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July 2013: The efficacy of interferon alfa in the treatment of polycythemia vera and essential thrombocythemia depends on concomitant somatic mutations outside the JAK-STAT pathway

The article of June was focused on the efficacy of interferon alpha in suppressing hematopoietic stem cells carrying the JAK2 (V617F) muation in a mouse model of polycythemia vera. It is now established that patients with myeloproliferative disorders may have concomitant somatic mutations outside the JAK-STAT pathway, in particular mutations of TET2, ASXL1, EZH2, DMT3A, and IDH1/IDH2. A group of American researchers studied 43 patients with polycythemia vera and 40 patients with essential thrombocythemia who had been enrolled in a phase II study on the use of pegylated interferon alfa [Quintas-Cardama et al. Pegylated interferon alfa-2a yields high rates of hematologic and molecular response in patients with advanced essential thrombocythemia and polycythemia vera. J Clin Oncol. 2009 Nov 10; 27 (32) :5418-24]. About 75% of these patients had achieved a complete hematologic response, and about half of them had obtained a molecular response to treatment. Sequencing of the genes TET2, ASXL1, EZH2, DMT3A, and IDH1/IDH2 showed that the presence of additional somatic mutations is relevant to response to treatment. In fact, patients who did not reach the molecular remission had a higher incidence of somatic mutations outside of the JAK-STAT pathway, and were more likely to acquire new mutations during therapy. The response to interferon alfa of patients with myeloproliferative disorders is therefore affected by the presence of additional somatic mutations involving TET2, ASXL1, EZH2, DMT3A, or IDH1/IDH2. (Go to abstract external link)

June 2013: Efficacy of interferon alpha in depleting hematopoietic stem cells carrying the JAK2 (V617F) mutation in a mouse model of polycythemia vera

Interferon alpha is a drug potentially very effective in the treatment of polycythemia vera. In the clinical trials conducted so far, interferon alpha has been shown not only to be able to induce hematologic remission of the disease, but also to significantly reduce the percentage of blood cells that carry JAK2 (V617F). The most interesting aspect, however, is the fact that some patients achieved a complete molecular remission. However, treatment with interferon alfa may have side effects: for this reason, ongoing clinical trials are assessing the efficacy and safety of pegylated interferon alpha. Clinical untis of the AGIMM project are participating in these trials. To better understand the mechanism of action of interferon alpha in polycythemia vera, a group of researchers from Boston, USA, and Brisbane, Australia, have studied its effect in a mouse model of polycythemia vera. This research has allowed the investigators to demonstrate that interferon alpha is able to suppress the most immature hematopoietic stem cells that carry JAK2 (V617F) and support the clonal hematopoiesis of polycythemia vera. This explains why the drug is able to give a molecular remission of the disease: in fact, it suppresses the cells that sustain the myeloproliferative disorder. This study represents a basis for future clinical trials aimed to study the combined use of interferon alfa and anti-JAK2 drugs in the treatment of polycythemia vera. (Go to abstract external link)

May 2013: Subcellular mislocalization of the transcription factor NF-E2 in erythroid cells discriminates pre-fibrotic primary myelofibrosis from essential thrombocythemia

The differential diagnosis between essential thrombocythemia and pre-fibrotic myelofibrosis is sometimes difficult and in many cases there is a discordance among pathologists in discriminating the two entities.The diagnosis relays on the morphology of the bone marrow biopsy; however, morphology can be ambiguously interpreted and the same bone marrow slide can be recognized by a pathologist as "essential thrombocythemia" and by another as "pre-fibrotic myelofibrosis". It was previously shown that the transcription factor NF-E2 is aberrantly expressed in the hematopoietic cells of patients with myeloproliferative disorders. Transcription factors are proteins that regulate the gene expression, and in turn the protein synthesis, playing an important role in cellular homeostasis. In this article, a group of german researchers, leaded by Heike Pahl, has shown, by means of immunohystochemical staining of bone marrow slides, that the transcription factor NF-E2 is mislocalized at the subcellular level in the erythroblasts of patients with essential thrombocythemia compared to those affected from prefibrotic myelofibrosis. The high degree of statistical significance and inter-observer concordance between independent pathologists in the association of NF-E2 localization and a specific type of disease suggest that NF-E2 localization can be a reliable diagnostic tool that distinguishes essential thrombocytemia from early, prefibrotic PMF, with important consequences for both therapeutic decisions and prognostic implications. (Go to abstract external link)

April 2013: A phase II study of Givinostat in combination with hydroxycarbamide in patients with polycythemia vera unresponsive to hydroxycarbamide monotherapy

Givinostat is a histone-deacetylase inhibitor that has shown activity in inhibiting proliferation of cells bearing the JAK2 V617F mutation, and also activity and good tolerability in patients with chronic myeloproliferative neoplasms. In this multicenter, open label, phase II study, 44 patients with polycythemia vera, unresponsive to the maximum tolerated dose of hydroxyurea , were treated with Givinostat (50 or 100 mg/die) in combination with the maximum tolerated dose of hydroxyurea. Complete or partial response was observed in about 50% of cases after 12 weeks of treatment, and control of pruritus in about 65%. The combination of these two drugs was well tolerated. Drug related adverse events include hyperkaliemia, thrombocytopenia and arrhythmia, all grade 2, anemia grade 3 and gastrointestinal disorders all grade <3. The most relevant clinical benefit observed in this study, besides a better control of cells count, is the reduction of pruritus. Givinostat showed only modest effect on spleen size, that suggest a different mechanism of action compared to JAK2 inhibitors. (Go to abstract external link)

March 2013: Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis.

Cardinal clinical features of myelofibrosis include progressive anemia and/or splenomegaly, frequently associated with debilitating constitutional symptoms (fatigue, night sweats, bone pain, pruritus and cough) and weight loss. The focus of this report is the single-center (Mayo Clinic) dose escalation phase of a phase 1/2 trial to determine the safety and tolerability of CYT387, a potent JAK-1/2 inhibitor, and to identify a therapeutic dose for the confirmation phase. CYT387 is well tolerated and produces significant anemia, spleen and symptom responses in MF patients. Among 33 patients who were red cell transfused in the month prior to study entry, 70% achieved a minimum 12-week period without transfusions (range 4.7 to >18.3 months). Most patients experienced constitutional symptoms improvement. Grade 3/4 adverse reactions included thrombocytopenia (32%), hyperlipasemia (5%), elevated liver transaminases (3%) and headache (3%). New-onset treatment-related peripheral neuropathy was observed in 22% of patients. A multiple-center dose-confirmation phase, which enrolled an additional 106 patients, was subsequently initiated at or below the maximum tolerated dose (MTD) of CYT387; analysis of these data are ongoing. (Go to abstract external link)

February 2013: Effect of Ruxolitinib Therapy on Myelofibrosis-Related Symptoms and Other Patient-Reported Outcomes in COMFORT-I: A Randomized, Double-Blind, Placebo-Controlled Trial

COMFORT-I (Controlled Myelofibrosis Study With Oral JAK Inhibitor Treatment–I) is a double-blind, placebo-controlled phase III study conducted in the USA, evaluating ruxolitinib in patients with intermediate-2 or high-risk myelofibrosis. The primary objective of the study was the statistically significant reduction in the size of the spleen in the group of patients treated with the drug, and this has been achieved and the results have been already published. Secondary endpoints included changes in symptoms of the disease, for\ the evaluation of which different questionnaires have been used: the modified MFSAF v2.0 (Myelofybrosis Symptoms Assessment Form), the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), the Patient Reported Outcomes Measurement Information System (PROMIS) Fatigue Scale, and the Patient Global Impression of Change (PGIC). After at least 24 weeks of therapy, 45.9% of patients treated with Ruxolitinib had achieved a reduction of at least 50% of the symptoms assessed with the MFSAF, against 5.3% of patients in placebo. A worsening of symptoms occurred in 3.9% of cases in the arm treated with ruxolitinib compared with 33.0% of those in the placebo arm. The other questionnaires used showed a subjective improvement in patients treated with the drug as compared with those in the placebo arm that was proportional to results measured with MFSAF, confirming that the latter questionnaire MFSAF is the most useful in patients with myelofibrosis. Of note, an improvement in symptoms was reported also in patients treated with the drug who did not achieve an optimal response in the reduction of the spleen volume. (Go to abstract external link)

January 2013: Allelic Expression Imbalance of JAK2 V617F Mutation in BCR-ABL Negative Myeloproliferative Neoplasms

Chronic Myeloproliferative Neoplasms (MPNs) are clonal stem cell disorders characterized by different clinical and cytomorphological phenotypes. These diseases are classified as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibosis (MF) according to their phenotipical features. The identification of the JAK2V617F mutation in MPNs allowed to better understand their pathogenesis and to have a useful and simple tool to define the diagnosis. To date several mechanisms for the contribution to phenotypic diversity were proposed, but it is still unclear how a unique single nucleotide mutation can cause three different diseases. The authors of this paper suggest that the allelic expression imbalance contributes to cause different clinical and cytomorphological phenotypes. This phenomenon consists in a preferential expression of one allele in respect to another due to the presence of a polymorphism able to activate expression or to an epigenetic mechanism of control. The experiments described in this paper show a three fold increase in the expression of the mutated allele compared to wild type allele in patients affected by ET and a two fold increase in patients affected by PV. No statistically significant difference was observed in MF patients, although the analysis need to be widened to a larger number of samples to validate this result. Moreover, the data obtained in this paper suggest that the allelic expression imbalance may not occur when the JAK2 V617F is in an homozygous status. In conclusion, the authors of this study propose allelic expression imbalance of JAK2 V617F mutant as another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs. (Go to abstract external link Full text - 340 kB)

December 2012: A prognostic model to predict thrombosis in 891 patients with WHO-defined essential thrombocythemia at diagnosis

The diagnostic criteria for essential thrombocythemia (ET) have been recently reviewed in the WHO classification. However, the risk of thrombosis in ET patients is variable and should be established since the time of diagnosis. Thus, a new prognostic model called IPSET-thrombosis (International Prognostic Score for ET-thrombosis) was developed, providing prognostic indications for the vascular risk based on certain risk factors present at diagnosis. In particular, age over 60 years, a history of previous thrombotic events, general cardiovascular risk factors (hypertension, diabetes, smoke) and the presence of JAK2 V617F mutation showed a significant impact on the thrombotic risk. The IPSET-thrombosis model was developed and validated in a cohort of 891 ET patients. A score to each risk factor was given, as follows: 2 for previous thrombosis and mutated JAK2 V617F and 1 for age over 60 years and at least one cardiovascular risk factor. On the basis of this score, different risk categories were calculated: for the low-risk category (score 0-1) the rate of thrombosis was 1% patients per year; for patients in the intermediate category (score 2) thrombosis rate was 2.3% patients per year and for patients in the high-risk category (score 3 or more) the probability to develop a thrombotic events increased up to 3,5% patients per year. In conclusion, the IPSET thrombosis model is an updated and efficient tool to predict thrombosis in patients with ET at diagnosis. (Go to abstract external link)

November 2012: Flt3 inhibitor AC220 is a potent therapy in a mouse model of myeloproliferative disease driven by enhanced wild-type Flt3 signaling

The Flt3 receptor tyrosine kinase is expressed at high levels on most myeloid and lymphoblastic leukemias, and for many years it has been considered a potential target for compounds that inhibit its kinase activity.
In this study, using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease driven by enhanced wild-type Flt3 signaling, it is demonstrated how this disease can be treated effectively with AC220 (also known as quizartinib). AC220 is the first Flt3 kinase inhibitor to show excellent potency, selectivity, and pharmacokinetic properties.
This mouse model exhibits an expanded population of multipotent progenitors. The daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence and significant loss of multipotent progenitors within 4 days, halted by a robust Flt3 ligand–induced recovery, preventing their further loss. Therefore, the counterbalance mediated by the Flt3 ligand provides an opposing force to AC220, creating a stable environment that allows the Flt3 progenitors to cycle at a rate that is remarkably similar to that of vehicle-treated mice.
The results of the present study indicates that patients with c-Cbl mutations, and those with similarly enhanced wild-type Flt3 signaling, may respond well to AC220.
Data indicate that AC220 may induce remission in patients with c-Cbl mutations and, therefore, may allow these patients time to recover sufficient strength for stem cell transplantation.
Monitoring Flt3 ligand production in clinical studies may provide an indication of a patient’s response to Flt3 inhibitors and guide the timing for administrating cytotoxic drugs. (Go to abstract external link)

October 2012: IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These enzymes catalyze the conversion of isocitrate to a-ketoglutarate (aKG) in an NADP+ dependent manner. The mutations affecting IDH1/IDH2 enzymes are gain-of-function mutations in that they drive the synthesis of the "oncometabolite" R-2-hydroxyglutarate (2HG). Tumor samples harboring these mutations had 2HG at levels up to 100-fold greater than controls. 2HG competitively inhibits tet methylcytosine dioxygenases (Tet2), which regulate DNA methylation, as well as JmjC domain containing histone demethylases. Accordingly, human AML cells with IDH1/IDH2 mutation show global DNA hypermethylation. However, it remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. In this study, the authors report the phenotype of mice expressing the mutant IDH1(R132H) in the hematopoietic system. These mice develop hematologic abnormalities, including anemia, splenomegaly, and extramedullary hematopoiesis. These mice are characterized by hematopoietic stem/progenitor expansion, including increased numbers of hematopoietic stem cells (HSCs) and lineage restricted progenitors. The authors found that HSCs from these mutant mice had significantly more methylated CpG sites compared to control cells, most notably at promoter and intragenic regions. Furthermore, myeloid cells from these mutant mice have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. These findings demonstrate that IDH1/2 mutations contribute to transformation in the hematopoietic system and advance our understanding of the links between IDH1/IDH2 mutations and leukaemogenesis. Moreover, the development of this model provides an avenue for preclinical testing of IDH1/IDH2 inhibitors, such that we learn whether this represents a potential therapy for the subset of patients harboring IDH mutations. (Go to abstract external link Full text - 800 kB)

September 2012: Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor

BACKGROUND:The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2. METHODOLOGY:In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. CONCLUSIONS: Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens. (Go to abstract external link)

August 2012: mir-433 is aberrantly expressed in myeloproliferative neoplasms and suppresses hematopoietic cell growth and differentiation

BCR-ABL negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and miRNA deregulation may play an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared to controls (p<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild-type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2. (Go to abstract external link)

July 2012: Disease characteristics and clinical outcome in young adults with essential thrombocythemia versus early/prefibrotic primary myelofibrosis

The essential trombocythemia (ET) is generally a disease that begins in the population between 55 and 65 years. To date there are few data on the clinical features and prognosis of patients with ET or primary myelofibrosis (PMF) during prefibrotic phase with thrombocytosis under the age of 40 years (representing approximately 20% of the population affected by TE ). In this study we have evaluated both the clinical features and the prognostic impact of 213 patients of which 178 (84%) with WHO-defined ET and 35 (16%) showing early PMF. Patients were followed for a mean period of 7.5 years. The values of platelets were higher in patients with early PMF. These patients also showed a lower value of hemoglobin, a white blood cell count and a higher value of lactate dehydrogenase (LDH) higher than in patients with ET. In patients with early PMF, there was a trend toward a greater number of thrombotic events, in particular arterial. The progression to overt myelofibrosis was observed more frequently in patients with PMF (9%) compared to patients with TE (3%). There were no reports of transformation to acute leukemia. Bleeding events were slightly higher in patients with early PMF than patients with TE although a statistical point of view this difference did not appear significant. Considering all these complications together, or thrombotic events, bleeding and transformation to myelofibrosis, were observed more frequently in PMF compared to ET (3.43% vs 1.29% patients/year). This value is statistically significant (p=.01). Multivariate analysis also revealed that the presence of the JAK2V617F mutation is an independent factor that predicts the possibility of adverse events mentioned. On the contrary, the degree of splenomegaly at diagnosis, the presence in the history of thrombotic events or bleeding, treatment with cytoreductive agents or aspirin did not affect these events. In conclusion, this study identifies a subgroup of young patients with early PMF with an increased risk of adverse events compared with patients with ET. (Go to abstract external link)

June 2012: A prognostic model to predict survival in 867 WHO-defined essential thrombocythemia at diagnosis: a study by the IWG-MRT

The diagnostic criteria for essential thrombocythemia (ET) have been recently reviewed in the WHO classification. However, among patients with ET the prognosis may vary and is not easy to determine since the time of diagnosis. It was therefore developed a new prognostic model called IPSET (International Prognostic Score for ET), that can provide prognostic indications based on certain risk factors present at diagnosis. In particular, the age over 60 years, the WBC count greater than 11x109/L and a history of previous thrombotic events have a significant impact on prognosis. In contrast, the presence of splenomegaly, the JAK2 mutational status and hemoglobin levels have no impact on prognosis. In this regard the presence of the mutation in the JAK2 gene is associated with an increased risk of arterial thrombosis in the WHO-TE. However, analysis of survival did not show any association with the JAK2 mutational status, although the JAK2 V617F mutation represents an important risk factor for thrombosis. The IPSET model was validated in a cohort of 867 patients. It was given a score to each risk factor: 2 for age and 1 to the leukocyte count and previous thrombotic events. On this total score, different risk categories were calculated: for the intermediate risk (total score 1) survival is 24.5 years; for patients with high risk (score 2 or 3) survival is 13.8 years. This study confirms the negative impact of the white blood cell count on prognosis. Although the IPSET model has not been designed to predict thrombotic events has proved useful in this sense. On the contrary does not seem able to predict the transformation in myelofibrosis. In conclusion, the model IPSET is an easy tool for predicting the prognosis in patients with essential thrombocythemia at diagnosis. (Go to abstract external link)

May 2012: Genetic analysis of patients with leukemic transformation of myeloproliferative neoplasms shows recurrent SRSF2 mutations that are associated with adverse outcome

Myeloproliferative neoplasms (essential thrombocythemia, polycythemia vera, primary myelofibrosis) have a variable risk of progression to secondary acute myeloid leukemia. This latter condition is less responsive to treatment than de novo acute myeloid leukemia, and therefore involves a poor prognosis. Previous studies had identified mutant genes associated with leukemic transformation of myeloproliferative neoplasms. In this study, Verstovsek and his collaborators performed a mutation analysis of 22 genes in 53 patients with myeloid neoplasms who had progressed to secondary leukemia: JAK2, MPL, IDH1, IDH2, FLT3, c-KIT, K/N/H-RAS, U2AF1, SF3B1, SRSF2, GATA2, RUNX1, EZH2, WT-1, DNMT3a, TP53, TET2, ASXL1, PTEN, ZRSR2. In addition to JAK2 and TET2 mutations, these investigators identified recurrent mutations in the SRSF2 gene, which encodes a core component of the RNA splicing machinery. Mutation frequency was about 20%, much higher than that observed in de novo acute myeloid leukemia or acute leukemia secondary to myelodysplastic syndrome. Furthermore, SRSF2 mutations were associated with worsened overall survival in patients with leukemic evolution of myeloproliferative neoplasm. These data underscore the pathophysiological relevance of mutations of splicing machinery in myeloid neoplasms, and suggest that SRSF2 mutations may guide novel therapeutic approaches for patients with myeloproliferative neoplasms who undergo leukemic transformation. (Go to abstract external link)

April 2012: Efficacy of vorinostat in a murine model of polycythemia vera

The very fact that about two thirds of patients with myeloproliferative neoplasm (essential thrombocythemia, polycythemia vera, primary myelofibrosis) carry the unique V617F mutation of JAK2 has led to the development of JAK2 kinase inhibitors. Unfortunately, these inhibitors are only partially effective, and some of them have been found to have dose-limiting toxicities in clinical trials. In the present study, Mohi and his collaborators studied the effect of vorinostat, a small-molecule inhibitor of histone deacetylase, against cells expressing JAK2 (V617F) and in an animal model of polycythemia vera. They found that vorinostat inhibited proliferation and induced apoptosis in cells expressing JAK2 (V617F). These effect were confirmed in the mouse model and in human JAK2 (V617F)-positive hematopoietic progenitors. More importantly, these investigators observed that vorinostat treatment normalized the peripheral blood counts and markedly reduced splenomegaly in Jak2 (V617F) knock-in mice, and also decreased the mutant allele burden. These observations suggest that vorinostat may have therapeutic potential for the treatment of JAK2 (V617F)-associated myeloproliferative neoplasms. (Go to abstract external link)

March 2012: A Double-Blind, Placebo-Controlled Trial of Ruxolitinib for Myelofibrosis

Ruxolitinib is an oral selective inhibitor of the JAK-1 and JAK-2 genes that has been recently approved in the U.S.A. for the treatment of intermediate- and high-risk myelofibrosis. In phase 1 and 2 studies, it was shown that treatment with ruxolitinib resulted in reduction of splenomegaly and improvement of symptoms related to the disease (including fatigue, pain, appetite loss, dyspnea), regardless the mutational status of JAK-2. In the March 1 issue of the New England Journal of Medicine, Srdan Verstovsek and collaborators report the results of a phase 3 study in which patients with intermediate or high-risk myelofibrosis were randomly assigned to twice daily administration of ruxolitinib or placebo. The primary endpoint of the trial was to assess the percentage of patients who obtain a 35% (or more) reduction of the spleen size (evaluated by magnetic resonance imaging) whereas the secondary endpoints were the assessment of the durability of response, of the changes in myelofibrosis-related symptoms and of the overall survival.
Forty-one point nine percent of the 155 patients who received the drug reached the primary endpoint, whereas only 0.7% of the 154 patients who received the placebo did. The difference was statistically significant. The reduction of spleen size was maintained for more than 48 weeks by 67% of the patients who had a spleen size reduction. Improvement of the disease-related symptoms was reached in 45.9% of patients treated with the drug and in 5.3% of the patients of the placebo group. A statistically significant improvement in the probability of overall survival was also reported in the group of patients who received ruxolitinib. The most common adverse events in the ruxolitinib group were anemia and thrombocytopenia; however, these events rarely led to the discontinuation of the therapy. In this group of patients, two transformations into acute myeloid leukemia were reported. Taken together, the results reported in this study confirm that ruxolitinib is an effective treatment for myelofibrosis, by reducing splenomegaly and ameliorating the clinical symptoms of the disease. It also appears to be associated with an improvement of overall survival. (Go to abstract external link)
In the same issue of the Journal a study by Claire Harrison and collaborators has compared the effects of oral ruxolitinib with the best available therapy in patients with myelofibrosis. (More informations external link).
A commentary by Ayalew Tefferi on JAK-2 inhibitors for the treatment of myeloproliferative neoplasms is published in the same issue of the Journal (More informations external link).

February 2012: The cell cycle regulator CDC25A is a target for the JAK2V617F oncogene

Since its discovery, the mutation V617F of the JAK2 gene has provided important understanding of the pathogenesis of chronic myeloproliferative neoplasms (MPNs). However, despite the large body of knowledge accumulated in the last years, many key-issues remain unexplained. For instance, it has been matter of debate whether the JAK2 V617F mutation could provide a proliferative advantage to the cells in which it occurred.
In the february issue of the Blood journal a french group shows how JAK2 V617F mutation is able to affect the cell cycle of hematopoietic cells of MPN patients. The researchers focus their attention on a protein, the CDC25A phosphatase, that plays a key role in the cell cycle regulation, allowing the progression of the cell through the G1 phase toward the S phase of the cell cycle as well as the activation of the DNA synthesis. Using both JAK2 mutated cell lines and primary hematopoietic cells both from MPN patients and mice expressing the V617F mutation, the authors show that this protein is overexpressed in cells bearing the mutation and that the higher the levels of the CDC25A protein the higher the level of the JAK2 signaling. Moreover, the authors convincingly show that the increase of the CDC25A levels is obtained through an increase of the translation of the corresponding mRNA, due to a prolonged activity of an elongation factor that depends on V617FJAK2 activity. The last and most promising finding of the paper is that the pharmacological inhibition of the CDC25A protein resulted both in a decreased proliferation of the clonogenic activity of MPN progenitor cells and in the impairment of the growth of EPO-independent BFU-E colonies in vitro. Notably, EPO-dependent growth of BFU-E was not affected by the CDC25A inhibitor, suggesting that "healthy" hematopoiesis is spared from the effects of the inhibitor.
This study has two important consequences in the field of MPNs: 1) it sets a role for the V617FJAK2 mutation in the regulation of the cell cycle and, in turn, of the proliferative potential of the mutated cells, and 2) identifies the CDC25A phosphatase as an attracting target for the design of novel drugs for MPN treatment. (Go to abstract external link)
An Inside Blood external link comment is linked to this article.

January 2012: Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and Jak2V617F in mice

STAT5A and STAT5B proteins (Signal Transducer and Activator of Transcription) are transcription factors that, within the cell, are activated by JAK proteins in response to numerous external stimuli, including erythropoietin and thrombopoietin. Their activation involves the expression of genes involved in cell growth, and previous studies showed that increased activation of STA5a/b can be documented in cells of patients with chronic myeloproliferative neoplasms and acute leukemia. In this study, in order to define the role of STAT5 in the development of chronic myeloid leukemia (CML) and polycythemia vera (PV), mice in which the expression of STAT5a/b could be quantitatively regulated (reduced to about 50% or abolished) were employed. Those mice were transplanted with cells expressing, respectively, the BCR/ABL rearrangement characteristic of CML or the JAK2V617F mutation characteristic of PV. The loss of one of the two alleles of STAT5a/b in mice with the rearrangement BCR/ABL leads to the development of a form of "attenuated" CML. In contrast, the total absence of STAT5a/b prevented the development of CML, despite the fact that cells continued to express BCR/ABL; however, it did not appear able to avoid that cells expressing BCR/ABL evolved in leukemia cells comparable to those of lymphoid blast crisis of CML. This suggests that STAT5a/b is necessary for the development of CML, but not of lymphoid blast crisis, indicating that these leukemic cells use also different signal transduction pathways, and that inhibition of STAT5a/b is not sufficient to eradicate the clone of BCR / ABL positive cells.
In the mouse model transplanted with JAK2V617F positive cells, erythrocytosis and leukocytosis, characteristic of PV, developed both in mice expressing STAT5a/b at normal levels and in those that expressed only one allele. The latter mice, however, had a lower degree of leukocytosis. The absence of expression of STA5a/b prevented the development of PV, even though the mice presented histological features in the bone marrow and spleen suggestive of evolution to myelofibrosis. This indicates that the lack of expression of STAT5a/b prevents the development of PV but not the evolution to myelofibrosis, suggesting a role of different signal transduction pathways activation in this process.
Data from this study provide a rationale for the development of STAT5a/b inhibitors for the treatment of CML and PV. (Go to abstract external link)

December 2011: Long-term follow-up of patients with portal vein thrombosis and myeloproliferative neoplasms

A chronic myeloproliferative neoplasm is one of the most frequent causes of portal vein thrombosis in patients without liver cirrhosis. The aim of this study was to describe the long-term outcome of patients with portal vein thrombosis and a myeloproliferative neoplasm. 44 patients were followed, with a median follow-up of 5.8 years. The median age at diagnosis of thrombosis was 48 years, and 70% of patients were female. 52% of patients were treated with oral anticoagulation, and 34% of these for an extended period. Gastrointestinal bleeding was a frequent complication, observed in 39% of cases, as well as a second thrombotic event (27%). 39% of patients died, and the most frequent causes of death were progression of the myeloproliferative disease (47%) and new thrombosis (18%), whereas no patients died of gastrointestinal bleeding.
In 70% of cases, thrombosis was the first manifestation of the myeloproliferative disease, stressing the importance of searching for a myeloprliferative disease in patients who develop portal vein thrombosis, especially in the light that mortality was related to myeloproliferative disease rather than complications of portal vein thrombosis itself. (Go to abstract external link)

November 2011: Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloproliferative neoplasms and other myeloid disorders. EZH2 mutations are associated with a poor prognosis and appear to be early events in the disease process, at least in some cases. Assuming that other components of PRC2 are mutated in these diseases, in this paper the researchers analyzed the genes that are part of this complex, such as SUZ12 and EED, and cofactors RBBP4 Jarid2. Were studied a total of 148 cases of myelodysplastic / myeloproliferative diseases and two cases of myeloproliferative neoplasm out of a series of 151 patients based on known molecular aberrations. SUZ12 mutations were found in 4 patients (1.4%), 2 with chronic myelomonocytic leukemia (a myelodysplastic syndrome/myeloproliferative) and two with chronic myeloproliferative neoplasm and known deletion in the SUZ12 adjacent region, and only one case with mutation of EED. No mutation was found in Jarid2 and RBBP4. SUZ12 and EED mutations resulted in reduced methyltransferase activity of the complex PRC2, and might therefore play a role in the pathogenesis of the disease. Further studies are needed to determine whether these mutations have prognostic significance or are involved in clinical manifestations of the disease. (Go to abstract external link)

October 2011: Pruritus in primary myelofibrosis: Clinical and laboratory correlates

Pruritus, that may be aquagenic or not, is a typical symptom of polycythemia vera, observed also in primary myelofibrosis (PMF). It can be so debilitating to result in avoidance of taking shower and sleep deprivation. The pathogenesis of this symptom is still largely unknown, although recent data suggested an association with the presence of the JAK2V617F mutation. In this study, the authors addressed this issue in a series of 566 patients with PMF, in which pruritus was present in 16% of cases. In 15 cases the itching was present long before the diagnosis of PMF. The symptom was significantly associated with leukocytosis and absence of the mutation MPLW515, and showed no correlation with platelet count, degree of anemia and splenomegaly, the karyotype, the prognostic classification system DIPSS-plus, the evolution in acute leukemia, age or gender. JAK2V617F and systemic symptoms were more frequent in patients with itching, but this association was of bordeline statistical significance. Serum levels of several cytokines were analysed but overall they resulted independent of the intensity of itching, possibly indicating that pruritus is not depend on an inflammatory process. Rather, it appears to be associated with leukocytes, possibly with substances secreted by these cells, but its nature still remains largely unclear. (Go to abstract external link)

September 2011: Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms

Myeloproliferative neoplasms are frequently associated to the V617F activating mutation of the JAK2 tyrosine kinase, which causes an overproduction of myeloid lineage cells. Several molecules targeting JAK2, including ruxolitinib (INCB018424), are currently in clinical development for treatment of these diseases. The authors of this paper conducted an in vitro study in order to identify additional JAK2 mutations other than V617F, which may confer resistance to ruxolitinib, thus being clinically relevant. To this aim, they introduced random mutations in the JAK2V617F gene and induced the expression of such mutants in murine cells in the presence of ruxolitinib. Indeed, if a mutation confers resistance to the drug, cells will proliferate even under treatment, otherwise cell growth will be inhibited. Further characterization of resistant cells led to the identification of 5 point mutations, which impair the binding between the JAK2V617F kinase and ruxolitinib, thus decreasing its efficacy. These mutations, as demonstrated by specific softwares, correspond or are close to the aminoacids involved in the interaction with the inhibitor and cause, beyond resistance, a proliferative advantage. Moreover, the researchers tested other JAK2 inhibitors, different from ruxolitinib, and showed cross-resistance of the mutants previously identified towards all drugs. However, an additional mutation was identified in a key position of the JAK2V617F kinase domain, which confers resistance to ruxolitinib but not to the other molecules tested. The results of this study are of great interest due to the potential wide use of JAK2 inhibitors for the treatment of all diseases characterized by the constitutive activation of JAK2. The data underline the need to carefully monitor the mutational status of JAK2 in patients that fail to respond to ruxolitinib and other JAK2 inhibitors, as well as the importance of alternative targeted therapies. (Go to abstract external link)

August 2011: Increased gene expression of histone deacetylases in patients with Philadelphia-negative chronic myeloproliferative neoplasms.

Histone deacetylases (HDACs) are enzymes involved in chromatin remodelling and play an important role in gene expression regulation. Histone acetylation converts chromatin into a more relaxed structure, which is associated with higher levels of gene transcription. This relaxation can be reversed by HDACs activity. Several evidences recently reported the alteration of HDACs expression levels in different types of cancer. Albeit the exact role of HDACs in cancerogenesis remains unclear, it has been attributed to their capability to down-regulate the expression of oncosoppressor genes. For this reason, HDACs inhibitors have been tested as anti-cancer agents and proved effective in inducing cell-cycle arrest, differentiation and apoptosis (programmed cell death) of neoplastic cells. Recently, increased HDACs activity has been demonstrated in stem cells from patients with Primary Myelofibrosis (PMF), suggesting a role of these enzymes in PMF pathogenesis or in the progression of Polycythemia Vera and Essential Thrombocythemia to Myelofibrosis. Different HDACs inhibitors (including Givinostat and Vorinostat) showed promising results in the treatment of this group of diseases, comprised among the Chronic Myeloproliferative Neoplasms (CMNs).
In this paper, the expression level of HDACs was evaluated in peripheral blood samples from 69 CMNs patients, in comparison with healthy donors, by global gene expression profiling (which allows to study more than 38000 genes); as a result, a pronounced deregulation of HDACs genes expression was highlighted. In particular, increased levels of HDAC6, HDAC9 and HDAC11 were found and, for the first time, decreased expression of HDAC7 was described. The exact role of the expression regulation of single enzymes in the pathogenesis of CMNs remains unclear, but their involvement in some physiopathological processes is already known. For example, increased levels of HDAC11 suppress the expression of interleukin-10, a cytokine involved in the inhibition of inflammation; also, deregulation of HDAC11 may contribute to dirupt the immunological balance towards a chronic inflammatory process. HDAC7 probably promotes apoptosis and its down-regulation could lead to accumulation of neoplastic cells. HDAC6 is a cytoplasmic protein recently shown to play a role in the proliferation of cancer cells. Moreover, HDACs may direcly deacetylate transcription factors such as NF-kB, HIF1-α, GATA1 and FOXP3, thus interfering actively with proteins of inflammation and proliferation.
In conclusion, this study point at the role of HDACs as promising targets for CMNs treatment. In particular, HDACs inhibitors have proved effective in regulating the chronic inflammatory process typical of these diseases, but their role in cell proliferation and apoptosis remains to be elucidated. (Go to abstract external link)

July 2011: TET2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation

Several studies on myeloid malignancies have identified recurrent alterations in the majority of patients with myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs) and acute myeloid leukemias (AMLs). Recently, many different lesions on TET2 gene have been identified as a novel class of mutations implicated in leukemogenesis. TET2 is an enzyme which chemically modifies DNA by converting 5-methylcytosine to 5-hydroxymethylcytosine, thus varying the DNA methylation state and as a consequence the transcriptional activity. In fact TET2 mutant protein observed in myeloid malignancies is deficient in its enzymatic function with consequent alteration of the blood cell transcriptional profile.
Although genetic and in vitro studies suggested a role for TET2 in regulation of hematopoietic development, the in vivo effects of TET2 loss on animal models have not previously been described.
In this paper, the authors demonstrated by inhibition of TET2 that this enzyme regulates the self-renewal and engraftment properties of Hematopoietic Stem Cell (HSC). In fact, TET2 deficiency in vivo leads to an increased self-renewal and engraftment ability. Furthermore, mice showed a significant splenomegaly, due to extramedullary hematopoiesis, and progressive defects in differentiation of monocytes and granulocytes. A detailed analysis of hematopoiesis revealed that mice had a progressive myeloproliferation associated with neutrophilia and a marked increase in monocyte counts; the presence of immature cells was detected both in bone marrow and in peripheral lymphoid tissue, coupled with a myeloid dysplasia. Collectively these data demonstrate that TET2 loss is associated with myeloproliferation in vivo.
Since the most common situation in patients with TET2 loss is the mutation of one out of two copies of the gene, similar experiments were performed in mice with just one mutated TET2 allele; by means of this approach the authors clearly demonstrated that this kind of alteration is an important pathogenetic event in malignant myeloproliferation.
In conclusion, the presented study implicates TET2 as a master regulator of normal and malignant hematopoiesis and sheds light on the neoplastic transformation mechanisms by a novel class of mutations found in myeloid malignancies. It is likely that therapies that modulate DNA methylation levels may represent a new therapeutic tool for malignancies characterized by loss of TET enzyme function. (Go to abstract external link)

June 2011: A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway

The JAK-STAT pathway is involved in signal transduction of many growth factors. Binding of these molecules to their receptors induces the activation of receptor-associated tyrosine kinases JAKs. The phosphorylation of substrates such as STAT proteins allows their translocation to the nucleus to transactivate gene expression.
Among the members of JAK tyrosine kinase family, JAK2 is characterized to be essential in erythropoiesis and its dysfunction has been implicated in myeloproliferative neoplasms (MPNs) and leukemias. In particular, the point mutation JAK2V617F occurs in a high number of MPN patients and it is involved in the pathogenesis of MPNs. Another protein kinase whose dysregulation is involved in tumorigenesis is CK2, as its expression and activity is upregulated in many blood tumors. Up to date, little is known about a potential cross-talk between CK2 and the JAK-STAT pathway that could represent another possible target for therapeutic strategies.
In this paper, the authors demonstrated by CK2 inhibition that a cytokine-induced JAK-STAT activation is dependent on the presence or activity of CK2. Moreover, the data showed that also the expression of SOCS-3, a downstream STAT target gene, is dependent on CK2 and that JAK2 is actually a target of CK2. Since a constitutively active mutated form of JAK2 (JAK2V617F) is involved in MPNs, the authors decided to study CK2-JAK2 interaction in JAK2V617F mutant-expressing erythroid leukemia cell line HEL. Endogenous JAK2V617F was demonstrated to be associated with CK2. Furthermore, CK2 inhibition suppressed autonomous JAK2V617F tyrosine phosphorylation and expression of the anti-apoptotic gene Bcl-xL causing a high apoptosis rate in HEL cells.
Finally, CK2 inhibition in mononuclear cells from polycithemia vera (PV) patients suppressed constitutive activation of JAK2, STAT5, STAT3 and ERK and it was able to induce apoptosis in 24 hours. Thus these experiments show that CK2 is required for constitutive activation of JAK2V617F signaling and cell survival either in a HEL cell or in primary cells from PV patients.
All these data indicate that CK2 could be an additional therapeutic target for JAK2V617F positive MPNs, and in particular CK2 inhibitors may be a useful therapeutic tool for malignances with an aberrant CK2, JAK and STAT activation. (Go to abstract external link)

May 2011: Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms

The amino acid mutation V617F in the JAK2 protein kinase is the main responsible for the development of myeloproliferative neoplasms (MPNs) in humans, such as Polycythemia Vera (PV), Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET). The mutation affects hematopoietic stem cells (HSCs), but the consequences of its expression in hematopoietic progenitors and stem compartments (HSPC) and its weight in myeloid differentiation are still unclear. The determination of the precise role of JAK2-V617F in these regions is essential in order to better understand the development of the disease and thus be able to design more specific drugs to hit the altered functions of the mutated form of JAK2.
This article then analyzed the effects of the mutation of JAK2 in hematopoietic stem cell compartment and in myeloid differentiation, in both bone marrow and in peripheral blood from patients with MPNs. The mutation does not appear to affect the total number of precursors in bone marrow hematopoietic compartment of patients with PV and ET. On the contrary, patients with primary myelofibrosis have a statistically significant increase compared with controls. On the other hand, there are no selective advantages on the ability of JAK2-V617F myeloid precursors to proliferate and differentiate in vitro in all three myeloproliferative disorders compared to wild-type cells. The effect of mutation is rather preponderant in "in vivo" expansion of hematopoietic compartments in more differentiated stages; in fact it is significantly higher at the level of erythroid and megakaryocytic compartments. The key issue is then the correlation between the degree of myeloid differentiation and the so-called "allele burden", ie the ratio between the JAK2 mutated and wild type hematopoietic cells: the greater is the allele burden, the higher is the degree of amplification of the terminally differentiated myeloid precursors. Less clear are data about the difference between homozygous or heterozygous JAK2-V617F mutation status; clones having both alleles mutated have a greater capacity for expansion either in hematopoietic progenitors and erythroid and megakaryocytic precursors compared with ones heterozygous for the mutation, although this feature is not confirmed in all patients. In conclusion, these data suggest that, despite the JAK2-V617F mutation has a fundamental effect and is sufficient for the development of myeloproliferation, there may be other yet unknown molecular mechanisms that contribute significantly to the modulation of the phenotype. This could well explain the limited effectiveness of JAK2 inhibitors in total eradication of leukemic stem cells, which are the main supporters of the development of human myeloproliferative neoplasms. (Go to abstract external link)

April 2011: JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation

Many clinical studies have shown that the V617F amino acid mutation in the JAK2 protein kinase is implicated in the development of myeloproliferative disorders, such as Polycythemia Vera (PV), Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET). Such mutation produces a constitutive activation of JAK2 kinase signaling pathway, which behaves in this way as an "ignition" for deranged cellular function delivering oncogenic signals. The exact molecular mechanism by which JAK2-V617F is able to develop a myeloproliferative phenotype, however, is still unclear. In this study published in "Cell" it is shown for the first time that mutated JAK2 not only acquires the ability to bind more strongly than the wild-type protein to one of its target, namely arginine methyl transferase PRMT5, but it also induces a greater degree of phosphorylation. As a result, highly phosphorylated PRMT5 down-regulates its ability to methylate some of its substrates, called histones, which act as specific regulators of the state of accessibility to DNA. This way, it could alter the gene expression profile of the entire hematopoietic cells in which mutation has occurred, promoting a myeloproliferative phenotype. By using CD34 + cells as a model it is shown in more detail that the reduction of PRMT5 activity promotes an increase in the number of CFU (Colony Forming Units) and of differentiated erythroid cells; on the contrary its over-expression significantly reduced colony formation, indicating that PRMT5 is able to negatively regulate the proliferation and expansion of erythroid progenitor cells. One added value of this study is that the molecular data obtained in a cellular model were confirmed directly in CD34 + cells taken from patients with Polycythemia Vera. Of the samples evaluated, only those with the V617F mutation in JAK2 kinase had increased phosphorylation of methyl-transferase PRMT5 compared to negative controls for the mutation. These results therefore indicate that phosphorylation of PRMT5 contributes to the myeloproliferative phenotype induced by mutation of JAK2, thus opening the possibility of introducing a new therapeutic target for patients with myeloproliferative disorders. (Go to abstract external link)

March 2011: Molecular and clinical features of the myeloproliferative neoplasm associated with JAK2 exon 12 mutations

It is known that about 95% of patients with Polycythemia Vera (PV) show the V617F mutation in exon 14 of the JAK2 gene. In some of the JAK2V617F negative PV patients Scott and colleagues described different types of mutations located in the JAK2 exon 12. This mutation has been evaluated in a series of 338 cases of patients with V617F-negative polycythemia and found in 4%. Since this is far less frequent than the V617F mutation, the clinical and biological characteristics of patients with exon 12 mutations were not yet fully known, although the mutations appeared to associate preferentially with isolated erythrocytosis. Current study analyzed 106 patients with PV harboring mutation in exon 12, collected in 13 different European centers. A total of 17 different mutations were identified in exon 12 of JAK2.
The study showed that 64% of patients with mutation in exon 12 presented with isolated erythrocytosis, 15% with erythrocytosis and leukocytosis (WBC> 10 x 109/L), 12% with erythrocytosis and thrombocytosis (platelets > 400 x 109/L) and 10% with erythrocytosis, leukocytosis, thrombocytosis. Eight-three percent of the patients had also a bone marrow characterized by hypercellularity. The majority of patients also presented erythropoietin levels lower than normal.
These observations lead to the recommendation to search for mutations in exon 12 of JAK2 in patients with erythrocytosis who have erythropoietin levels lower than normal and absence of the V617F mutation.
Statistical analysis showed that patients with the mutation in exon 12 are characterized by higher hemoglobin levels than patients with V617F mutation and lower levels of platelets and leukocytes. Finally, the risk of thrombosis, bleeding, evolution to post-PV myelofibrosis and transformation to acute leukemia was similar in the comparison between two cohorts of patients with mutation of JAK2 exon 14 and exon 12.
Risk factors for thrombotic events that are commonly considered to be significant for patients with V617F mutation, ie age and history of previous thrombotic events, resulted significant also for patients characterized by the presence of JAK2 exon 12 mutations. (Go to abstract external link)

February 2011: Safety and Efficacy of TG101348, a Selective JAK2 Inhibitor, in Myelofibrosis

About half of patients with myelofibrosiscarry the V617F mutation of JAK2 gene. In this study the authors report the results of a multicenter phase I study for the treatment of patients with primary myelofibrosisor myelofibrosissecondary to polycythemia vera or essential thrombocythemia, at high or intermediate risk, with a new JAK2 inhibitor drug called TG101348, administered once a day. The study was conducted in several American centers. The study enrolled a total of 59 patients, including also a group of patients who had gradually increase of the dose to determine the maximum tolerateddose. This dose was determined to be 680 mg per day. The main toxicity observed at higher doses was the increase of the enzyme amylase, reversible and asymptomatic. Seventy-three percent of patients included in the protocol continued therapy for at least 6 cycles, with limited side effects including nausea, vomiting, diarrhea, anemia and thrombocytopenia. As a result of treatment, most patients reported a rapid improvement in symptoms such as fatigue, itching, night sweats, the feeling of early satiety. Thirty-nine percent also obtained a rapid reduction in spleen size after 6 cycles and 47% after 12 cycles. The majority of patients with leukocytosis and thrombocytosis at the beginning of therapy achieved a normalization of blood parameters after 6-12 cycles of therapy. Finally, a significant result was the gradual reduction in the amount of JAK2V617F mutant allele burden, that is a marker for the amount of disease. The conclusions of the study are therefore that this new inhibitor of JAK2 appears very promising amongnewly available drugs for the treatment of chronic myeloproliferative disorders, and that it appears to be well tolerated with reversibleside effects upon discontinuation of the drug. In addition, this drug seems to have a powerful impact not only on symptoms but also on the burden of disease in patients with myelofibrosis. (Go to abstract external link)

January 2011: Clonal endothelial progenitor cells and vascular complications in myeloproliferative neoplasms

Our comprehension of the pathophysiology of myeloproliferative neoplasms has been revolutionized in 2005 by the discovery of the unique JAK2 (V617F) mutation. This led to the identification of several genes (including JAK2, MPL, LNK, TET2, IDH1/IDH2, EZH2, ASXL1 and TP53) whose somatic mutations play a role in the molecular pathogenesis and/or progression of these disorders. Fewer studies have investigated in these years the cellular basis of myeloproliferative neoplasms. In December 2001, Dr. Barosi ad his coworkers published a seminal study in this field [Blood. 2001 Dec 1;98(12):3249-55], showing that the absolute number of CD34-positive hematopoietic cells was increased in peripheral blood from patients with primary myelofibrosis. This observation led to the concept of abnormal stem cell trafficking as a cellular mechanism of human disease. A few weeks ago, Dr. Teofili and coworkers published an interesting study indicating that endothelial progenitor cells are clonal and exhibit the JAK2 (V617F) mutation in a subset of patients with myeloproliferative neoplasm. These Italian investigators evaluated circulating endothelial colony forming cells (ECFCs) in 42 patients with polycythemia vera, essential thrombocythemia or primary myelofibrosis. The growth of colonies was obtained in 22 patients and the highest levels were found in patients with primary myelofibrosis. In 5 patients who had experienced thromboembolic complications, ECFCs were clonal as evaluated by X-chromosome inactivation patterns and carried JAK2 (V617F). This study confirms previous observations by the AGIMM investigators showing that ECFCs may belong to the myeloproliferative clone [PLoS One. 2010 Dec 9;5(12):e15277], and suggests that abnormal endothelial progenitor cells may contribute to the thrombophilic state observed in patients with myeloproliferative neoplasm. (Go to abstract external link)

December 2010: Somatic mutations of the TET2 gene disrupt TET2 enzymatic activity and favor myeloid tumorigenesis

In the WHO classification of tumors of hematopoietic and lymphoid tissues, myeloid neoplasms include myeloproliferative neoplasms, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms and acute myeloid leukemia. These disorders are associated with deregulated production of myeloid cells and may have overlapping features. In particular, phenotypic shifts may be observed among myeloid malignancies, and both myelodysplastic syndromes and myeloproliferative neoplasms may progress to acute myeloid leukemia. Two years ago, Delhommeau and coworkers conducted a study to identify a candidate tumor-suppressor gene common to patients with myeloid neoplasms. They found somatic mutations of the gene TET2 in about 15% of myeloid neoplasms [N Engl J Med. 2009 May 28;360(22):2289-301]. TET2 defects were present in hematopoietic stem cells and preceded the JAK2 (V617F) mutation in some patients with myeloproliferative neoplasm. However, the function of the TET2 protein was unknown at that time, and the gene TET2 was just known to be a close relative of the gene encoding TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA [Science. 2009 May 15;324(5929):930-5]. In a recent collaborative study, Dr. Rao (Harvard Medical School, Boston), Dr. Maciejewski (Taussig Cancer Institute, Cleveland) and coworkers demonstrated a strong correlation between myeloid neoplasms and loss of TET2 catalytic activity, with bone-marrow samples from patients carrying TET2 mutations having low levels of 5hmC in genomic DNA [Nature. 2010 Dec 9;468(7325):839-43]. In addition, depletion of Tet2 in mouse hematopoietic precursors altered their differentiation towards monocyte/macrophage lineages in culture, indicating that TET2 is required for normal myeloid differentiation. Thus, this study suggests that loss of TET2 catalytic activity may impair myeloid differentiation and favor myeloid tumorigenesis in humans through a mechanism of epigenetic deregulation. (Go to abstract external link)

November 2010: JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

Most of the diagnostic tests rely on genomic DNA for the search of JAK-2 mutations, including the common V617F mutation and the less frequent exon 12, 13 and 14 genomic alterations. Usually the genomic DNA is derived from peripheral blood granulocytes. However, this approach, although well established in the usual practice, underestimates the frequency of a recently described deletion of JAK2 exon 14, which determines the synthesis of a truncated JAK-2 protein. In fact, by means of direct sequencing, a complete deletion of exon 14 was described in less than 1% of patients with Ph-MPNs. In this paper, published few months ago in PLosOne, the Authors have looked for exon 14 deletion in a relatively large cohort of patients with MPNs, using a sensitive RT-PCR-based assay with fluorescent fragment, taking advantage from a previous own observation showing that RNA allows more sensitive detection of mutations than does DNA at early stage of disease. In addition, the approach reported by the Authors can be performed not only on cell derived but also on plasma derived RNA. Nine out of 61 patients (15%) with clinically confirmed MPNs harbored an exon 14 deletion, whereas 51 out of 183 patients (27%) with de-identified diagnosis harbored the same deletion. Of these 51 patients, 20 resulted to be positive also for the JAK-2V617F mutation while 31 not. In all cases harboring the mutation, the truncated protein was also detected. In none of the 46 healthy control subjects the deletion was found. These results indicate that a complete deletion of exon 14 is a common event in MPN, possibly more frequent in patients without the V617F JAK-2 mutation. Therefore, the search for this deletion can have diagnostic relevance and could play a role in the pathogenesis and progression of MPNs, in particular those without the V617F JAK-2 mutation. (Go to abstract external link Full text - 670 kB)

October 2010: Physiological JAK2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells

The JAK-2V617F mutation represents an ideal candidate for a molecular targeted therapy of MPNs. However, early data from clinical trials based on the administration to MPN patients of JAK-2 inhibitors (see the september paper of the month in our web site) have resulted in the amelioration of some clinical symptoms of the disease, such as splenomegaly and systemic symptoms such as fever, pruritus, weight loss) but not on the natural history of the disease, calling into question the actual capacity of these drugs to cure the disease. An in depth-study of the effects of JAK-2 inhibitors on the disease-initiating population would take great advantage from the availability of an animal model in which the JAK-2V617F allele is expressed at physiological levels both in hematopoietic stem and progenitor cells, as it happens in human patients. Up to now such a model was not available. Mullaly and coll at the Dana-Farber Cancer Institute, Boston MA, have generated a murine model of MPN clinically resembling a human polycythemia vera. This was obtained by the conditional knock-in of a JAK-2V617F allele into mouse ESCs, under the control of its endogenous promoter. The Authors show that the physiological expression of the JAK-2V617F allele greatly drives the hematopoietic stem cell differentiation and expansion toward the myeloid lineage, in particular the erythroid and at a lesser extent the megakaryocytic compartments, resulting in a clinical and pathological picture of polycythemia vera. They also show that the disease is transplantable in healthy, sublethally irradiated mice and that this is due to the more immature compartment of hematopoietic stem cells but is indipendent from the more mature stem and progenitor cells. Interestingly, by means of experiments of competitive repopulation of the bone marrow, they show that the disease is initiated by the more immature stem cells regardless the presence of the JAK-2V617F mutation, which is able to confer only a very moderate proliferative advantage to the stem cells that harbor the mutation, suggesting that other factors, behind the JAK-2V617F mutation, such as for instance TET-2 mutations are required to initiate the disease. Finally, and in agreement with this observation, they show that the stem cell compartment, which is the ultimate target for a curative therapy of MPNs, is resistant to treatment with a JAK-2 inhibitor. This model, besides the novel and interesting insights on the pathogenesis of MPNs, represents also an ideal tool for evaluating new therapeutic strategies, not only based on specific inhibitors of JAK-2 but also on drugs that can exert their effect on the malignant stem cell population, independently fro the presence of the JAK-2 mutation(s). (Go to abstract external link, go to editorial external link)

September 2010: Safety and efficacy of JAK1 & JAK2 inhibitor, INCB018424, in myelofibrosis

About 50% of patients with myelofibrosis carry a V617F mutation of the JAK2 gene, but even in cases that lack the mutation an abnormal activation of cell signaling pathways via JAK/STAT has been documented; therefore, drugs that target the JAK/STAT pathway are supposedly active even in JAK2 wild-type patients. In this paper published in the New England Journal of Medicine the results of a phase 1-2 study with a drug inhibitor of JAK1 and JAK2, called INCB018424, in patients with high risk myelofibrosis, both primary and secondary to polycythemia vera or essential thrombocythemia, are reported. The study was conducted in several American centers, coordinated by S. Verstovsek of MD Anderson, Houston. Most of the 153 patients enrolled in the study achieved a rapid improvement of symptoms, and more than half of the cases experienced a reduction of at least 50% of the spleen volume, which resulted in an improvement in fatigue, systemic symptoms, gastrointestinal disorders. It was reassuring that the efficacy was similar in patients without mutation of the JAK2 gene. Improvement of anemia was observed in 14% of cases, but a similar percentage developed drug-related anemia. The treatment was generally well tolerated, with few and low-grade toxicities. Two additional phase 3 protocols (COMFORT 1 and COMFORT 2) are currently ongoing in the United States and several European locations, including Florence, Pavia and Bergamo, which all are part of the AGIMM group. The publication of the study has been accompanied by an editorial, signed by AM Vannucchi, Florence, whose title emphasizes how these results are encouraging and represent a breakthrough in the treatment of myelofibrosis: "From palliation to targeted therapy in myelofibrosis". However, at the same time, it must be underlined that treatment with the JAK1/JAK2 inhibitor did not result in a meaningful reduction of the proportion of mutated cells. Therefore, the search for a therapy capable of "eradicating" the disease is still open. (Go to abstract external link, go to editorial external link)

August 2010: A pilot study of the Histone-Deacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic myeloproliferative neoplasms

Histone deacetylase are proteins that regulate the expression of genes responsible for proliferation, differentiation and cell death, as well as a variety of other cellular functions. Inhibitors of these proteins have been developed and studied for their antitumor activity; among them, Givinostat has antiproliferative and proapoptotic activity against cells of several hematological malignancies. A previous in-vitro study showed a specific activity of this drug against cells bearing the JAK2V617F mutation, a recurrent mutation in myeloproliferative neoplasms. In this paper, the group of Bergamo leaded by Prof. A. Rambaldi reports results of the first trial on patients with polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) positive for the JAK2V617F mutation. Ffity-four percent of patients with PV and ET achieved a clinical response, and 19% of MF patients a major response, according to the European leukemia Net response criteria. In most patients there was the disappearance of pruritus, while reduction of splenomegaly was obtained in 75% of patients with PV or ET and in 38% of those with MF. Side effects reported were mild to moderate and reversible upon discontinuation. These first encouraging results represent the basis for a subsequent clinical trial in patients with PV, which is currently ongoing in some Italian hematologic centres. (Go to abstract external link)

July 2010: Treatment with the Bcl-xL Inhibitor ABT-737 in Combination with Interferon a Specifically Targets JAK2V617F Positive Polycythemia Vera Hematopoietic Progenitor Cells

Recent studies showed that treatment of patients affected by polycythemia vera with interferon alpha is effective in inducing not only clinical-hematological response, but also molecular response, with progressive reduction until disappearance of JAK2V617F mutant allelic burden. Although pegylated interferon formulations could be better tolerated than conventional ones, about one third of patients referred important collateral effects. Because an inhibitor of anti-apoptotic proteins Bcl-2 and Bcl-XL, named ABT-737, has previously been shown to be active against cells of patients with polycythemia vera, in this study researchers from Mount Sinai School of Medicine in New York have analyzed the effects of combining low doses of these drugs. Using in-vitro systems, it was demonstrated that such a combination was as effective as high doses of interferon alone, providing the rational for future use of a combination schedule in clinical trials. (Go to abstract external link)