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Barosi G.,Center for the Study of Myelofibrosis
Best Practice and Research: Clinical Haematology | Year: 2014

Essential thrombocythemia (ET) and primary myelofibrosis (PMF), together with polycythemia vera (PV) are Phildelphia-negative (Ph-neg) classical myeloproliferative neoplasms (MPN). ET has been traditionally identified by thrombocytosis and absence of relevant bone marrow (BM) fibrosis, while PMF by BM reticulin or collagen fibrosis with megakaryocyte hyperplasia and dysplasia, and extramedullary hematopoiesis. These diagnostic profiles have been challenged since 2001 when the World Health Organization (WHO) has included in the domain of PMF a new category of patients, namely early/prefibrotic MF, characterized by the absence of relevant reticulin fibrosis in BM, dual megakaryocyte and granulocyte proliferation, and megakaryocyte dysplasia. This review is focused on summarizing the diagnostic uncertainties of early/ prefibrotic myelofibrosis, recent advances in our understanding of the biology of the variant, and the accompanying translational implications. © 2014 Elsevier Ltd. All rights reserved.


Dragoni S.,University of Pavia | Laforenza U.,University of Pavia | Bonetti E.,Center for the Study of Myelofibrosis | Lodola F.,University of Pavia | And 7 more authors.
Stem Cells and Development | Year: 2013

Endothelial colony-forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) that are capable of acquiring a mature endothelial phenotype. ECFCs are mainly mobilized from bone marrow to promote vascularization and represent a promising tool for cell-based therapy of severe ischemic diseases. Vascular endothelial growth factor (VEGF) stimulates the proliferation of peripheral blood-derived ECFCs (PB-ECFCs) through oscillations in intracellular Ca2+ concentration ([Ca2+]i). VEGF-induced Ca2+ spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release and store-operated Ca2+ entry (SOCE). The therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has also been shown in recent studies. However, VEGF-induced proliferation of UCB-ECFCs is faster compared with their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel 3 (TRPC3) that mediates diacylglycerol-dependent Ca2+ entry. The present study aimed at investigating whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca 2+ response to VEGF. We found that VEGF induces oscillations in [Ca2+]i that are patterned by the interaction between InsP3-dependent Ca2+ release and SOCE. Unlike PB-ECFCs, VEGF-evoked Ca2+ oscillations do not arise in the absence of extracellular Ca2+ entry and after pharmacological (with Pyr3 and flufenamic acid) and genetic (by employing selective small interference RNA) suppression of TRPC3. VEGF-induced UCB-ECFC proliferation is abrogated on inhibition of the intracellular Ca2+ spikes. Therefore, the Ca 2+ response to VEGF in UCB-ECFCs is shaped by a different Ca 2+ machinery as compared with PB-ECFCs, and TRPC3 stands out as a promising target in EPC-based treatment of ischemic pathologies. © Mary Ann Liebert, Inc.


Barosi G.,Center for the Study of Myelofibrosis
Expert Review of Hematology | Year: 2012

Conventional drugs for myelofibrosis are driven by clinical needs, primarily anemia and splenomegaly. With these therapies, stem cell transplantation remains the only potentially curative approach. The discovery that mutations affecting JAK2 or MPL lead to activation of the intracellular JAK-STAT signaling pathway, and that other mutations (TET2, EZH2, ASXL1, IDH1 and IDH2) interfere with the normal machinery of epigenetics, has prompted to the development of therapies targeted at controling the major disease mechanisms. JAK2 ATP competitive inhibitors (ruxolitinib, lestaurtinib, SAR302503, SB1518 and CYT387) or drugs that indirectly inhibit the JAK-STAT pathway (everolimus) have documented major effects on splenomegaly and its constitutional symptoms. Epigenetic drugs (demethylating agents and histone deacetylase inhibitors) have displayed only minor effects on the disease symptoms. Relenting disease progression remains an unmet clinical need. © 2012 Expert Reviews Ltd.


Rumi E.,Fondazione IRCCS Policlinico San Matteo | Rumi E.,University of Pavia | Pietra D.,Fondazione IRCCS Policlinico San Matteo | Pascutto C.,Fondazione IRCCS Policlinico San Matteo | And 21 more authors.
Blood | Year: 2014

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decisionmaking, but should also be considered in designing clinical trials. © 2014 by The American Society of Hematology.


Dragoni S.,University of Pavia | Guerra G.,University of Molise | Pla A.F.,University of Turin | Bertoni G.,University of Pavia | And 11 more authors.
Journal of Cellular Physiology | Year: 2015

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca2+ signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca2+-permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca2+ entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays amaster signalling role inmature endothelium, by controlling both vascular remodelling and arterial pressure.Wefound thatEPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca2+ signals uniquely in presence of extracellular Ca2+. GSK- and PMA-induced Ca2+ elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La3+ and Gd3+ dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels. © 2014 Wiley Periodicals, Inc.


Barosi G.,Center for the Study of Myelofibrosis | Rosti V.,Center for the Study of Myelofibrosis | Gale R.P.,Imperial College London
OncoTargets and Therapy | Year: 2015

The recent approval of molecular-targeted therapies for myeloproliferative neoplasm-associated myelofibrosis (MPN-MF) has dramatically changed its therapeutic landscape. Ruxolitinib, a JAK1/JAK2 tyrosine kinase inhibitor, is now widely used for first- and second-line therapy in persons with MPN-MF, especially those with disease-related splenomegaly, intermediate- or high-risk disease, and constitutional symptoms. The goal of this work is to critically analyze data supporting use of ruxolitinib in the clinical settings approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). We systematically reviewed the literature and analyzed the risk of biases in the two randomized studies (COMFORT I and COMFORT II) on which FDA and EMA approval was based. Our strategy was to apply the Grading of Recommendation, Assessment, Development and Evaluation (GRADE) approach by evaluating five dimensions of evidence: (1) overall risk of bias, (2) imprecision, (3) inconsistency, (4) indirectness, and (5) publication bias. Based on these criteria, we downgraded the evidence from the COMFORT I and COMFORT II trials for performance, attrition, and publication bias. In the disease-associated splenomegaly sphere, we upgraded the quality of evidence because of large effect size but downgraded it because of comparator choice and outcome indirectness (quality of evidence, low). In the sphere of treating persons with intermediate- or high-risk disease, we downgraded the evidence because of imprecision in effect size measurement and population indirectness. In the sphere of disease-associated symptoms, we upgraded the evidence because of the large effect size, but downgraded it because of comparator indirectness (quality of evidence, moderate). In conclusion, using the GRADE technique, we identified factors affecting the quality of evidence that were otherwise unstated. Identifying and evaluating these factors should influence the confidence with which physicians use ruxolitinib in persons with MPN-MF. © 2015 Barosi et al.


Barosi G.,Center for the Study of Myelofibrosis
Current Hematologic Malignancy Reports | Year: 2015

New targeted therapies administered in phase II and phase III studies have produced substantial improvements in outcomes of myelofibrosis (MF). However, strong documentation that the new agents modify the natural history of the disease is lacking, and a number of therapeutic indications of new drugs remain unaddressed. Overall survival (OS) improvement is the major goal of next-generation clinical trials in MF. This may be attained if an adequate population of patients and an unambiguous design of the trial will be selected. Another goal is preventing disease progression in early MF: this requires a controlled clinical trial with an accessible endpoint and a clinically relevant definition of disease progression. Improvement in the documentation of responsiveness of patient-reported outcomes (PROs) will allow to use them as a critical endpoint of new trials. Finally, exploiting the clinical utility of biomarkers should become a major goal of future clinical experimentation in MF. © 2015, Springer Science+Business Media New York.


Barosi G.,Center for the Study of Myelofibrosis | Vannucchi A.M.,University of Florence | De Stefano V.,Catholic University | Pane F.,CEINGE Biotecnologie Avanzate | And 5 more authors.
Leukemia Research | Year: 2014

This article presents the results of group discussion among experts from SIE, SIES and GITMO societies aimed at highlighting unmet challenges in the management of Ph-neg myeloproliferative neoplasms (MPNs). The issues analyzed were: diagnosis of prefibrotic myelofibrosis; diagnosis of Ph-neg MPNs in the setting of splanchnic vein thrombosis (SVT); management of low-risk PV and low-risk ET patients with JAK2V617F mutation; molecular biomarkers in the prognostic evaluation of myelofibrosis (MF); ruxolitinib therapy in low-risk MF; therapy in patients with SVT-associated Ph-neg MPN; indications of splenectomy in MF. For each of these issues, proposals for advancement in clinical research were addressed. © 2013 Elsevier Ltd.


Potenza D.M.,University of Pavia | Guerra G.,University of Molise | Avanzato D.,University of Turin | Poletto V.,Center for the Study of Myelofibrosis | And 7 more authors.
Cell Calcium | Year: 2014

Hydrogen sulphide (H2S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca2+ concentration ([Ca2+]i) is central to endothelial proliferation and may be triggered by both VEGF and H2S. Albeit VEGFR-2 might serve as H2S receptor, the mechanistic relationship between VEGF- and H2S-induced Ca2+ signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H2S donor, stimulates pro-angiogenic Ca2+ signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca2+]i in Ea.hy926 cells. NaHS-induced Ca2+ signals in Ea.hy926 cells did not require extracellular Ca2+ entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP3) signalling pathway. Moreover, the Ca2+ response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca2+ signals were suppressed by dl-propargylglycine (PAG), which blocks the H2S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca2+]i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca2+ oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H2S-evoked elevation in [Ca2+]i is essential to trigger the pro-angiogenic Ca2+ response to VEGF in mature endothelial cells, but not in their immature progenitors. © 2014 Published by Elsevier Ltd.


Guglielmelli P.,University of Florence | Lasho T.L.,Mayo Medical School | Rotunno G.,University of Florence | Score J.,Wessex Reg Genetics Laboratory | And 23 more authors.
Leukemia | Year: 2014

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF. © 2014 Macmillan Publishers Limited.

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