Decreasing circ_0014614 promotes the differentiation of bone marrow flineage cells into megakaryocytes in essential thrombocythemia via activiation of miR-138-5p/caspase3 axis.

BACKGROUND: Circular RNAs (circRNA) are pivotal in hematological diseases. Previous study showed that circ_0014614 (circDAP3) was significantly underexpressed in bone marrow-derived exosomes from essential thrombocythemia (ET) patients, affecting the differentiation of bone marrow lineage cells into megakaryocytes. METHODS: Fluorescence in situ hybridization (FISH) was used to display circ_0014614's primary cytoplasmic location in K562 cells. Cytoscape software was used to predict the circRNA-miRNA-mRNA networks, and their expression at the cellular level was detected by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was utilized to detect the expression levels of circ_0014614，miR-138-5p and caspase3 mRNA. Western blot was used to determine the protein levels of GATA-1, RUNX-1, NF-E2, CD41 and caspase3. The proliferation of K562 cells was assessed using the Cell Counting Kit-8 (CCK-8) Assay. Furthermore, the interplay between miR-138-5p and circ_0014614 or caspase3 was elucidated through a Dual-luciferase reporter assay. RESULTS: FISH assay indicated circ_0014614's primary cytoplasmic location in K562 cells. In ET bone marrow and K562 cells, circ_0014614 and caspase3 were down-regulated, whereas miR-138-5p saw a significant surge. Overexpressing circ_0014614 curtailed K562 cells' proliferation and differentiation. Further, circ_0014614 targeted miR-138-5p, with heightened miR-138-5p levels counteracting circ_0014614's inhibition. MiR-138-5p further targeted caspase3, and caspase3 silencing neutralized suppressed miR-138-5p's effects on K562 cell differentiation. CONCLUSION: Circ_0014614 was down-regulated in ET bone marrow and bone marrow lineage cells, and upregulating circ_0014614 can inhibit bone marrow lineage cells' proliferation and differentiation into megakaryocytes. Mechanistically, circ_0014614 functioned as ceRNA via sponging miR-138-5p and alleviated the inhibitory effect of miR-138-5p on its target caspase3, which potentially deters tumor activity in ET.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Top advances of the year: Myeloproliferative neoplasms.

The rapid pace of drug development in hematology has led to multiple approvals for myelofibrosis (MF) and polycythemia vera (PV) in recent years. Moreover, there are many innovative agents and combinations being explored for myeloproliferative neoplasms (MPNs). In the past year, there have been several advances in MF, PV, and essential thrombocythemia. In MF, investigational approaches are focusing on strategies to optimize inhibition of signal transduction (including JAK inhibition), modify epigenetics, enhance apoptosis, target DNA replication, transform host immunity, and/or alter the tumor microenvironment. In PV, ropeginterferon alfa-2b has been introduced to the market in the United States, and data continue to accumulate to support the safety and efficacy of this treatment. Hepcidin mimesis is also emerging as a novel way to treat erythrocytosis. In essential thrombocythemia, ropeginterferon alfa-2b is being evaluated, as are therapies to modify epigenetics and inhibit CALR. The enhanced focus on MPNs brings hope that our field can improve morbidity and mortality in this group of diseases.

Unraveling the connection between calreticulin and myeloproliferative neoplasms via calcium signaling.

Mutations in the form of insertions and deletions (INDEL) in the calreticulin gene lead to essential thrombocythemia (ET) which is characterized by the formation of thrombosis. However, the connection between calreticulin INDEL and ET remains largely elusive. Through combined molecular dynamics simulation, clustered regularly interspaced short palindromic repeats (CRISPR) and calcium imaging studies on the wild type and mutated isoforms of calreticulin, the mechanism underlying the calreticulin INDEL induced ET was investigated at the molecular level. Our results demonstrate that mutations in exon-9 could lead to significant conformational variations of calreticulin structure and thereby reducing its interaction with calcium ions due to decreased electrostatic contributions. The consequence of mutations on calreticulin's structural integrity was revealed by identifying the key residues and their roles in calcium binding. Furthermore, mutations implemented by CRISPR-Cas9 in exon-9 showed diminished calcium signaling in HEK-293T cells, which agree well with our in-silico findings. The current study might help in understanding the variations of molecular interactions between calreticulin's exon-9 and calcium ions during physiological and pathological conditions. The results might also provide useful information for designing novel therapeutic approaches targeting ET.

Essential thrombocythemia manifesting as livedoid and purpuric skin lesions: Report of two cases and literature review.

Essential thrombocythemia is a chronic myeloproliferative syndrome which usually runs its course as an asymptomatic elevated platelet count. Cutaneous manifestations secondary to microcirculation abnormalities are rare but can represent a helpful diagnostic clue in order to prevent major thromboembolic events. We report two cases of heterogeneous livedoid and "net-like" skin lesions in the context of essential thrombocythemia with identical histopathologic findings (medium-sized blood vessels with luminal obliteration by eosinophilic material, mostly positive for the platelet marker CD61, without vasculitis). In conclusion, we seek to raise awareness of the clinicopathological features of essential thrombocythemia to allow for prompt diagnosis and treatment.

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.

Essential thrombocythaemia progression to the fibrotic phase is associated with a decrease in JAK2 and PDL1 levels.

It has been postulated that the changes in the molecular characteristics of the malignant clone(s) and the abnormal activation of JAK-STAT signaling are responsible for myeloproliferative neoplasm progression to more advanced disease phases and the immune escape of the malignant clone. The continuous JAK-STAT pathway activation leads to enhanced activity of the promoter of CD274 coding programmed death-1 receptor ligand (PD-L1), increased PD-L1 level, and the immune escape of MPN cells. The aim of study was to evaluate the PDL1 mRNA and JAK2 mRNA level in molecularly defined essential thrombocythaemia (ET) patients (pts) during disease progression to post-ET- myelofibrosis (post-ET-MF). The study group consisted of 162 ET pts, including 30 pts diagnosed with post-ET-MF. The JAK2V617F, CALR, and MPL mutations were found in 59.3%, 19.1%, and 1.2% of pts, respectively. No copy-number alternations of the JAK2, PDL1, and PDCDL1G2 (PDL2) genes were found. The level of PD-L1 was significantly higher in the JAK2V617F than in the JAK2WT, CALR mutation-positive, and triple-negative pts. The PD-L1 mRNA level was weakly correlated with both the JAK2V617F variant allele frequency (VAF), and with the JAK2V617F allele mRNA level. The total JAK2 level in post-ET-MF pts was lower than in ET pts, despite the lack of differences in the JAK2V617F VAF. In addition, the PD-L1 level was lower in post-ET-MF. A detailed analysis has shown that the decrease in JAK2 and PDL1 mRNA levels depended on the bone marrow fibrosis grade. The PDL1 expression showed no differences in relation to the genotype of the JAK2 haplotypeGGCC_46/1, hemoglobin concentration, hematocrit value, leukocyte, and platelet counts. The observed drop of the total JAK2 and PDL1 levels during the ET progression to the post-ET-MF may reflect the changes in the JAK2V617F positive clone proliferative potential and the PD-L1 level-related immunosuppressive effect. The above-mentioned hypothesis is supported by The Cancer Genome Atlas (TCGA) data, confirming a strong positive association between CD274 (encoding PD-L1), CXCR3 (encoding CXCR3), and CSF1 (encoding M-CSF) expression levels, and recently published results documenting a drop in the CXCR3 level and circulating M-CSF in patients with post-ET-MF.

Macrophage frequency in the bone marrow correlates with morphologic subtype of myeloproliferative neoplasm.

Bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF, n = 18), essential thrombocythemia (ET, n = 14), polycythemia vera (PV, n = 12), and Philadelphia chromosome-positive chronic myeloid leukemia (CML, n = 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (p = 0.017) and control BM (p < 0.001). Similar findings were observed by CD163 staining (PMF vs. CML: p = 0.017; PMF vs. control: p < 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (p = 0.009) and reactive cases (p < 0.001). PMF had higher frequencies of macrophages than PV (CD68: p < 0.001; CD163: p < 0.001) and ET (CD68: p < 0.001; CD163: p < 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome-negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.

Heterogeneity of the bone marrow niche in patients with myeloproliferative neoplasms: ActivinA secretion by mesenchymal stromal cells correlates with the degree of marrow fibrosis.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

Clinical and molecular predictors of fibrotic progression in essential thrombocythemia: A multicenter study involving 1607 patients.

The current retrospective study involving a total of 1607 patients was designed to identify clinical and molecular variables that were predictive of inferior myelofibrosis-free survival (MFS) in WHO-defined essential thrombocythemia (ET), utilizing three independent patient cohorts: University of Florence, Italy (n = 718); Mayo Clinic, USA (n = 479) and Policlinico Gemelli, Catholic University, Rome, Italy (n = 410). The Florence patient cohort was first examined to identify independent risk factors for MFS, which included age > 60 years (HR 2.5, 95% CI 1.3-4.9), male sex (2.1, 1.2-3.9), palpable splenomegaly (2.1, 1.2-3.9), CALR 1/1-like or MPL mutation (3.4, 1.9-6.1) and JAK2V617F variant allele frequency > 35% (4.2, 1.6-10.8). Subsequently, an operational molecular risk category was developed and validated in the other two cohorts from Mayo Clinic and Rome: "high molecular risk" category included patients with JAK2V617F VAF >35%, CALR type 1/1-like or MPL mutations; all other driver mutation profiles were assigned to "low molecular risk" category. The former, compared to the latter molecular risk category, displayed significantly higher risk of fibrotic transformation: Florence cohort with respective fibrotic transformation risk rates of 8% vs. 1.2% at 10 years and 33% vs. 8% at 20 years (p < 0.001; HR 6.1; 95% CI 3.2-11.7); Mayo Cohort, 16% vs. 7% at 10 years and 44% vs. 25% at 20 years (p < 0.001; HR 2.5; 95% CI 1.6-4.1); and Rome cohort 7.8% vs. 4.6% at 10 years and 31.2% vs. 7.1% at 20 years (p = 0.007, HR 2.7; 95% CI 1.3-5.8). The present study provides practically useful risk signals for fibrotic transformation in ET and facilitates identification of patients who require close monitoring and appropriate counseling.

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

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

Use of pegylated interferon in young patients with polycythemia vera and essential thrombocythemia.

Myeloproliferative neoplasms (MPN) are rare disorders in young patients, and because of this, standardized treatment recommendations are not available. Pediatric patients are more frequently treated with hydroxyurea than interferon, yet there are no data suggesting this is the best practice. Current treatment guidelines for adults suggest using interferon as upfront therapy in young patients. We reviewed the cases of 13 young patients with polycythemia vera or essential thrombocythemia, who were treated with interferon. Extreme thrombocytosis was well controlled and the medication was tolerated by many. Our work shows the need for prospective studies evaluating interferon in our youngest patients with MPN.

Total Platelet Transcriptomics and Its Network Analysis by RNA-Seq and miRNA-Seq and PCA Application in Essential Thrombocythaemia.

Differentiating the aetiology of thrombocytosis is limited yet crucial in patients with essential thrombocythaemia (ET). MicroRNAs (miRNAs) regulate haematopoiesis and lineage commitment; aberrant expression of miRNAs plays an important role in myeloproliferative neoplasms. However, the miRNA profile has been poorly explored in ET patients compared to patients with reactive thrombocytosis (RT). A total of 9 samples, including 5 ET patient samples, 2 RT patient samples, and 2 healthy control samples, were analysed in this study. We produced 81.43 million reads from transcripts and 59.60 million reads from small RNAs. We generated a comprehensive miRNA-mRNA regulatory network and identified unique 14 miRNA expression patterns associated with ET. Among the 14 miRNAs, miR-1268a was downregulated in ET and showed an inverse correlation with its 8 putative target genes, including genes associated with thrombus formation and platelet activation (CDH6, EHD2, FUT1, KIF26A, LINC00346, PTPRN, SERF1A, and SLC6A9). Principal component analysis (PCA) showed ET and non-ET groups well clustered in space, suggesting each group had a distinctive expression pattern of mRNAs and miRNAs. These results suggest that the significant dysregulation of miR-1268a and its 8 target genes could be a unique expression of platelet mi-RNAs and miRNA/mRNA regulatory network in ET patients.

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

Evaluation of platelet function in essential thrombocythemia under different analytical conditions.

Background. Studies of platelet aggregation (PA) in essential thrombocythemia (ET) reported contrasting results, likely due to differences in analytical conditions.Objective. We investigated platelet aggregation using different techniques and analytical conditions.Patients and Methods. PA was studied by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP) and impedance aggregometry in PRP and whole blood (WB). ADP, collagen, thrombin receptor activating peptide (TRAP-14) and adrenaline were used as agonists. Since ET patients (n = 41) were on treatment with aspirin (100 mg/d), healthy controls (n = 29) were given aspirin (100 mg/d) for 5 days before testing: therefore, thromboxane A2-independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca2+] or lepirudin (L) [physiological Ca2+]; platelet count was adjusted to 250 x 109/L in a set of C-PRP (adjusted C-PRP) and left unmodified in the other samples.Results. Results of PA in 17 ET patients who were poor responders to aspirin (high serum thromboxane B2 levels) were not included in the analysis. With LTA, PA in ET was lower than in controls in adjusted C-PRP and normal in native C-PRP and L-PRP. With impedance aggregometry, PA in L-PRP and L-WB tended to be higher in ET than in controls. Platelet serotonin and ADP contents were reduced in ET. The percentages of circulating platelets expressing P-selectin and platelet-leukocyte hetero-aggregates were higher in ET.Conclusions. Analytical conditions dramatically affect in vitro PA of ET patients, which appears defective under the least physiological conditions and normal/supranormal under conditions that are closer to the physiological.

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.

Ruxolitinib vs best available therapy for ET intolerant or resistant to hydroxycarbamide.

Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis, and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase 2 trial of ruxolitinib (JAK1/2 inhibitor) vs best available therapy (BAT) in ET and polycythemia vera patients resistant or intolerant to HC. Here, findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 and 52 patients randomized to receive ruxolitinib or BAT, respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P = .40). At 2 years, rates of thrombosis, hemorrhage, and transformation were not significantly different; however, some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were 2 complete molecular responses (CMR) and 1 partial molecular response in CALR-positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in 1 CMR patient, presumably because of the emergence of a different clone, raising questions about the relevance of CMR in ET patients. Grade 3 and 4 anemia occurred in 19% and 0% of ruxolitinib vs 0% (both grades) in the BAT arm, and grade 3 and 4 thrombocytopenia in 5.2% and 1.7% of ruxolitinib vs 0% (both grades) of BAT-treated patients. Rates of discontinuation or treatment switching did not differ between the 2 trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET. This trial was registered at www.isrctn.com as #ISRCTN61925716.

JAK2 inhibitors for myeloproliferative neoplasms: what is next?

Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

Emerging treatments for classical myeloproliferative neoplasms.

There has been a major revolution in the management of patients with myeloproliferative neoplasms (MPN), and in particular those with myelofibrosis and extensive splenomegaly and symptomatic burden, after the introduction of the JAK1 and JAK2 inhibitor ruxolitinib. The drug also has been approved as second-line therapy for polycythemia vera (PV). However, the therapeutic armamentarium for MPN is still largely inadequate for coping with patients' major unmet needs, which include normalization of life span (myelofibrosis and some patients with PV), reduction of cardiovascular complications (mainly PV and essential thrombocythemia), prevention of hematological progression, and improved quality of life (all MPN). In fact, none of the available drugs has shown clear evidence of disease-modifying activity, even if some patients treated with interferon and ruxolitinib showed reduction of mutated allele burden, and ruxolitinib might extend survival of patients with higher-risk myelofibrosis. Raised awareness of the molecular abnormalities and cellular pathways involved in the pathogenesis of MPN is facilitating the development of clinical trials with novel target drugs, either alone or in combination with ruxolitinib. Although for most of these molecules a convincing preclinical rationale was provided, the results of early phase 1 and 2 clinical trials have been quite disappointing to date, and toxicities sometimes have been limiting. In this review, we critically illustrate the current landscape of novel therapies that are under evaluation for patients with MPN on the basis of current guidelines, patient risk stratification criteria, and previous experience, looking ahead to the chance of a cure for these disorders.

Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms.

Philadelphia-negative classical myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 revision of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues includes new criteria for the diagnosis of these disorders. Somatic mutations in the 3 driver genes, that is, JAK2, CALR, and MPL, represent major diagnostic criteria in combination with hematologic and morphological abnormalities. PV is characterized by erythrocytosis with suppressed endogenous erythropoietin production, bone marrow panmyelosis, and JAK2 mutation. Thrombocytosis, bone marrow megakaryocytic proliferation, and presence of JAK2, CALR, or MPL mutation are the main diagnostic criteria for ET. PMF is characterized by bone marrow megakaryocytic proliferation, reticulin and/or collagen fibrosis, and presence of JAK2, CALR, or MPL mutation. Prefibrotic myelofibrosis represents an early phase of myelofibrosis, and is characterized by granulocytic/megakaryocytic proliferation and lack of reticulin fibrosis in the bone marrow. The genomic landscape of MPNs is more complex than initially thought and involves several mutant genes beyond the 3 drivers. Comutated, myeloid tumor-suppressor genes contribute to phenotypic variability, phenotypic shifts, and progression to more aggressive disorders. Patients with myeloid neoplasms are at variable risk of vascular complications, including arterial or venous thrombosis and bleeding. Current prognostic models are mainly based on clinical and hematologic parameters, but innovative models that include genetic data are being developed for both clinical and trial settings. In perspective, molecular profiling of MPNs might also allow for accurate evaluation and monitoring of response to innovative drugs that target the mutant clone.