Philadelphia Chromosome-negative Myeloproliferative Disorders Research Articles

Higher tumor mutational burden and PD-L1 expression correlate with shorter survival in hematologic malignancies.

The prognostic implications of tumor mutational burden (TMB) and programmed death ligand 1 (PD-L1) expression are poorly studied in hematologic malignancies. This study aimed to better understand the characteristics and prognostic value of TMB and PD-1/PD-L1 in hematologic malignancies. This real-world study was conducted among patients with hematologic malignancies who had next-generation sequencing (NGS) (Foundation Medicine) at the University of California San Diego Moores Cancer Center (2014-2018). TMB was measured by NGS. PD-L1 expression (tumor proportion score, TPS) was measured by immunohistochemistry (classified as high (⩾50%), low (1-49%), and negative (<1%)). Data was curated from the electronic medical records. In 388 evaluable patients, the most common diagnoses were B-cell non-Hodgkin lymphoma (NHL) (35%) and Philadelphia chromosome-negative myeloproliferative disorders (16%). Median TMB was 1.6 mutations/Mb (range, 0-46.83). Forty-eight patients (12%) had TMB ⩾10 mutations/Mb, 90% of which were B-cell or T-cell NHL. In 85 samples with available PD-L1 scores, 11 were high; 26, low; and 48, no tumor cell expression. PD-L1 TPS positive (⩾1%) was most common in T-cell NHL (7/9 (77%) cases) followed by B-cell NHL (21/51 (41%) cases). TMB ⩾4 mutations/Mb and PD-L1 score ⩾1% were significantly associated with shorter overall survival (OS) from diagnosis, with hazard ratio (HR) = 1.46 (p = 0.02, 95% confidence interval (CI) 1.05-2.03) and HR = 2.11 (p = 0.04, 95% CI 1.04-4.30), respectively; the relationship was more pronounced when PD-L1 ⩾50% versus <50% was used (HR = 2.80, p = 0.02, 95% CI 1.19-6.59). Higher TMB and higher PD-L1 positivity correlation were significant but weak (Pearson correlation coefficient R 2 = 0.04, p = 0.04). TMB ⩾4 mutations/Mb and positive PD-L1 TPS are poor prognostic factors, correlating with shorter OS across hematologic malignancies. ClinicalTrials.gov NCT02478931.

Covert Brain Infarcts in Patients with Philadelphia Chromosome-Negative Myeloproliferative Disorders.

Backgrounds and Purpose. Philadelphia chromosome-negative myeloproliferative disorders (Ph-negative MPD) are a rare group of hematological diseases, including three distinct pathologies: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). They most often manifest with thrombotic complications, including cerebrovascular events. Covert brain infarcts (CBIs) are defin ed as predominantly small ischemic cerebral lesions that are detected using magnetic resonance imaging (MRI) in the absence of clinical stroke events. The relationship between MPD and CBIs remains unclear. Methods. Included in the study were 103 patients with the diagnosis of Ph-MPD (according to WHO 2016 criteria) (median age—47 (35; 54) years; 67% female). In total, 38 patients had ET, 42 had PV, and 23 had PMF. They underwent clinical examination, routine laboratory analyses (complete blood count), brain MRI, ultrasound carotid artery, flow-mediated dilatation (as a measure of endothelial dysfunction—FMD). Results. Overall, 23 patients experienced an ischemic stroke (as per MRI and/or clinical history), of which 16 (15.5%) could be classified as CBIs. The rate of CBIs per MPD subtype was statistically non-significant between groups (p = 0.35): ET–13.2%, PV–21.4%, and PMF–8.7%. The major vascular risk factors, including arterial hypertension, carotid atherosclerosis, and prior venous thrombosis, were not associated with CBIs (p > 0.05). Age was significantly higher in patients with CBIs compared to patients without MRI ischemic lesions: 50 (43; 57) years vs. 36 (29; 48) (p = 0.002). The frequency of headaches was comparable between the two groups. CBIs were associated with endothelial dysfunction (OR - 0.71 (95% CI: 0.49–0.90; p = 0.02)) and higher hemoglobin levels (OR—1.21 (95% CI: 1.06–1.55); p =0.03). Conclusions. CBIs are common in patients with Ph-negative MPD. Arterial hypertension and carotid atherosclerosis were not associated with CBIs in this group of patients. The most significant factors in the development of CBIs were endothelial dysfunction (as measured by FMD) and high hemoglobin levels. Patients with Ph-negative MPD and CBIs were older and had more prevalent endothelial dysfunction.

Clinicopathologic Characteristic and Natural History of Patients with Non Hereditary, Non Polycythemia Vera (NH/NPV) Persistent Polycythemia

Introduction;Polycythemia Vera (PV), is one of the most common Philadelphia chromosome negative myeloproliferative disorders. The identification of Janus Kinase (JAK) 2 mutations and the publication of 2016 revision of WHO classification of myeloid neoplasms has improved diagnostic accuracy of PV. The lower cut-off values of Hemoglobin (Hb) and hematocrit (Hct) used in WHO diagnostic criteria, however, resulted in increased number of adult patients undergoing further evaluation for PV leading to identification of patients with acquired, idiopathic Persistent polycythemia (PP) who not meet WHO criteria for PV despite extensive work up. The clinicopathologic features, disease course, treatment and outcomes of these patients has not been well characterize. This study seeks to described clinicopathologic and molecular profile of cohort of adult patients with Non Hereditary, Non-Polycythemia Vera (NHNPV) Persistent Polycythemia.Methodology;Patients with PP diagnosed and followed up in Fresno community regional medical center/University of San Francisco (UCSF) Fresno, from January 2010 to December 2020 were reviewed. Patients who didn't meet WHO diagnostic criteria for PV after full evaluation including bone marrow biopsies were included in the study. Those with incomplete evaluation, clearly attributable secondary causes of polycythemia or erythropoietin (EPO) level above institutional laboratory reference range (2-18), were excluded from the study. Patients with polycythemia diagnosed at pediatric age were also excluded. The study was approved by institutional review board of UCSF Fresno.Results;129 subjects with JAK 2 Negative PP were reviewed of which 63 had EPO levels within or below the normal range (2-18).Of 36 patients who met the study inclusion and exclusion criteria, majority were males (70% N= 28) and non-Hispanic (56% N=20) with median age at diagnosis of 48.5 years (range 18-70 years). Half of the patients (N=18) have no identifiable etiology of PP. In patients with associated conditions, the most common is non-oxygen requiring clinical OSA (36%) and tobacco smoking (19.4%). Of note, all these patients have EPO level less than 10 which represents the median value of the local institutional defined normal range for EPO. Four (11%) patients have non-iron overloaded hemochromatosis gene mutation (1 Homozygous H63D HFE gene, 2 heterozygous mutation and one with unspecified mutation type).Average Hb and Hct at diagnosis was (17.26+/- 2.23) and (49.76+/- 6.34) respectively. Mean EPO level 7.05+/-2.75 with less than third of patients 8 (22%) with level less or equal to in 5 units. There is no statistically significance difference between mean diagnostic EPO level between males and females in the study (7.20+/-2.89 vs.6.51+/-2.26) and so is the Hct though males have numerically higher levels.The most common bone marrow morphology includes normocellularity (51.76+/-10.36) with erythroid hyperplasia (25%) or megakaryocyte hyperplasia without dysplasia (17%) and grade 1/3 fibrosis 6/36.Abnormal mutations were identified in 17 patients whose bone marrow biopsies were evaluated with limited or extensive next generation sequencing (NGS). Of those patients with available results, 6 patients (35% out of those with NGS) have identifiable previously well described genetic mutations {FANCA S1311fs*1, HGF R134H, TP53 splice site 97-2A>G, KMT2A (Gain), MLL3 (KMT2C) and AFF4, ASXL1 c.1934dupG/TrpfsX12 (p. Gly646)}.9 patients had variance of unknown significant (VUS) mutations (Table 1) with average of 6.2 mutation per patient. Only 1 VUS (TSC1 K587R) was reported in 2 different patients (#7, #9)Mild Myeloproliferative neoplasia (MPN) associated symptoms was reported in 22% of patient with Intermittent phlebotomy (72% N= 26) and aspirin (75% N=27) was the treatment for majority of patients. One patient died during the follow up period. No patient progressed to other MPN, myelofibrosis or AML during the study period.Conclusion;With improved diagnostic criteria and accuracy of PV, increased number of patients are diagnosed with NHNPV Persistent polycythemia. None of the mutation or VUS identified from extensive evaluation is recurrent in our cohort of patient. Longer follow up is needed to delineate clinical significance and prognosis of these mutation in patients with NHNPV persistent Polycythemia. [Display omitted] DisclosuresAbdulhaq: BMS, Alexion, Oncopeptides, Morphosys, Pfizer, Norvartis: Honoraria; Oncopeptides, Alexion, Amgen: Speakers Bureau; Morphosys, BMS, Amgen: Membership on an entity's Board of Directors or advisory committees.

Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML), also known as chronic myelogenous leukemia, chronic myelocytic leukemia, and chronic granulocytic leukemia, is characterized by the expansion of myeloid progenitor cells at various stages of maturation, their premature release into the circulation, and a tendency to home to extramedullary sites. Symptoms at presentation reflect the increase in mass and turnover of the leukemic cells, although as many as 50% of patients are asymptomatic at diagnosis and come to attention through unexpected findings on routine blood tests. In treatment, the ongoing development of established and novel tyrosine kinase inhibitors (TKIs) has made for closer to normal life spans in patients diagnosed with CML. This review serves as an overview of CML, detailing its epidemiology and etiology, pathophysiology, diagnosis, treatment (including an assessment of the latest clinical trials involving TKIs), and management of patients with advanced phases. Figures show BCR-ABL signaling pathways and mechanisms of resistance to imatinib. Tables list stages of CML, differential diagnosis of CML and Philadelphia chromosome–negative myeloproliferative disorders, a summary of pivotal phase III trials of approved TKIs for the treatment of front-line or relapsed CML, response evaluation to TKIs used as first-line therapy, and a summary of important phase II trials of second- and third-generation TKIs after previous TKI failure. This review contains 2 highly rendered figures, 5 tables, and 87 references.

Budd–Chiari syndrome in very young adult patients with polycythemia vera

Polycythemia vera is a Philadelphia chromosome-negative myeloproliferative disorder with incidence of 1% under the age of 25. The Budd-Chiari syndrome (BCS) is a well known complication of polycythemia vera even in children, and characterized by occlusion of hepatic outflow. A computerized archive search of medical records at Sheba Medical Center of the past three decades of patients with polycythemia vera and BCS under the age of 25 years was performed. A work-up for JAK2 V617F mutation and thrombophilia was done. Medical charts and imaging tests were carefully reviewed. Three patients under the age of 22 were finally recruited. Two of those were found in life-threatening condition and improved clinically following treatment with bivalirudin, a direct thrombin inhibitor. It is conceivable that bivalirudin contributed to a favorable outcome of those patients in comparison to historical outcome previously reported. In conclusion, polycythemia vera in the young is not a mild disease since BCS, which is one of its complication, can be fatal even in those age group unrelated to the presence of hereditary thrombophilia. Once BCS occurs, we would suggest giving a trial with bivalirudin before an invasive procedure is planned.

Leukaemic transformation of Philadelphia chromosome‐negative myeloproliferative neoplasms: are Asian patients different?

Leukaemic transformation (LT) is rare in the natural history of Philadelphia(Ph) chromosome-negative myeloproliferative disorders (MPD), and has a dismal prognosis. Little literature is available on Asian patients. The aim of this study is to report a single institution experience of Asian patients who developed acute leukaemia after being diagnosed and treated for Ph chromosome-negative MPDs, and to compare the findings of this series with similar studies from the literature. Patients were recruited from the MPD registry of Singapore General Hospital, Department of Hematology. Clinical data including treatment modalities and duration of use in myeloproliferative phase, latency to LT, characteristics of leukaemia, chemotherapy administered and survival after LT were examined. Over a 29-year period from 1980 to 2009, there were 22 Asian patients with LT of Ph chromosome-negative MPD of which four had polycythaemia vera (PV), nine had essential thrombocythaemia (ET), seven had myelofibrosis (MF) and two had unspecified MPD at diagnosis. Primary treatment modality was Hydroxyurea (HU) during MPD phase. Median latency to LT was 14 years for PV, 10 years for ET and 1 year for MF. Median age at LT diagnosis was 67.5 years. Nine patients had complex cytogenetics, with abnormalities of chromosomes 5 and 7 being common. Overall, median survival was 2 months after LT. Eight patients who received induction chemotherapy had a median survival of 2.5 months. Survival was independent of MPD type and treatment administered. None received stem cell transplantation. LT of Ph chromosome-negative MPD is rare and uniformly fatal. Despite chemotherapy, survival was poor, and patients succumbed to refractory disease and infections. Asian patients did not have a more favourable outcome. It remains to be investigated whether upfront stem cell transplant may be a treatment option.

JAK2 kinase inhibitors and myeloproliferative disorders

The pathophysiology of Philadelphia-chromosome negative myeloproliferative disorders has significantly advanced with the discovery of JAK2V617F. The prevalence of JAK2V617F mutation has made it a much anticipated target for inhibition; this review will update and assess progress. Many agents have been studied in preclinical trials, of which few have entered clinical trials. Data from the clinical trials are limited and mostly in the form of abstracts and reviews. The prevalence of the JAK2V617F mutation in the classic Philadelphia-chromosome negative myeloproliferative disorders has made it a much anticipated target for inhibition. Present in greater than 90% of patients with polycythemia vera and approximately 50% of patients with essential thrombocythemia and primary myelofibrosis, it has been hoped that targeted inhibition of JAK2V617F would achieve similar disease control as imatinib mesylate has produced in chronic myeloid leukemia. However, JAK2V617F in the Philadelphia-chromosome negative myeloproliferative disorders, unlike bcr/abl tyrosine kinase in chronic myeloid leukemia, is not a causative but rather a secondary somatic mutation. As the JAK2 inhibitors move into phase III clinical trials, their efficacy and role in therapy is becoming clearer; however, there are still many questions needing answers.

Molecular markers guide diagnosis and treatment in Philadelphia chromosome-negative myeloproliferative disorders (Review)

The Philadelphia negative chronic myeloproliferative neoplasms are hematological disorders with several diagnostic challenges. Due to recent molecular findings, the WHO classification of Tumors of Hematopoietic and Lymphoid Tissue 2008 reorganized the field of chronic myeloproliferative diseases. Thus, specific molecular markers provide important information for current diagnostic strategies. This review highlights the important diagnostic tools in classical and atypical myeloproliferative neoplasms mainly the JAK2V617F mutation, the Mpl receptor, Polycythemia rubra vera 1 (PRV1), platelet-derived growth-factor receptor alpha (PDGFRA), platelet-derived growth-factor receptor beta (PDGFRB), fibroblast growth-factor receptor 1 (FGFR1) and c-kit tyrosine kinase. A description of the origin, clinical correlations and role in diagnosis and therapy is provided for each of these molecular markers.

Pivotal role of mast cells in pruritogenesis in patients with myeloproliferative disorders

AbstractPruritus is a common symptom in patients with Philadelphia chromosome–negative myeloproliferative disorders (MPDs). The pathophysiology of MPD-associated pruritus is unclear. We have demonstrated that MPD mast cells (MCs) are involved by the malignant process. In the present study, we explored the hypothesis that MCs play an important role in the development of pruritogenesis in MPDs. We found that MPD MCs released significantly greater amounts of pruritogenic factors, including histamine, leukotrienes, and interleukin-31 (IL-31) than normal MCs. Elevated levels of IL-31 were also observed in MPD CD3+ cell-conditioned media. MPD MCs exhibited increased migratory behavior in response to stem cell factor or interleukin-8, which was associated with increased filamentous-actin content. Furthermore, the presence of pruritus in MPDs was statistically correlated with a greater number of MCs being generated by CD34+ cells, a greater number of MC colonies being formed by CD34+ cells, decreased apoptosis and prostaglandin D2 release by cultured MCs, and higher plasma levels of IL-31. These data demonstrate that functional abnormalities of MPD MCs probably lead to pruritogenesis in patients with MPDs. These studies provide cellular and molecular targets for the development of antipruritus drugs for patients with MPDs.

The impact of JAK2 non-receptor tyrosine kinase mutation on the mobilization of hematopoietic stem cells into peripheral blood of patients with Philadelphia chromosome-negative myeloproliferative disorders

The impact of JAK2 non-receptor tyrosine kinase mutation on the mobilization of hematopoietic stem cells into peripheral blood of patients with Philadelphia chromosome-negative myeloproliferative disorders

Preclinical Characterization of the JAK-2 Inhibitor, SGI-1252

Discovery of somatic mutation of JAK-2 (G1849T that produces JAK-2V617F) in the hematopoietic cells of patients with Philadelphia chromosome negative myeloproliferative disorders (Ph−MPDs) was a watershed event that not only provided new insights into the pathobiology of polycythemia vera, essential thrombocytosis and primary myelofibrosis but also identified a potential target for therapy. Herein we report the results of preclinical studies designed to characterize the activity of a novel inhibitor of JAK-2. The compound, SGI-1252, developed by SuperGen (Dublin, CA) incorporates with high affinity into the ATP-binding site of JAK-2. SGI-1252 was tested against a panel of 75 kinases and was found to have significant activity against only FLT-3, TYK-2 and the SRC family members, ABL, LCK, YES, in addition to JAK-2 and JAK-1. SGI-1252 has an IC50 for JAK-2 of 5.4 nM with an IC50 for JAK-2V617F of 19.7 nM. The inhibitor also effectively blocks the activity of JAK-1 (IC50 14.8 nM) but has little JAK-3 inhibitory activity (IC50 1,700 nM). SGI-1252 is a potent inhibitor of STAT-5 phosphorylation (EC50 76.2 nM) and was also found to block the JAK-2 dependent expression of the anti-apoptotic protein, BCL-XL (EC50 778 nM). Drug treatment of a murine cell line (FDCP) transfected with either human wild-type JAK-2 or JAK-2G1849Tgenerated IC50 values of 83 nM and 108 nM, respectively, and SGI-1252 treatment of human cell lines, HEL, UKE-1 and SET-2, that express mutant JAK2 in different copy numbers, gave IC50 values of 472 nM, 83 nM and 63 nM, repectively. When tested in ex-vivo expanded native human erythroid progenitor cells from 17 patients with Ph−MPDs (10 PV and 7 MF), SGI-1252 showed an IC50 of ~100 nM, regardless of the JAK-2V617F allele burden. Using a flow cytometric assay, SGI-1252 was shown to induce apoptotic cell death in a concentration dependent manner. Luminex technology allows for concurrent quantitative analysis of multiple proteins from the same tissue source, and this technology was used to investigate simultaneously the effects of SGI-1252 on total and phospho ERK1/2, total and phospho STAT3, phospho STAT5, caspase 3, cleaved PARP and GAPDH (control) in untreated and drug treated cells at IC50 and IC80 concentrations. Significant in vivo efficacy of SGI-1252 was also observed using HEL and MV-4-11 xenograft models when compared to treatment with vehicle or daunorubicin. Using a murine model, we found that SGI-1252 has high oral bioavailability and is well tolerated with a five-day repeat maximum dose of at least 900 mg/kg. Together, these studies demonstrate that SGI-1252 is a potent inhibitor of JAK-2 dependent proliferation in both JAK-2V617F positive cell lines and in ex vivo expanded erythroid progenitors derived from patients with JAK-2V617F positive Ph−MPDs. Moreover, our studies show that the effects of SGI-1252 are mediated by blocking both JAK-2 dependent anti-apoptoic pathways and JAK-2 dependent proliferative pathways. Using the orally available form of the compound, pharmacokinetic, pharmacodynamic and toxicity studies in mice suggest that serum concentration of the drug well above the predicted therapeutic range can be achieved without significant hematological toxicity. Based on these preclinical experiments, SGI-1252 appears to be an excellent candidate for phase I/II studies in patients with Ph−MPDs.

Characterization of JAK2 V617F Allele Burden in Advanced Myelofibrosis (MF) Patients: No Change in V617F:WT JAK2 Ratio in Patients with High Allele Burdens despite Profound Clinical Improvement Following Treatment with the JAK Inhibitor, INCB018424

Background: Activating mutations in the pseudokinase domain of JAK2 occur at a high frequency in Philadelphia chromosome-negative myeloproliferative disorders (MPDs). Increasing JAK2 V617F allele burden has been shown to correlate with disease severity (bone marrow dysfunction, organomegaly and constitutional symptoms), which is consistent with exaggerated JAK2 signaling playing a central role in MPDs. INCB018424 is a potent and selective JAK1/2 inhibitor currently being evaluated in the clinic for the treatment of MPDs including primary myelofibrosis (PMF), post-polycythemia vera and essential thrombocythemia myelofibrosis (Post-PV/ET MF).Methods: The percentage of mutant JAK2 V617F relative to wild-type JAK2 was determined in peripheral blood using a quantitative single-nucleotide extension assay. The assay is based on the single nucleotide extension of a DNA primer that anneals adjacent to the V617F mutation. The percentage of JAK2 V617F in bone marrow was determined using pyrosequencing as previously described. The relationship between JAK2 V617F allele burden and clinical phenotype was assessed with samples obtained at study entry. JAK2 V617F allele burden was assessed in nineteen PMF and Post-PV/ET MF patients treated with 25 mg BID INCB018424 for a minimum of three months using peripheral blood and bone marrow.Results: There is an excellent correlation in the JAK2 V617F allele burden between samples obtained from bone marrow and peripheral blood, suggesting that the fraction of cells bearing the mutant clone remains stable during hematopoiesis in this patient group. The mean %V617F values were 59%, 80% and 86% for Post-ET MF (n=2), Post-PV MF (n=8) and PMF (n=9) patients. V617F allele burden correlated with spleen size, absolute neutrophil count (ANC) and Body Mass Index (BMI) in this patient population (R2 values if 0.61, 0.17 and 0.44, respectively), with a higher allele burden predicting larger spleen size, a higher ANC and a lower BMI. Eighteen of the nineteen evaluable JAK2 V617F-positive patients had baseline JAK2 V617F values above 50%, with the mean %V617F value for all evaluable patients being 81%, implying that the majority of hematopoietic stem cells contain the mutant allele in this patient population. During INCB018424 therapy (at least 3 months of treatment) in these patients, profound improvements in clinical endpoints including reduction or resolution of splenomegaly and constitutional symptoms are observed, yet only a modest decrease in JAK2 V617F allele burden is seen in both bone marrow and peripheral blood (13% and 9 % decrease from baseline, respectively, at 3 months).Conclusions: While the patient group is small and heterogeneous in nature, clinical correlates of V617F allele burden observed in myelofibrosis patients in this study are similar to those reported in PV. In addition, despite profound clinical improvements in these patients, V617F allele burdens do not change dramatically suggesting that the clinical activity of INCB018424 might involve inhibition of downstream signaling in cells harboring activating JAK2 mutations rather than changing the allele burden.

The JAK2V617F tyrosine kinase mutation identifies clinically latent myeloproliferative disorders in patients presenting with hepatic or portal vein thrombosis

Clinically latent myeloproliferative disorders (MPDs) are important causes of what would otherwise be considered idiopathic hepatic (HVT) or portal vein thrombosis (PVT). They may be difficult to diagnose initially because the peripheral blood count may be normal at the time of thrombosis. A strong association between an activating mutation of the gene encoding one of the Janus kinase family of tyrosine kinases (JAK2(V617F)) and the Philadelphia chromosome-negative MPDs has been identified. We have studied 19 patients with unexplained HVT or PVT and tested for JAK2(V617F). Fourteen (74%) of the 19 patients were heterozygous for JAK2(V617F) but did not meet diagnostic criteria for a MPD at the time of presentation with thrombosis. Prolonged follow-up established the presence of an overt MPD in 13 of the 14 patients after a median duration of 38 months. We recommend testing for JAK2(V617F) in all patients with unexplained HVT or PVT, to identify latent MPDs and prevent potential complications.

Circulating angiogenic monocyte progenitor cells are reduced in JAK2V617F high allele burden myeloproliferative disorders

The clinical course of patients with Philadelphia chromosome negative myeloproliferative disorder is frequently complicated by thrombotic events. Post-natal vasculogenesis has been proposed to play a critical role in angiogenesis by acting through a hierarchy of endothelial progenitor cells. Some endothelial progenitor cells have been shown to share a number of features associated with monocytes while other more primitive progenitor cells produce endothelial cells in vitro exclusively. The cells which share features of monocytes and endothelial cells have been termed angiogenic monocytes. Reduced levels of angiogenic monocyte progenitor cells have been reported to be predictive of atherosclerotic disease progression. Angiogenic monocyte progenitor cells were assayed in vitro from the peripheral blood mononuclear cells of myeloproliferative disorder patients. Angiogenic monocyte colonies were plucked and analyzed for endothelial cells and hematopoietic cell markers, JAK2V617F and their ability to incorporate into vascular endothelium following their transplantation into non-obese diabetic, severe combine immunodeficient mice. Myeloproliferative disorder angiogenic monocyte colonies that were detected were uniformly JAK2V617F positive and produced cells that expressed phenotypic markers characteristic of both monocytes and endothelial cells. Reduced numbers of angiogenic monocyte colonies were present in the blood of myeloproliferative disorder patients with a high JAK2V617F burden (> 50%), ( p < 0.01). Transplanted angiogenic monocytes were able to contribute to the vascular endothelium of non-obese diabetic, severe combine immunodeficient mice. These studies suggest that reduced numbers of circulating angiogenic monocyte progenitors contribute to the propensity to develop thrombotic complications in myeloproliferative disorder patients.

A phase I/II study of INCB018424, an oral, selective JAK inhibitor, in patients with primary myelofibrosis (PMF) and post polycythemia vera/essential thrombocythemia myelofibrosis (Post-PV/ET MF)

7004 Background: Mutations in JAK2 and MPL have been recognized in Philadelphia chromosome-negative myeloproliferative disorders, including PV, ET and PMF, making Janus associated kinase (JAK) a therapeutic target for these diseases. INCB018424 is a potent, selective and orally bioavailable JAK inhibitor with an excellent safety profile supporting its clinical evaluation. Methods: A phase I/II trial of INCB018424 given orally BID is being conducted in patients with PMF and Post-PV/ET MF. Primary end points in this study are responses according to the International Working Group (IWG) consensus criteria for treatment response in myelofibrosis with myeloid metaplasia (IWG-MRT). Results: Overall characteristics of the 32 patients enrolled include median age of 65 yrs; 67% males; 47% PMF, 38% post-PV and 15% post ET; and 87% with JAK2V617F mutation. The starting dose of 25 mg PO BID was demonstrated to be the maximum tolerated dose (MTD) (N = 6 pts). Two patients had grade 4 thrombocytopenia in the 50 mg PO B...

Philadelphia Chromosome–Negative Myeloproliferative Disorders: An Historical Perspective

Philadelphia Chromosome–Negative Myeloproliferative Disorders: An Historical Perspective

INCB018424, an Oral, Selective JAK2 Inhibitor, Shows Significant Clinical Activity in a Phase I/II Study in Patients with Primary Myelofibrosis (PMF) and Post Polycythemia Vera/Essential Thrombocythemia Myelofibrosis (Post-PV/ET MF).

Background: A mutation in the Janus tyrosine kinase 2 gene (JAK2 V617F) has been recognized in Philadelphia chromosome-negative myeloproliferative disorders, including PV (∼95% of patients), ET (∼50%) and PMF (∼50%). INCB018424 is a potent and orally bioavailable selective JAK2 inhibitor with >80-fold selectivity against a broad panel of kinases, including JAK3. INCB018424 potently inhibits JAK2 V617F mediated signaling and malignant cell survival in vitro and in vivo in mice. Preclinical safety studies with INCB018424 demonstrated an excellent safety profile with no off-target toxicities.Methods: A phase I/II trial of INCB018424 given orally BID is being conducted in patients with PMF and Post-PV/ET MF. Both JAK2 V617F and JAK2 wild type patients are eligible. PK and PD data are being collected. Responses are being evaluated using the International Working Group (IWG) consensus criteria for treatment response in myelofibrosis with myeloid metaplasia, for the IWG for Myelofibrosis Research and Treatment (IWG-MRT) (Tefferi et al., Blood , 108, 1497–1503, 2006).Results: The initial dose of 25 mg PO BID resulted in a rapid and marked reduction in splenomegaly. All 3 patients in the first cohort of patients achieved a reduction in spleen size that is consistent with a clinical improvement response provided it is sustained for 8 weeks; spleen sizes of 25, 22, and 7 cm below the left costal margin have decreased to 8, 10, and 0 cm in the first month of therapy. At two months follow-up, the patient with a baseline spleen size of 22 cm is now down to 2 cm. All 3 patients also noted significant symptomatic improvement. The second cohort of 3 patients started therapy at an increased dose (50 mg PO BID) and at one week follow-up, the initial two patients (one JAK2 wild type, one JAK2 V617F) had decreases in spleen size from 22 cm to 17 cm and from 22 cm to 16 cm, respectively. No dose-limiting toxicities or other significant adverse events occurred in any patients to date. PK/PD analyses demonstrated that administration of INCB018424 resulted in plasma drug concentrations sufficient to markedly inhibit JAK2, as shown by suppression of phosphorylated STAT3 (a substrate of JAK2) in whole blood cells in all 3 patients in the first cohort. The 3 patients in the first cohort were all JAK2 V617F mutation positive: percentages of JAK2 V617F positive whole blood cells before therapy were 79%, 49% and 91%, and after one month of therapy, they were 59%, 48% and 78%, respectively.Conclusions: Initial results show marked reduction in splenomegaly and symptomatic improvement, without significant toxicity. The percentage of blood cells with JAK2 V617F mutation appears to be decreasing in patients on therapy. Updated results on current and future patients will be presented at the meeting.

Analysis of Mutant cMPL in Philadelphia Chromosome-Negative Myeloproliferative Disorders (Ph−MPDs) Using a Novel High-Sensitivity Assay.

cMPL is a gene encoding for the thrombopoietin receptor that is essential for thrombopoiesis and contributes to pluripotent hematopoietic stem cell expansion. A gain of function cMPL mutation, MPLW515L, was identified in myeloid cells from patients with primary myelofibrosis (PMF). Subsequent studies identified a second gain of function mutation, MPLW515K, in PMF and essential thrombocytosis (ET). The prevalence of MPLW515L and MPLW515K mutations was 5% in PMF and 1% in ET. No mutant cMPL was detected in Polycythemia Vera (PV). The methods utilized in these assays were sensitive to mutant frequencies of >3–5%. We developed a rapid, sensitive, quantitative real time PCR assay based on a unique primer design wherein allelic discrimination was enhanced by the synergistic effect of a mismatch in the −1 position, and a locked nucleic acid nucleoside at the −2 position of the allele-specific primers. An assay of similar design can detect G1849T mutation of JAK2 in <0.1% mutant allele in peripheral blood granulocyte (Nussenzveig Exp Hematol 2007 3:32). We hypothesized that a similar high sensitivity assay would increase detection of mutant cMPL in Ph−MPDs. We analyzed genomic DNA from peripheral blood granulocytes of 197 MPD patients and found that 10/197 (5.1%) carried one of the two cMPL mutations. Further, 5 of these patients were also JAK2V617F positive. cMPL mutations were detected in 1/78 (1.3%) PV patients, 3/56 (5.4%) ET patients, 4/49 (8.2%) PMF patients, and 2/11 (18%) MPD-Unspecified patients. W515L accounted for 9/10 cases, with W515K accounting for only 1. Of the ten positive samples, five (including the patient with PV) had ≤1% mutant alleles. To confirm the validity of our assay, we tested DNA from 96 normal controls. Neither W515L nor W515K was detected (p=0.03 compared to samples from the Ph−MPD patients). Additionally, when DNA from megakaryocytic colonies from a patient with 0.70% mutant alleles was analyzed, 12.5% of colonies were found to be heterozygous for cMPLW515L. These studies demonstrate the sensitivity and accuracy of our assay and show that cMPL activating mutations are more common in ET than previously reported. Mutant allele frequency appears greater in megakaryocytic cultures perhaps indicating a proliferative advantage for the cMPL-mutant clone. That mutant cMPL and JAK2V617F can be found in the same patient demonstrates the molecular heterogeneity of Ph−MPDs and emphasizes the need for prospective studies designed to determine the relationship between genotype and clinical phenotype. Scott J. Samuelson and Sabina Swierczek contributed equally to this project.

Pegylated Interferon-alfa-2a (PEG-IFN-α-2A; PEGASYS™) for Essential Thrombocythemia (ET) and Polycythemia Vera (PV): An Update of an Ongoing Phase II Study.

Therapy for patients with high-risk Philadelphia chromosome-negative myeloproliferative disorders (MPDs) involves the use of cytoreductive agents such as recombinant human interferon-alpha (IFN-α), hydroxyurea (HU), and anagrelide (AG). Despite the significant activity of IFN-α in MPDs, therapy with this agent is frequently hindered by poor tolerance and inconvenient dosing schedules. PEG-IFN-α is formulated by covalently attaching polymers of ethylene glycol to the native IFN-α molecule, which results in decreased renal excretion and increased serum half-life that allows for weekly administration with acceptable toxicity. Based on the superior pharmacokinetic profile of PEG-IFN-α-2a relative to conventional IFN-α, we designed a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) for patients with ET or PV. A total of 50 patients have been enrolled and treated thus far (22 ET, 28 PV). Median age is 53 years (range, 23–77) and time from diagnosis to PEG-IFN-α-2a 64 months (range, 1–348). Prior therapies (median 2; range 0–6) included HU (n=33), AN (n=22), phlebotomy (n=27), IFN-α (n=8: 5 oral and 3 sc), other (n=10). PEG-IFN-α-2a was the initial therapy in 4 patients. The JAK2 V617F mutation was detected in 11 (50%) of 22 ET and in 26 (93%) of 28 PV patients. Six (12%) patients had abnormal cytogenetics. Initial starting dose of PEG-IFN-α-2a was 450 mcg/week, but that was modified to the current starting dose of 90 mcg/week. Dose modifications are allowed according to response or toxicity. Patients are currently receiving 450 mcg (n=1), 360 mcg (n=1), 270 mcg (n=5), 180 mcg (n=11), 135 mcg (n=8), 90 mcg (n=9), and 45 mcg (n=5). After a median follow-up of 11 months (range, 2–28), 47 (94%) patients have responded. Complete response (CR) was achieved by 46 (92%) patients (for ET: platelets <440x109/L, off HU and AG, in the absence of thromboembolic events; for PV: Hb <15 g/dL, off HU and AG, no phlebotomy, with disappearance of splenomegaly) whereas 1 (2%) patient with PV had a partial response ([PR]; no phlebotomy, off HU and AG, but still palpable spleen). The mutant JAK2 V617F to total JAK2 ratio was determined by PCR (n=37) and by a quantitative pyrosequencing assay (n=30) prior to PEG-IFN-α-2a. JAK2 V617F mutational analysis was subsequently performed after 6 and 12 months into PEG-IFN-α-2a therapy in 20 and 10 patients, respectively. Nine (30%) of the 30 patients assessable for JAK2 V617V quantitation had >10% reduction in JAK2 V617F expression, including 2 (7%) in whom the mutant allele became undetectable. In addition, 4 (13%) patients had a 5%–10% reduction. JAK2 V617F quantitation has not been repeated yet in 9 patients. PEG-IFN-α-2a was well tolerated in most patients. Twenty-two episodes of grade 3–4 toxicity were reported, including neutropenia (n=11), elevated transaminases (n=4), and anemia, thrombocytopenia, depression, fatigue, infection, cardiac, and pain in 1 case each. Ten patients were taken off study, including 6 (12%) due to therapy-related toxicities: grade 3 neutropenia (n=1), fatigue (n=1), depression (n=1), ischemic retinopathy (n=1), dyspnea (n=1),and diarrhea (n=1). In summary, therapy with PEG-IFN-α-2a results in remarkable clinical activity and acceptable toxicity profile in patients with ET or PV. Significant reduction of JAK2 V617F allele burden occurred in a proportion of responders, suggesting selective targeting of the malignant clone.

Epigenetic control of PRV-1 expression on neutrophils

Polycythemia rubra vera-1 (PRV-1) is a GPI-linked protein that is expressed on a subgroup of neutrophils. The number of PRV-1-expressing neutrophils increases in pregnancy and sepsis, or after administration of granulocyte colony-stimulating factor. Expression of the PRV-1 gene is also increased in patients with polycythemia vera (PV) and essential thrombocythemia (ET). We investigated whether DNA methylation of the PRV-1 gene has a role in regulation of transcription and expression of the PRV-1 protein. We compared the level of methylation of the PRV-1 gene and expression of the PRV-1 mRNA in normal neutrophils expressing PRV-1 to those that are PRV-1-negative. We also studied PRV-1 methylation and mRNA expression in patients with Philadelphia chromosome-negative myeloproliferative disorders and in an in vitro model of DNA demethylation. We found that methylation of CpG dinucleotides close to initiation codon of the PRV-1 gene was inversely related to expression of PRV-1 in normal neutrophils. Furthermore, overexpression of the PRV-1 gene in PV and ET is associated with a decrease in methylation of this gene. Among patients with PV and ET, methylation of the PRV-1 gene is also inversely correlated with the presence of the JAK2(V617F) somatic mutation. In an in vitro model, exposure of KG1 and KG1a cells to a DNA demethylating agent caused a decrease in methylation of the PRV-1 gene and increased its mRNA level. DNA methylation regulates PRV-1 expression under physiologic and pathologic conditions.