inflammation may provoke the generation and expansion of myeloid-derived suppressors cells (MDSCs) that highly influence the adaptive immune response. Although peripheral blood MDSC levels are elevated, their frequency in the bone marrow of MPNs patients is not well elucidated yet. Our results indicated increased levels of total (T)-MDSCs (CD33+HLA-DR-/low) and polymorphonuclear (PMN)-MDSCs (CD33+/HLA-DRlow/CD15+/CD14-) in the bone marrow and peripheral blood of all three types of MPNs malignancies. However, these bone marrow MDSCs-increased frequencies did not correlate with the clinical parameters, such as hepatomegaly, leukocytes, hemoglobin, or platelet levels, or with JAK2 and CALR mutations. Besides, bone marrow MDSCs, from ET, PV, and PMF patients, exhibited immunosuppressive function, determined as T-cell proliferation inhibition. Notably, the highest T-MDSCs and PMN-MDSC levels were found in PMF samples, and the increased MDSCs frequency strongly correlated with the degree of myelofibrosis. Thus, these data together indicate that the immunosuppressive MDSCs population is increased in the bone marrow of MPNs patients and may be implicated in generating a fibrotic microenvironment.

Inhibition of proinflammatory signaling impairs fibrosis of bone marrow mesenchymal stromal cells in myeloproliferative neoplasms.

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been identified as a major cellular source of fibrosis, the exact molecular mechanism and signaling pathways involved have not been identified thus far. Here, we show that BM-MSCs contribute to fibrosis in myeloproliferative neoplasms (MPNs) by differentiating into αSMA-positive myofibroblasts. These cells display a dysregulated extracellular matrix with increased FN1 production and secretion of profibrotic MMP9 compared to healthy donor cells. Fibrogenic TGFß and inflammatory JAK2/STAT3 and NFκB signaling pathway activity is increased in BM-MSCs of MPN patients. Moreover, coculture with mononuclear cells from MPN patients was sufficient to induce fibrosis in healthy BM-MSCs. Inhibition of JAK1/2, SMAD3 or NFκB significantly reduced the fibrotic phenotype of MPN BM-MSCs and was able to prevent the development of fibrosis induced by coculture of healthy BM-MSCs and MPN mononuclear cells with overly active JAK/STAT signaling, underlining their involvement in fibrosis. Combined treatment with JAK1/2 and SMAD3 inhibitors showed synergistic and the most favorable effects on αSMA and FN1 expression in BM-MSCs. These results support the combined inhibition of TGFß and inflammatory signaling to extenuate fibrosis in MPN.

Correlation between leukocyte-platelet aggregates and thrombosis in myeloproliferative neoplasms.

INTRODUCTION: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. METHODS: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. RESULTS: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P < .05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89 ng/mL vs 286.39 ng/mL, P = .034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR = 1.561, 95% CI: 1.007-2.420, P = .046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR = 1.975, 95%CI: 1.215-3.212, P = .006) for thrombosis occurrence. CONCLUSION: Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms.

BACKGROUND: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. AIMS: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. RESULTS: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors-endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)-in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. CONCLUSION: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.

Cardiovascular Safety of Anagrelide Hydrochloride versus Hydroxyurea in Essential Thrombocythaemia.

Essential thrombocythaemia (ET) is a rare myeloproliferative neoplasm. This multicentre, Phase 3b, randomised, open-label, non-inferiority study investigated the cardiac safety, efficacy and tolerability of first-line treatment with anagrelide or hydroxyurea in high-risk ET patients for up to 3 years. Eligible patients aged ≥ 18 years with a diagnosis of high-risk ET confirmed by bone marrow biopsy within 6 months of randomisation received anagrelide (n = 75) or hydroxyurea (n = 74), administered twice daily. Treatment dose for either compound was titrated to the lowest dose needed to achieve a response. Planned primary outcome measures were change in left ventricular ejection fraction from baseline over time and platelet count at Month 6. Planned secondary outcome measures were platelet count change from baseline at Months 3 and 36; percentage of patients with complete or partial response; time to complete or partial response; number of patients with thrombohaemorrhagic events; and changes in white blood cell count or red blood cell count over time. Neither treatment altered cardiac function. There were no significant differences in adverse events between treatment groups, and no reports of malignant transformation. The incidence of disease-related thrombotic or haemorrhagic events was numerically higher in anagrelide-treated patients. Both treatments controlled platelet counts at 6 months, with the majority of patients experiencing complete or partial responses. In conclusion, these results suggest that long-term treatment with anagrelide is not associated with adverse effects on cardiac function. This is one of the few studies using left ventricular ejection fraction assessment and central biopsy reading to confirm the diagnosis of ET.Trial registration number: Clinicaltrials.gov NCT00202644.

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation.

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. METHODS: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. RESULTS: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. CONCLUSIONS: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.

IL6 inhibition of inflammatory S100A8/9 proteins is NF-κB mediated in essential thrombocythemia.

This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34+ cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-κB, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-κB and inflammation signalling in MPN-derived CD34+ cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-κB and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. SIGNIFICANCE OF THE STUDY: We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-κB and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.

CD56-Positive Acute Myeloid Leukemia Following Treatment of Hairy Cell Leukemia with Cladribine - Report of 2 Cases and Review of the Literature.

Treatment of hairy cell leukemia (HCL) with alfa-interferon and purine analogs significantly prolongs survival in these patients. However, with life prolongation, an increased risk of secondary malignancies has been reported. Acute myeloid leukemia (AML), as a second malignancy after HCL treatment is extremely rare and has been reported in only 12 cases so far. We here report additional 2 cases of CD56+ AML developed after sustained clinical remission of HCL achieved with cladribine (2 and 6 years after, respectively). The first patient refused chemotherapy and shortly thereafter died. The second patient responded to chemotherapy and was successfully allo-transplanted. Three years later, the patient is in stable clinical remission, which is a unique case in the literature. In conclusion, it is not clear whether development of AML in HCL patients is caused by mutagenic potential of the applied chemotherapy or by immune suppression/ perturbations as a characteristic of the underlying disease.

IL-6 stimulation of DNA replication is JAK1/2 mediated in cross-talk with hyperactivated ERK1/2 signaling.

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.

ß-catenin and PPAR-γ levels in bone marrow of myeloproliferative neoplasm: an immunohistochemical and ultrastructural study.

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: ß-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 ß-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 ß-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 ß-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-γ immunopositive megakaryocytes characterized by abnormal maturation.

Moxetumomab pasudotox in relapsed/refractory hairy cell leukemia.

This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.

Recommendations for the diagnosis and treatment of patients with polycythaemia vera.

OBJECTIVES: To present the Central European Myeloproliferative Neoplasm Organisation (CEMPO) treatment recommendations for polycythaemia vera (PV). METHODS: During meetings held from 2015 through 2017, CEMPO discussed PV and its treatment and recent data. RESULTS: PV is associated with increased risks of thrombosis/thrombo-haemorrhagic complications, fibrotic progression and leukaemic transformation. Presence of Janus kinase (JAK)-2 gene mutations is a diagnostic marker and standard diagnostic criterion. World Health Organization 2016 diagnostic criteria for PV, focusing on haemoglobin levels and bone marrow morphology, are mandatory. PV therapy aims at managing long-term risks of vascular complications and progression towards transformation to acute myeloid leukaemia and myelodysplastic syndrome. Risk stratification for thrombotic complications guides therapeutic decisions. Low-risk patients are treated first line with low-dose aspirin and phlebotomy. Cytoreduction is considered for low-risk (phlebotomy intolerance, severe/progressive symptoms, cardiovascular risk factors) and high-risk patients. Hydroxyurea is suspected of leukaemogenic potential. IFN-α has demonstrated efficacy in many clinical trials; its pegylated form is best tolerated, enabling less frequent administration than standard interferon. Ropeginterferon alfa-2b has been shown to be more efficacious than hydroxyurea. JAK1/JAK2 inhibitor ruxolitinib is approved for hydroxyurea resistant/intolerant patients. CONCLUSIONS: Greater understanding of PV is serving as a platform for new therapy development and treatment response predictors.

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

PURPOSE: Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. METHODS: We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. RESULTS: We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34+ cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV. CONCLUSIONS: From our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

The WHO diagnostic criteria for polycythemia vera-role of red cell mass versus hemoglobin/hematocrit level and morphology.

Regarding diagnosis of polycythemia vera (PV), discussion persists about hemoglobin (Hb) and/or hematocrit (Hct) threshold values as surrogate markers for red cell mass (RCM) and the diagnostic impact of bone marrow (BM) morphology. We performed a retrospective study on 290 patients with PV (151 males, 139 females; median age 65 years) presenting with characteristic BM features (initial biopsies, centralized evaluation) and endogenous erythroid colony (EEC) formations. This cohort included (1) a group of 229 patients when following the 2008 versus 256 patients diagnosed according to the 2016 World Health Organization (WHO) guidelines, all presented with increased RCM; (2) masked PV patients with low Hb (n = 143)/Hct (n = 45) recruited from the 2008 WHO cohort; (3) a cohort of 17 PV patients with elevated diagnostic Hb/Hct levels but low RCM; and (4) nine PV patients with increased RCM, opposing low Hb/Hct values. All patients were treated according to current PV guidelines (phlebotomies 87%, hydroxyurea 79%, and acetylsalicylic acid 87%). Applying the 2016 WHO criteria significantly increased concordance between RCM and Hb values compared with the 2008 WHO criteria (90 vs. 43% in males and 83 vs. 64% in females). Further analysis of the WHO 2016 PV cohort revealed that increased RCM is associated with increased Hb/Hct (93.8/94.6%). Our study supports and extends the diagnostic impact of the 2016 revised WHO classification for PV by highlighting the importance of characteristic BM findings and implies that Hb/Hct threshold values may be used as surrogate markers for RCM measurements.

Predictive parameters for imatinib failure in patients with chronic myeloid leukemia.

OBJECTIVE: Until recently, imatinib was the standard first-line treatment in chronic myeloid leukemia (CML). The inclusion of nilotinib and dasatinib as first-line options in CML raised a debate on treatment selection. The aim of our study was to analyze predictive parameters for imatinib response as the first-line treatment of CML patients. METHODS: The study included 168 consecutive patients with chronic phase Philadelphia-positive CML who were diagnosed and treated with Imatinib 400 mg once daily at a single university hospital. Numerous parameters were analyzed in terms of imatinib response including comorbidities as well as occurrence of second malignancies. RESULTS: After the median follow-up of 87 months in 61 patients (36.3%), the imatinib failure was verified. Cox regression analysis identified hepatomegaly (p = 0.001), leukocytosis ≥ 100 × 109/l (p = 0.001), blood blasts ≥ 1% (p = 0.002), and the presence of additional cytogenetic aberrations (p = 0.002) as predictors of Imatinib failure. Based on these findings, a new prognostic model was developed according to which imatinib failure had 17% (8/47) of patients in low risk, 34.9% (30/86) of patients in intermediate risk, and 76.7% (23/30) of patients in high-risk group (HR = 3.973, 95% CI for HR 2.237-7.053, p < 0.001). CONCLUSION: The new score allows better selection of patients who are suitable for treatment with imatinib and may guideline the clinical decision for front-line treatment of CML.

Bone marrow microvessel density and plasma angiogenic factors in myeloproliferative neoplasms: clinicopathological and molecular correlations.

Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry "hot spot" method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF > PV > ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: ρ = 0.491, p < 0.001; CD105-MVD: ρ = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs.

Angiogenic factors are increased in circulating granulocytes and CD34+ cells of myeloproliferative neoplasms.

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 TGFß 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.

[Treatment by bloodletting in the past and present].

INTRODUCTION: Therapeutic bloodletting has been practiced at least 3000 years as one of the most frequent methods of treatment in general, whose value was not questioned until the 19th century, when it was gradually abandoned in Western medicine, while it is still practiced in Arabic and traditional Chinese medicine. CONTENT: In modern medicine bloodletting is practiced for very few indications. Its concept was modeled on the process of menstrual bleeding, for which it was believed to"purge women of bad humours. "Thus, bloodletting was based more on the belief that it helps in the reestablishment of proper balance of body "humours" than on the opinion that it serves to remove excessive amount of blood as well as to remove toxic "pneumas" that accumulate in human body. It was indicated for almost all known diseases, even in the presence of severe anemia. Bloodletting was carried out by scarification with cupping, by phlebotomies (venesections), rarely by arteriotomies, using specific instruments called lancets, as well as leeches. In different periods of history bloodletting was practiced by priests, doctors, barbers, and even by amateurs. In most cases, between one half of liter and two liters of blood used to be removed. Bloodletting was harmful to vast majority of patients and in some of them it is believed that it was either fatal or that it strongly contributed to such outcome. In the 20th century in the "Western"medicine bloodletting was still practiced in the treatment of hypertension and in severe cardiac insufficiency and pulmonary edema, but these indications were later abandoned. CONCLUSION: Bloodletting is still indicated for a few indications such as polycythemia, haemochromatosis, and porphyria cutanea tarda, while leeches are still used in plastic surgery, replantation and other reconstructive surgery, and very rarely for other specific indications.

Macrophage migration inhibitory factor is an endogenous regulator of stress-induced extramedullary erythropoiesis.

Macrophage migration inhibitory factor is a well-known proinflammatory cytokine that is released during systemic stress response. Although MIF can affect erythrocyte production, the role of this cytokine in stress-induced erythropoiesis is completely unknown. To extend our previous findings showing that chronic psychological stress stimulates extramedullary erythropoiesis, here we examined whether MIF is involved in the control of stress-induced erythropoietic response. Adult male C57BL/6 wild-type (WT) and MIF-KO (knock-out) mice were subjected to 2-h daily restraint stress for either 7 or 14 consecutive days. The number of erythroid progenitors and CD71/Ter119 profile of erythroid precursors were analyzed in the bone marrow and spleen. Additionally, MIF protein expression was assessed in WT mice. Our results demonstrated that chronic restraint stress enhanced the number of both erythroid progenitors and precursors in the spleen. Stress-induced increase in the number of splenic late erythroid progenitors as well as in the percentage of CD71(+)Ter119(+)-double-positive precursors was significantly more pronounced in MIF-KO mice compared to WT animals. Furthermore, repeatedly stressed WT animals demonstrated an augmented MIF expression in the spleen. Unlike the spleen, the bone marrow of chronically stressed WT mice exhibited less prominent changes in erythropoietic stress response and no significant alteration in MIF expression. In addition, MIF deficiency did not influence the bone marrow erythropoiesis in stressed animals. These findings suggest that MIF regulates extramedullary erythropoiesis by inhibiting an overexpansion of splenic immature erythroid cells during chronic stress and indicate a novel role for this cytokine under chronic stress conditions.