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.

Colony-Forming Cell Assay Detecting the Co-Expression of JAK2V617F and BCR-ABL1 in the Same Clone: A Case Report.

BCR-ABL1-negative myeloproliferative disorders and chronic myeloid leukaemia are haematologic malignancies characterised by single and mutually exclusive genetic alterations. Nevertheless, several patients co-expressing the JAK2V617F mutation and the BCR-ABL1 transcript have been described in the literature. We report the case of a 61-year-old male who presented with an essential thrombocythaemia phenotype and had a subsequent diagnosis of chronic phase chronic myeloid leukaemia. Colony-forming assays demonstrated the coexistence of 2 different haematopoietic clones: one was positive for the JAK2V617F mutation and the other co-expressed both JAK2V617F and the BCR-ABL1 fusion gene. No colonies displayed the BCR-ABL1 transcript alone. These findings indicate that the JAK2V617F mutation was the founding genetic alteration of the disease, followed by the acquisition of the BCR-ABL1 chimeric oncogene. Our data support the hypothesis that a heterozygous JAK2V617F clone may have favoured the bi-clonal nature of this myeloproliferative disorder, generating clones harbouring a second transforming genetic event.

Comparison of JAK2V617F -positive essential thrombocythaemia and early primary myelofibrosis: The impact of mutation burden and histology.

An accurate histological diagnosis may distinguish essential thrombocythaemia (ET) from early primary myelofibrosis (early-PMF), which is associated with worse outcome. Outcome of ET is also negatively affected by the presence of the JAK2V617F mutation. To investigate the impact of JAK2V617F mutation burden and histology on outcome, we collected 475 WHO-diagnosed ET (69.2%) or early-PMF JAK2V617F -positive patients followed in 4 Italian haematology centers. JAK2V617F allele burden was ≤50% in 90% and 87% of ET and early-PMF patients, respectively (P = .34). During follow-up, 32 (9.7%) ET and 18 (12.3%) early-PMF patients experienced 59 thrombotic events, and 27 patients (5.6%) and 6 (1.2%) patients evolved to myelofibrosis and acute leukemia, respectively. At last contact, 28 (5.8%) patients had died. In early-PMF compared to ET, the 10-year mortality rates (6.7% and 4.3%, P = .73), leukemic transformation rates (1.4% and 1.2%, P = .45), and thrombosis rates (16.7% and 12.2%, P = .12) were comparable. Only progression to overt myelofibrosis at 10 years was significantly worse (11.4% and 1.5%, P = .004). In multivariate analysis, a higher (>50%) JAK2V617F burden was significantly correlated with fibrotic progression and histology. Considering JAK2V617F -positive disease, a higher (>50%) JAK2V617F burden and histological classification are independent prognostic risk factors for disease progression. These findings reinforce the need for standardized detection of this mutation.

The prognostic relevance of serum lactate dehydrogenase and mild bone marrow reticulin fibrosis in essential thrombocythemia.

The 2016 World Health Organization (WHO) diagnostic criteria for myeloproliferative neoplasms (MPN) underscore the prognostically-relevant distinction between essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF). In addition, leukocytosis has been identified as an important prognostic marker in otherwise WHO-defined ET. However, controversy remains regarding the objectivity of morphologic criteria in distinguishing ET from pre-PMF and the precise prognostic cutoff values for leukocytosis. Serum lactate dehydrogenase (LDH) level might be a biologically more accurate measure of leukocyte turnover and a more sensitive marker of pre-PMF, in otherwise WHO-defined ET. In the current study of 183 consecutive patients with WHO-defined ET, the presence of grade 1 bone marrow (BM) fibrosis did not affect presenting clinical or laboratory features; in contrast, increased serum LDH at diagnosis was associated with leukocytosis (p = .002), thrombocytosis (p < .001), palpable splenomegaly (p = .03) and higher international prognostic score (IPSET) (p = .002); serum LDH did not correlate with BM fibrosis, JAK2/CALR/MPL or TET2/ASXL1 mutations. In univariate analysis, risk factors for survival included age ≥60 years (p = .002; HR 10.2, 95% CI 2.3-44.6), male sex (p = .02; HR 3.2, 95% CI 1.2-8.2), leukocyte count ≥15 × 109 /L (p = .007; HR 4.7, 95% CI 1.5-14.6), and increased serum LDH (p = .002; HR 3.7, 95% CI 1.5-9.1), but not BM fibrosis (p = .17). In multivariable analysis, age, sex and serum LDH remained significant; serum LDH also remained significant, in the context of IPSET (p = .003) and in patients with leukocytosis (p = .003). We conclude that serum LDH level carries an independent prognostic value for survival in ET and might represent a biologically more accurate surrogate for leukocytosis.

Refractory Arterial Insufficiency Associated with Janus Kinase 2 Positive Essential Thrombocythaemia.

Essential thrombocythaemia (ET) is one of the severe rare clonal haematologic stem cell disorders that encompass myeloproliferative neoplasms. ET has a well-described association with peripheral arterial thrombosis, which presents a challenging clinical presentation. Further understanding into the underlying pathophysiology of thrombosis in ET has been made following the identification of the Janus Kinase 2 (JAK2) mutation, which is thought to confer a prothrombotic phenotype. Here we present a case of refractory arterial insufficiency associated with JAK2-positive ET.

JAK2V617F leads to intrinsic changes in platelet formation and reactivity in a knock-in mouse model of essential thrombocythemia.

The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.

JAK2 p.V617F detection and allele burden measurement in peripheral blood and bone marrow aspirates in patients with myeloproliferative neoplasms.

Detection of the JAK2 p.V617F mutation and measurement of its allele burden can be performed using both peripheral blood (PB) and bone marrow (BM) samples from patients with myeloproliferative neoplasms (MPNs). However, the diagnostic accuracy of detecting the JAK2 p.V617F mutation and quantifying its allele burden in PB and BM samples has not been systematically compared. We retrospectively analyzed 388 patients with MPN who had been tested for JAK2 p.V617F allele burden using both PB and BM samples within 3 months of each other. The sensitivity and specificity of detecting JAK2 p.V617F in PB when compared with BM were both 100%. Furthermore, the JAK2 p.V617F allele burden measured in PB and BM were equivalent by linear regression analysis (R(2) = 0.991; P < .0001). We therefore conclude that PB is a reliable source for testing for the JAK2 p.V617F mutation and quantifying its allele burden in patients with MPN.

Open-label study of oral CEP-701 (lestaurtinib) in patients with polycythaemia vera or essential thrombocythaemia with JAK2-V617F mutation.

JAK2-V617F is central to the pathogenesis of myeloproliferative neoplasms. We examined whether lestaurtinib decreased JAK2-V617F allele burden and evaluated its clinical benefits and tolerability in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET). This phase 2, open-label, multicentre study was designed to detect ≥15% reduction in JAK2-V617F allele burden in 15% of patients. Eligible patients received lestaurtinib 80 mg twice daily for 18 weeks and could participate in a 1-year extension phase of treatment. Of 39 enrolled patients, 27 (69%) had PV; 12 (31%) had ET. While the pre-specified responder rate of 15% was not met, lestaurtinib modestly reduced JAK2-V617F allele burden and reduced spleen size in a subset of patients. Of 37 patients in the full efficacy analysis, 5 (14%) responded clinically. Every patient had ≥1 adverse event, most commonly gastrointestinal (95%). Fifteen patients (38%) experienced serious adverse events; 23 (59%) withdrew due to adverse events. This is the first reported study of JAK2-inhibitor treatment in patients with PV/ET and highlights both the need for further studies to assess the role of JAK2 inhibition in treatment of PV/ET and the use of JAK2-V617F as a biomarker for response. This trial was registered at www.clinicaltrials.gov as NCT00586651.

Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients.

In patients not meeting the required hematocrit (HCT) or hemoglobin (Hb) thresholds according to BCSH and the WHO diagnostic criteria, the diagnosis of masked polycythemia vera (mPV) has been proposed. A comparison of HCT or Hb values with the expression of JAK2V617F, JAK2 exon 12, and CALR mutations in strictly WHO-defined 257 overt PV and 140 mPV (59 mPV according to BCSH) and 397 patients with essential thrombocythemia (ET) was performed. Hb and HCT thresholds of mPV patients were significantly higher than JAK2V617F ET (P < 0.0001). The best cut-off for Hb to discriminate JAK2-mutated ET from PV was 16.5 g/dL for males and 16.0 g/dL for females. For HCT, this was 49% in males and 48% in females. The proportion of patients correctly classified as ET or PV when regarding Hb or HCT levels was 95% in males and 93% in females and 94% in both males and females, respectively.

JAK2V617F monitoring in polycythemia vera and essential thrombocythemia: clinical usefulness for predicting myelofibrotic transformation and thrombotic events.

The JAK2V617F allele burden has been identified as a risk factor for vascular events and myelofibrotic transformation in polycythemia vera (PV) and essential thrombocythemia (ET). However, all previous studies have evaluated a single time point JAK2V617F measurement. Therefore, the frequency and the clinical significance of changes in the JAK2V617F mutant load occurring during the disease evolution remain unknown. In the present study, JAK2V617F monitoring was performed during the follow-up of 347 patients (PV = 163, ET = 184). According to their JAK2V617F evolutionary patterns, patients were stratified as stable < 50% (n = 261), stable ≥50% (n = 52), progressive increase (n = 24) and unexplained decrease (n = 10). After a 2,453 person-years follow-up, a total of 59 thrombotic events, 16 major hemorrhages, and 27 cases of myelofibrotic transformations were registered. At multivariate analyses, patients with a persistently high (≥50%) or unsteady JAK2V617F load during follow-up had an increased risk of myelofibrotic transformation (Incidence rate ratio [IRR]: 20.7, 95% CI: 6.5-65.4; P < 0.001) and a trend for a higher incidence of thrombosis (IRR: 1.7, 1-3.3; P = 0.05) than patients with a stable allele burden below 50%. In conclusion, JAK2V617F monitoring could be useful in patients with PV and ET for predicting disease's complications, especially myelofibrotic transformation.

Inhibition of related JAK/STAT pathways with molecular targeted drugs shows strong synergy with ruxolitinib in chronic myeloproliferative neoplasm.

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia-negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F-positive cell lines, HEL and Ba/F3 (JAK2V617F EPOR) , and in primary mononuclear and bone marrow CD34-positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50 )(PV)  = 15 nmol/l], as well as sorafenib (IC50 PV=8µmol/l), KNK437 (IC50 PV=100µmol/l ), and perifosine (IC50 PV=15µmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)(PV)  < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2-related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.

JAK2V617F mutation and hydroxyurea treatment as determinants of immature platelet parameters in essential thrombocythemia and polycythemia vera patients.

Immature platelets (IPFs), which are hemostatically more active than mature platelets, have been found elevated in essential thrombocythemia and polycythemia vera, 2 myeloproliferative neoplasms (MPN) characterized by an increased risk of thrombosis. It is not known whether the IPF levels are influenced by pathogenetic factors, including JAK2V617F mutational status, or by treatment regimen. To address this point, in 46 essential thrombocythemia and 38 polycythemia vera consecutive patients, we measured IPF and correlated the results to JAK2V617F mutation and myelosuppressive treatment with hydroxyurea. This analysis provides 2 new elements regarding IPF and MPN. The first finding is that the JAK2V617F mutation is linked to the quantity of IPF in patients with MPN, which might contribute to the prothrombotic phenotype in these patients. The second finding is that IPF is susceptible to myelosuppressive treatment, which may additionally explain the favorable effect of hydroxyurea therapy on MPN outcome as well as the associated thrombotic risk.

Increased basal intracellular signaling patterns do not correlate with JAK2 genotype in human myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are associated with recurrent activating mutations of signaling proteins such as Janus kinase 2 (JAK2). However, the actual downstream signaling events and how these alter myeloid homeostasis are poorly understood. We developed an assay to measure basal levels of phosphorylated signaling intermediates by flow cytometry during myeloid differentiation in MPN patients. Our study provides the first systematic demonstration of specific signaling events and their comparison with disease phenotype and JAK2 mutation status. We demonstrate increased basal signaling in MPN patients, which occurs in both early and later stages of myeloid differentiation. In addition, the pattern of signaling is not correlated with JAK2 mutation status and signaling intensity is poorly correlated with mutant JAK2 allele burden. In contrast, signaling differences are detected between different MPN disease phenotypes. Finally, we demonstrate that signaling can be inhibited by a JAK2-selective small molecule, but that this inhibition is not JAK2 V617F specific, because MPN patients with mutant JAK2, wild-type JAK2, and control patients were inhibited to a similar degree. Our data suggest that, in addition to JAK2 mutations, other factors contribute significantly to the MPN phenotype, results that are relevant to both the pathogenesis and therapy of MPN.

Aberrant expression of signaling proteins in essential thrombocythemia.

Dysregulated expression of signaling proteins may contribute to the pathophysiology of essential thrombocythemia (ET). This study aimed to characterize protein expression in ET and to correlate the dysregulated proteins with phenotypes and prognosis of ET patients. The expression of 128 proteins in peripheral blood neutrophils from 74 ET patients was assessed and compared with those from 29 healthy subjects and 35 polycythemia vera (PV) patients using protein pathway array. Fifteen proteins were differentially expressed between ET patients and normal controls. These dysregulated proteins were involved in the signaling pathways related with apoptosis and inflammation. Our results showed a significant overlap in protein expression between ET patients with JAK2V617F mutation and PV patients. In addition, nine proteins were associated with JAK2V617F mutation status in ET patients. Furthermore, estrogen receptor beta (ERß) and Stat3 were independent risk factors for subsequent thrombosis during follow-up on multivariable analysis. Our study shows a broad dysregulation of signaling protein in ET patients, suggesting their roles in ET pathogenesis. The expression levels of ERß and Stat3 could be promising predictors of subsequent thrombosis in ET patients.

The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Myeloproliferative neoplasm induced by constitutive expression of JAK2V617F in knock-in mice.

The Jak2(V617F) mutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2(V617F) in knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2(V617F) in mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.

Blood tests may predict early primary myelofibrosis in patients presenting with essential thrombocythemia.

According to World Health Organization (WHO)-defined criteria, patients presenting clinically as essential thrombocythemia (ET) may show early primary myelofibrosis (PMF) with accompanying thrombocythemia [1]. Previous clinicopathological studies revealed that laboratory parameters like gender-matched hemoglobin (Hb), white blood cell (WBC) count, and particularly lactate dehydrogenase (LDH) values are significantly different in PMF [2]. By strictly applying the WHO criteria, our investigation was aimed to study sensitivity and specificity of these features in an exploratory cohort of 536 patients and to validate the results on an independently recruited series of 321 strictly corresponding patients. The discriminatory power of these parameters (Hb, WBC, and LDH) was tested by plotting their receiver operating characteristic curves. The best performance was found for LDH (areas under the curve, AUC 5 0.7059). WBC and Hb had superimposable curves, with AUC of 0.6279 and 0.6257, respectively. A diagnostic algorithm was generated by applying these parameters in a stepwise fashion. Nearly half of the patients could be correctly allocated to WHO-defined ET or early PMF in both cohorts investigated. It is important to note that this result does not substitute bone marrow morphology with hematological parameters, however, in clinical practice may alert physicians to get more suspicious of early PMF in a patient presumably presenting with ET.

JAK2 and genomic instability in the myeloproliferative neoplasms: a case of the chicken or the egg?

The myeloproliferative neoplasms (MPNs) are a particularly useful model for studying mutation accumulation in neoplastic cells, and the mechanisms underlying their acquisition. This review summarizes our current understanding of the molecular defects present in patients with an MPN, and the effects of mutations targeting Janus kinase 2 (JAK2)-mediated intracellular signaling on DNA damage and on the elimination of mutation-bearing cells by programmed cell death. Moreover, we discuss findings that suggest that the acquisition of disease-initiating mutations in hematopoietic stem cells of some MPN patients may be the consequence of an inherent genomic instability that was not previously appreciated.