Negative Primary Myelofibrosis Research Articles

The Clinical Characteristics and Outcomes of JAK2/ CALR/MPL Mutation Related Myeloproliferative Neoplasms - A Single Centre Study from Malaysia

INTRODUCTION: The pathogenesis of classical myeloproliferative neoplasm (MPN) driven by JAK2, CALR and MPL gene mutations affects the clinical course and survival. This study aimed to determine the prevalence of driver mutations in MPN patients and their association with clinical presentations and outcomes. MATERIALS AND METHODS: We conducted a retrospective study involving patients with classical MPN diagnosed from 2002 to 2019. The patient's clinical and laboratory information, as well as outcomes, were collected and reviewed. RESULTS: 267 patients with classical MPN were recruited into the study. Majority of these patients were Chinese (46.5%), followed by Malay (40.1%) and Indian (12.7%). Most of the patients had essential lthrombocythaemia (ET) (57.3%), followed by polycythaemia vera (PV) (30.0%) and primary myelofibrosis (PMF) (12.7%). JAK2V617F mutation was detected in PV (87.5%), ET (68.0%), and PMF (67.6%) patients whereas CALR mutation was present in 15.0% of ET and 8.8% of PMF patients and MPL mutation was present in 0.7% and 5.9% of ET and PMF patients respectively. CALR-mutated ET patients were less likely to develop vascular events compared to JAK2V617F mutated patients (Odds ratio 0.301, 95% confidence interval 0.097–0.939, p=0.039). As for clinical outcomes, triple negative PMF patients had shorter median overall survival than those with JAK2V617F mutation (24.0 months vs. 161.0 months, p=0.017). CONCLUSION: Majority of classical MPN patients were Chinese with ET being the most common MPN subtype. The mutation profiles, clinical features, and survival outcomes were comparable to previous reports. Mutation studies are therefore important for prognostication and should be performed routinely.

Obliteration of liver sinusoids through platelet aggregates associated to extramedullary haematopoiesis in myeloid neoplasms.

Herein is reported a series of five patients with myeloid neoplasms presenting hepatic complications in whose liver biopsy revealed obstruction of sinusoids by platelet aggregates associated to liver extramedullary haematopoiesis. Indication of liver biopsies was jaundice, unexplained hepatomegaly or portal hypertension. Haematological disorders were classified according to the World Health Organisation. Molecular profile was established in all cases as well as grade of liver extramedullary haematopoiesis and myelofibrosis. The patients were four men and one woman aged from 50 to 82years. Two patients had myeloproliferative neoplasm (triple negative primary myelofibrosis and JAK2-mutated essential thrombocytopenia), two patients had unclassifiable myelodysplastic/myeloproliferative neoplasm and one patient had chronic myelomonocytic leukaemia type 1. Liver biopsies revealed platelet aggregates occluding sinusoids in association with extramedullary haematopoiesis grade 1 in one patient, grade 2 in two patients and grade 3 in two patients. Two of these patients presented co-existing liver fibrosis due to chronic alcoholic consumption and ischemic heart failure. These five patients died from 2 to 23months after liver biopsy due to acute myeloblastic leukaemia (three patients), portal hypertension (one patient) or other causes (acute heart failure). Intrahepatic sinusoidal microthromboses through platelet aggregates might cause portal hypertension or liver deficiency in patients with myeloid neoplasms, independently of JAK2 mutational status and grade of extramedullary haematopoiesis.

Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET).
 Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR.
 Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations.
 Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Show Safety and Early Clinical Signal in Primary Myelofibrosis

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Show Safety and Early Clinical Signal in Primary Myelofibrosis

PS1449 DISSECTING THE THERAPEUTIC MECHANISM OF RUXOLITINIB IN JAK2V617F MEDIATED MPNS REVEALED AN INHIBITION OF BOTH ONCOGENIC AND NON-ONCOGENIC CELLS BY DOWN REGULATION OF INFLAMMATORY CYTOKINE LEVELS

Background: Ruxolitinib is a potent JAK2/JAK1 specific inhibitor approved by the FDA and EMA for the treatment of primary myelofibrosis (PMF). The primary clinical benefits of JAK2 inhibitors in PMF patients are reduction in spleen size, alleviation of constitutional symptoms and a significant improvement in the quality of life. However, no significant improvement of bone marrow fibrosis has been demonstrated so far, and the effect on the JAK2V617F allelic burden is modest. In addition, the beneficial clinical effects were also achieved in JAK2V617F negative PMF patients. Aims: It is unclear at the moment whether Ruxolitinib primarily blocks proliferation of the malignant clone or exerts its effects by inhibiting bystander normal hematopoietic cells or bone marrow niche cells. By clinical investigation it is not possible to dissect between these two modes of inhibitor action. However, JAK2V617F variants which are resistant towards clinically used inhibitors can be expressed in mice. Such a mouse model would allow investigation of the mode of action of the JAK2 inhibitors in PMF patients. Methods: So far no inhibitor resistant JAK2 mutations have been reported in patients. Therefore, a comprehensive cell based screening strategy was used to identify Ruxolitinib resistant JAK2V617F variants. Results: Using cell based screening method, we were able to identify seven specific mutations which confer resistance to Ruxolitinib while retaining the kinase activity. For this study, we used JAK2V617F+L902Q and JAK2V617F+L983F variants since these two variants were highly resistant towards the Ruxolitinib. In order to determine the disease phenotype of JAK2V617F+L902Q and JAK2V617F+L983F we transplanted retrovirally infected JAK2V617F and JAK2V617F+L902Q and JAK2V617F+L983F bone marrow into recipient mice. Similar to JAK2V617F recipients, JAK2V617F+L902Q and JAK2V617F+L983F transplanted mice showed an increase in WBC, HCT, RBC, HB and reticulocyte values. Histopathological analysis revealed that the myeloproliferative phenotype resembles PV with a proliferation of all three lineages albeit with only a moderate increase in megakaryopoeisis. Grade II bone marrow fibrosis was observed in JAK2V617F+L902Q and JAK2V617F+L983F after 2–3 months similar to JAK2V617F mice. Ruxolitinib treatment lead to a decrease in spleen size, length and WBC number in JAK2V617F mice however, surprisingly, JAK2V617F+L902Q and JAK2V617F+L983F mice also decreased the spleen size, length and WBC number after Ruxolitinib treatment. FACS analysis of different hematopoietic cell population from peripheral blood, spleen and bone marrow results suggested that Ruxolitinib mainly act on eGFP+ neutrophils and also on eGFP− neutrophils, B and T cells in JAK2V617F, JAK2V617F+L902Q and JAK2V617F+L983F MPN mouse models. Finally, analysis of pro inflammatory cytokines results from Ruxolitinib treated mice results showed significant down regulation of several cytokines including TNF-alpha, MCP-1, IL-6, INF-beta, IL-27 and IL-23 in both JAK2V617F and JAK2V617F+L902Q MPN mouse models suggesting that ruxolitinib mediated clinical benefits might also be due to the down regulation of inflammatory cytokines by inhibition of JAK1/2 signaling in both hematopoietic as well as non-hematopoietic cells. Summary/Conclusion: Taken together, our results clearly suggest that identification of novel inhibitors that specifically act on malignant cells and spare the non-malignant cells which play a major role in normal hematopoiesis will improve the clinical efficacy in PMF patients.

PB2201 PROGRESS AND SURVIVAL IN TRIPLE NEGATIVE PRIMARY MYELOFIBROSIS PATIENTS

Background: Primary myelofibrosis (PMF) is a Philadelphia negative myeloproliferativ neoplasm characterized by a clonal myeloid expansion which determines progressive bone marrow fibrosis resulting in inefficient hematopoiesis. Diagnosis is based both on bone marrow morphology but also clinical aspects. There were described several mutations such as those in JAK2 V617F, MPL exon 10 and CALR exon 9 indel genes in aproximately 90% of the patients while the rest of 10% were so-called triple negative. Aims: To evaluate survival and prognosis in triple negative PMF patients. Methods: In this retrospective study, 58 patients diagnosed with PMF were enrolled from January 2014 to December 2018. Data were analyzed with SPSS 24. Results: The median age was 58 years. The male to female ratio was 3:1. Of the total 58 patients studied, 30 (51,72%) carried JAK2 (V617F), 14 (24,14%) showed positivity for CALR exon 9 indel, 4 (6,9%) had MPL (exon 10) mutation and 10 (17,24%) were so-called triple negative PMF. Median overall survival was 3,8 years in CALR mutant patients, 2,1 years in those presenting mutation in JAK2 gene, 2,3 years in MPL mutant patients and 1,6 years in triple negative patients. An analysis of the frequency of blast transformation at 1 year, revealed that it was identifyied in 71% of triple negative patients, 22% of JAK2 mutant patients and in 3% of both CALR and MPL mutant patients. Summary/Conclusion: This study reveals the importance of genetic analysis not only for diagnosing PMF but also for risk stratifying, prognos estimation and furthermore treatment choice. Triple negative PMF patients had a higher incidence of blast transformation compared with those presenting mutations.

Triple Negative Myelofibrosis and Myelodysplastic Syndrome with Fibrosis: Clinico-Biological Characterization and Correlation with Gene Mutations

Abstract Introduction: Triple negative primary myelofibrosis (TN-PMF) and myelodysplastic syndromes with fibrosis (F-MDS) are rare entities, often difficult to distinguish each other. Currently, no specific molecular markers allowing a precise differential diagnosis are available. In this sense, next generation techniques (NGS) might be useful to distinguish between both entities and to refine prognosis. Methods: Thirty-nine patients with TN-PMF (n=16) or F-MDS (n=23) were analyzed, Targeted NGS was performed in 28 cases (10 TN-PMF and 18 F-MDS) using the Sophia Genetics Myeloid Tumor Solution Panel including the following genes: ABL1, ASXL1, BRAF, CALR, CBL,CEBPA, CSF3R,CSNK1A1,DNMT3A, ETV6, EZH2, FLT3,HRAS, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, WT1 and ZRSR2. High molecular risk (HMR) was defined by the presence of any mutation in ASXL1, SRSF2, EZH2 or IDH1/2 genes. Clinical and biological data at diagnosis were compared among both groups of patients, including bone marrow cytogenetics, bone marrow histology, and the results from NGS analysis. The probabilities of survival and progression to acute leukemia were estimated by the Kaplan Meier method using the log rank test for comparisons. Results: Median age at diagnosis for the whole series was 63 years (range 42-87). Cytogenetic abnormalities were detected in 16 (57%) out of 28 assessable patients, being classified as low risk (n=17), intermediate risk (n=5), and high risk (n=6) There were no significant differences among TN-PMF and F-MDS regarding age, sex, hematological values, spleen size or cytogenetic risk or the number of blasts in peripheral blood or bone marrow. Mutations were detected by NGS in 26 out of 28 (93%) patients. Median number of mutations was 3 (Range: 1-7). Most frequent mutated genes were: ASXL1 n=10 (36%), TET2 n=7 (25%), SRSF2 n=6 (21%), DNMT3A n=6 (21%), U2AF1 n=5 (18%), ETV6 n=4 (14%), SETBP1 n=3 (11%), RUNX1 n=3 (11%), EZH2 n=2 (7%), TP53 n=2 (7%), ZRSR2 n=2 (7%) and KMT2A n=2 (7%). Mutations in either TET2, U2AF1, SETBP1, TP53 or RUNX1 genes were observed in only 1 patient with TN-PMF in contrast with 12 out of 18 (67%) F-MDS cases (p=0.006). HMR mutations were detected in 12 cases (43%) with 7 of them (21%) carrying two or more HMR mutations. There were no significant differences among TN-PMF and F-MDS regarding the total number of mutations per case or in the presence of HMR mutations. Median survival was 1.6 and 3.7 years for patients with TN-PMF and F-MDS, respectively (p=0.4). Leukocyte count > 25x109/L and presence of blasts ≥ 1% in peripheral blood were associated with a significantly lower survival. High risk cytogenetics and ≥ 2 HMR mutations were associated with a tendency towards lower survival. Median survival was 1.6 years in patients with ≥ 3 mutations in comparison with 8.3 years in those with < 3 mutations, respectively (p=0.03). Progression to acute myeloid leukemia was observed in 11 patients, resulting in a probability of 32% at 3 years. There were no significant differences in the probability of progression to acute leukemia among TN-PMF and F-MFDS. Time to acute leukemia was significantly shorter in patients with ≥ 3 mutations (3-year probability 45% versus 16%, p=0.039). Conclusions: TN-PMF and F-MDS showed a high rate of mutations in myeloid genes with TET2, U2AF1, SETBP1, TP53 or RUNX1 being more frequently mutated in F-MDS. Despite survival being short in both entities, NGS allowed the identification of a subgroup of patients with especially poor prognosis characterized by the presence of ≥ 3 mutations. Disclosures Hernandez Boluda: Novartis: Consultancy; Incyte: Consultancy. Cervantes:Hospital Clinic Barcelona: Employment; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 37: An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome

Abstract Introduction: Activating Janus kinase 2 (JAK2) mutations have been described in myeloproliferative neoplasms (MPNs) since 2005. Since its initial discovery, the JAK2 V617F mutation has been described in more than 95% of patients with polycythemia vera (PV) and in 50-60% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Subsequent efforts were made to identify molecular mutations in MPN cases lacking the V617F mutation and further mutations in the exon 12 domain of the JAK2 were identified in PV patients. However, there have been only a few studies evaluating the presence of these JAK2 exon 12 mutations in PMF patients. Case Report: A 60-year-old Malay gentleman with no known prior medical history of note presented at our Emergency Department with a 1-week history of bipedal edema and exertional dyspnea. Physical examination revealed bibasal coarse crepitations with moderate hepatosplenomegaly. Laboratory investigations revealed normochromic, normocytic anemia, thrombocytopenia, hypoalbuminemia, and elevated creatinine levels. A spot urine sample showed a protein to creatinine ratio of 93 mg/mmol with an estimated total urinary protein (TUP) of 0.8 g/day. Further workup of his renal impairment, which included an autoimmune screen, hepatitis, and humman immunodeficiency tests, were all unremarkable. A myeloma screen revealed elevated immunoglobulin G levels of 29.18 g/L but with no monoclonal bands detected. Transthoracic echocardiography showed mild pulmonary hypertension with a pulmonary artery systolic pressure (PASP) of 45 mmHg, but the ejection fraction was normal at 65%. He was also noted incidentally to have a prolonged prothrombin time (PT), activated partial thromboplastic time (APTT), and thrombin clotting time. Fibrinogen levels were normal and a lupus anticoagulant was not detected. Bone marrow aspiration and biopsy results were consistent with myelofibrosis and diffuse coarse reticulin fibers of grade 4 were reported. Quantitative BCR-ABL messenger ribonucleic acid (mRNA), JAK2 V617F, and calreticulin (CALR) mutations were negative. However, a JAK2 H538QK539L (mutation leading to a substitution of glutamine for histidine at position 538 and leucine for lysine at position 539) in exon 12 was detected. Incidentally, iron stores were absent on the bone marrow aspirate sample. Ultrasound (US) and computed tomography (CT) scans showed the presence of hepatosplenomegaly with portal hypertension with no evidence of any venous thrombosis. A nonobstructive left renal calculi was seen but no other renal abnormality was detected. He was started on low-salt diet, fluid restriction, diuretic, and iron supplements. Within 1 month, his hemoglobin increased from 7.7g/dL to 14 g/dL with a rise in hematocrit to 52.1%. His iron supplements were stopped and he required 3 sessions of venesection in total to control his hematocrit. Subsequently, he was commenced on hydroxyurea for cytoreduction. His spleen reduced in size and his proteinuria normalized after 10 months. However, he developed worsening anemia and hydroxyurea had to be stopped. On follow-up, his hemoglobin increased to 12 g/dL with a baseline platelet count of > 100 X 109/L. Throughout this period, his bipedal edema remained chronic but he was not consistent with his renal follow-ups. Within 12 months, his proteinuria started increasing and is now within the nephrotic range. Discussion: We describe here a patient who was diagnosed with a JAK2 V617F negative PMF. Subsequent analysis revealed that he was harboring a JAK2 exon12 mutation instead. This was associated with a deranged coagulation profile and proteinuria which responded to hydroxyurea. This patient was never diagnosed with PV previously and thus did not meet the criteria for post-PV myelofibrosis. Also, we believe that this is the first case of a JAK2 exon12 mutation occurring in a myelofibrotic patient. Given the recent description of MPN-related glomerulopathy and the response of this patient to hydroxyurea, we believe further investigations into the associations of triple-negative (JAK2 V617F, myeloproliferative leukemia virus oncogene (MPL) and CALR negative) PMF with JAK2 exon12 mutations and the associated organ involvements may increase our knowledge of the pathogenesis of this rare condition. Citation Format: Moon Ley Tung, Hersharan Kaur Sran. An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 37.

Calr Vs. JAK2 Vs. MPL Mutations in the Progression of Philadelphia-Negative Myeloproliferative Neoplasms

Abstract Objective: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by proliferation in one or more myeloid lineages. The classical Philadelphia (Ph) chromosome-negative MPN includes polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). All of these diseases are associated with an increased risk of thrombo-hemorrhagic complications, progression to myelofibrosis or acute leukemia transformation, which remains the primary source of mortality. Mutations in JAK2 (JAK2 V617Fand JAK2 exon 12), MPL (MPL W515L/K) and CALR have been identified as the driver of MPN, and thus included as the major diagnostic criteria for MPN in the 2008 WHO diagnostic criteria. It has been reported that patients bearing similar JAK2 mutations may present different phenotypes of MPNs. In addition, these gene mutations are involved in heterogeneity in clinical manifestations and different natural histories with progression to post polycythemia vera/essential thrombocytosis myelofibrosis (post-PV/ET MF) and acute myeloid leukemia (AML). The main purpose of the current study is to investigate the effects of recurrent gene mutations (CALR/JAK2/MPL) on the overall survival (OS) and progression with a cohort of 1353 Chinese patients with different subtypes of MPNs. Methods: In the current study, a cohort of 1353 MPN patients were screened for mutations in JAK2 (exon 12), CALR (exon nine), and MPL exon (ten). MPN patients were divided into seven groups according to the clinical diagnosis and mutations: CALR mutated PMF or ET patients, JAK2 mutated PV, ET or PMF patients, and triple (CALR / JAK2 / MPL) negative PMF or ET patients. Overall survival (OS) was calculated from the time of diagnosis of MPN to death or the last follow-up. Myelofibrosis-free survival (MFS) and leukemia-free survival (LFS) were calculated from the time of diagnosis of PV/ET/PMF to the occurrence of post-ET/PV MF, leukemia, death or the last follow-up. Results: Multivariate analysis indicates that triple-negative mutation of CALR/JAK2/MPL is an independent risk factor of poor OS (HR = 2.47, 95%CI: 1.17-5.22; p= 0.018) as well as leukemia-free survival (LFS, HR = 2.83, 95%CI: 1.19-6.72; p= 0.013) in patients with PMF, but not those with ET. Compare to JAK2 mutation, CALR mutations do not correlate with significantly better OS (p= 0.59) or LFS (p= 0.748) in PMF patients. Interestingly, CALR mutated ET patients have a trend of worse myelofibrosis-free survival rate (MFS) than triple-negative patients, while similar to that of patients with JAK2 mutation (p= 0.93).The multivariable analyses, including age and sex,reveal that JAK2 mutated PV patients (HR = 2.51, 95%CI: 1.55-4.05; p Conclusion: Triple-negative mutation is a worse prognostic factor in PMF patients; JAK2 mutated PV patients have a worse MFS compared toET patientswith JAK2 mutation. Disclosures No relevant conflicts of interest to declare.

Analysis of DNA methyltransferase 3A gene mutations in patients with Philadelphia-negative myeloproliferative neoplasms

Context: Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) from a group of disorders characterized by dysregulated JAK-STAT functionality, abnormal hematopoiesis, as well as increased production of proliferative cytokines. In addition to JAK2V617F mutation, additional gene alterations that are involved in epigenetic mechanisms, particularly de novo DNA methyltransferase 3A (DNMT3A), have been described in Ph-negative MPN biology. Aims: The aim of this study is to evaluate the H/C/S/P mutations in codon R882 of DNMT3A gene among patients with Ph-negative MPNs. Subjects and Methods: This study was conducted on 64 newly diagnosed patients with PV, ET, and PMF who referred to Shafa Hospital, Ahvaz, Iran. In the beginning, 5 mL whole blood was drawn from each patient, and the DNMT3A R882 codon mutations were investigated following the isolation of peripheral blood mononuclear cells by DNA amplification protocol using polymerase chain reaction and DNA sequencing techniques. Results: The R882H G > A mutation, which results in an amino acid substitution at position 882 of DNMT3A gene from arginine (R) to histidine (H), was observed in two patients (3.1%) with JAK2V617F positive PV and JAK2V617F negative PMFs. Conclusions: Based on the results, DNMT3A-R882 mutations occur at a low frequency in patients with Ph-negative MPNs. To the best of our knowledge, this is the first study to specifically estimate the prevalence of DNMT3A mutations among Ph-negative MPN patients living in the Middle East. It is recommended to investigate these mutants as a secondary defect along with common major complications in such patients.

A Novel CSF3R Mutation Uncovers the Importance of Membrane-Proximal N-Glycosylation for Receptor Regulation

Abstract Mutations in CSF3R (aka GCSFR) occur in the majority of patients with Chronic Neutrophilic Leukemia (CNL) and also are rarely found in adult and pediatric Acute Myeloid Leukemia. The most common CSF3R mutation in CNL is T618I (aka T595I), a point mutation in the membrane-proximal extracellular domain that causes ligand independence. Mutations that lead to a premature stop in the cytoplasmic domain are also found in CNL and result in increased expression of CSF3R on the cell surface. Understanding the biology of other novel mutations in CSF3R may lead to insight into both receptor biology and oncogenesis. The CSF3R N610H mutation was identified in a patient with a myeloproliferative neoplasm, which was most consistent with a JAK2, CALR, MPL mutation negative primary myelofibrosis. This patient had a history of mild leukocytosis for several years with most recent white blood cell counts between 13.3 and 15.3 x 103/uL. A bone marrow biopsy revealed 90% cellularity with mildly increased reticulin fibrosis, increased myeloid to erythroid ratio, no overt dysplasia, and less than 5% blasts. The cells were karotypically normal with a micro deletion of the 3' end of PDGFRB (5q) identified by FISH at 59%. The patient has had minimal symptoms with no anemia or thrombocytopenia and is currently being monitored but not receiving any intervention. The patient's bone marrow was next-generation sequenced using a 42-gene myeloid malignancy targeted mutation hotspot panel which revealed a mutation at N610H in CSF3R at a 50% mutant allele frequency. Given its proximity to the most common CSF3R mutation found in Chronic Neutrophilic Leukemia (T618I, aka T595I), we were interested in understanding whether the N610H mutation might contribute to disease biology. N610 (also known as N586 in the traditional numbering system that does not include the signal peptide) is part of an N-X-T motif, which is a consensus sequence for N-linked glycosylation. Haniu et al (Biochemistry 1996) demonstrated that N610 is one of 8 sites that are N-glycosylated on CSF3R. We confirm by mass spectrometry (MS) based analysis that N610 is occupied with a bisecting complex N-glycan (in vitro). We further found that the N610H mutation and a more conservative N610Q substitution are highly activating in CSF3R, leading to cytokine-independent growth in the murine Ba/F3 cell line. Furthermore, like the T618I mutation, these mutations render the receptor ligand-independent. N610H and N610Q lead to a robust increase in downstream signaling through the JAK/STAT pathway as demonstrated by an increase in the levels of phospho-STAT3. The loss of N-glycosylation in the membrane-proximal region of CSF3R may therefore increase ligand-independent receptor activation and promote oncogenesis. This study highlights the insight that rare human mutations can provide into the relationship between receptor structure and function. Disclosures Radich: Ariad: Consultancy; Novartis: Consultancy, Research Funding; Gilliad: Consultancy; Incyte: Consultancy. Bertozzi:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Catalent Biologics: Membership on an entity's Board of Directors or advisory committees; Verily: Membership on an entity's Board of Directors or advisory committees; Enable Bioscience: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Oh:CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Background:The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome.Material and Methods:Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9 mutations in 100 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U).Results:The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF.Conclusions:The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms.

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34+ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34+ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Incidence and Clinical Features of Calreticulin mutation in Patients with Jak2V617F-Negative Essential Thrombocytosis and Primary Myelofibrosis from Singapore

Abstract Background: Slightly less than half of the patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF) do not have specific gene mutations. Recent discovery of CALR mutation in JAK2V617F-negative ET and PMF patients have accounted for 20-25% of ET and PMF patients, and therefore redefining diagnostic approach to ET or PMF. Here we reported the incidence of CALR mutation and clinical features of clinically diagnosed Jak2V617F-negative ET and PMF in Singapore. Method: We retrospectively identified 140 cases of Jak2V617F-negative ET and PMF from two healthcare institutions in Singapore since 2006. Sanger sequencing was performed for the targeted detection of CALR exon 9 mutations. Clinicopathologic correlation was carried out by assessing relevant clinical data and the CALR mutation status, and compared with JAK2-positive cases (25 cases of ET and 9 cases of PMF). Results: The Jak2V617-negative ET/PMF patients were ethnic Chinese (76.2%), Malay (13.9%) and others (9.9%). CALR mutations were detected in 32.8% (40/122) of Jak2V617-negative ET and 55.6% (10/18) Jak2V617-negative PMF patients. Higher platelet count was observed in CALR-positive ET patients with median platelet count of 1056.5 x 109/L and 637.5 x 109/L for CALR-positive and -negative patients respectively (p<0.001). CALR-positive ET also required higher doses of hydroxyurea to maintain platelet count below 600 x 109/L (p=0.038). There was no difference in thrombotic events, presence of splenomegaly, presenting WBC count or hemoglobin (Hb) level between CALR-positive and -negative ET. However, when compared to Jak2-positive ET, Jak2-negative ET had a significantly lower incidence of thrombotic events (Jak2-negative ET, 8.2%; Jak2-positive ET, 50.0%, p<0.001), and lower Hb level (p<0.001). The incidence of thrombotic events in CALR-positive ET and PMF were 10.3% and 10.0% respectively. There was also no significant difference in presenting WBC count, platelet count, Hb level, presence of splenomegaly, or thrombotic event between CALR-positive vs -negative PMF. Lactic acid dehydrogenase (LDH) level at diagnosis however, was significantly higher in CALR-positive ET/PMF when compared to CALR-negative group (p=0.011). There was also significantly higher incidence of elevated peripheral blasts of >1% in CALR-positive ET/PMF (12% in CALR-positive and 3.3% in CALR-negative group, p=0.045). Blast transformation occurred in 6% of patients with CALR-positive PMF while only 1.1% in CALR-negative PMF. Besides the two commonly reported mutations (CALR52del, 30.8%; CALR5ins, 33.3%) in our screening of the CALR exon 9 in Jak2V617F-negative ET and PMF patients, 7 other novel mutations (c.1098_1137delinsTTTCTT, c.1102_1104delAAG, c.1122_1155delinsGGGCCAGGCACTTGTCG, c.1129_1153delinsCTTTGCGTTTCTTTT, c.1132_1155delinsTGTCG, c.1139delA, c.1141delG) were also found individually. All mutant CALR proteins possess an altered C-terminus with a longer peptide stretch caused by a disrupted reading frame due to these frameshift mutations. Conclusion: CALR mutation is common in Jak2V617F-negative PMF and ET in this multiracial Singaporean cohort, and has similar frequency when compared with Western report. It was associated with higher platelet count in ET, higher level of LDH and possibly a higher risk of blast transformation in PMF. 7 novel CALR mutations were identified and more subtypes of CALRmutation variants are expected to be uncovered in the near future. Disclosures No relevant conflicts of interest to declare.

Safety and Efficacy of INCB018424, a JAK1 and JAK2 Inhibitor, in Myelofibrosis

Myelofibrosis is a Philadelphia chromosome–negative myeloproliferative neoplasm associated with cytopenias, splenomegaly, poor quality of life, and shortened survival. About half of patients with myelofibrosis carry a gain-of-function mutation in the Janus kinase 2 gene (JAK2 V617F) that contributes to the pathophysiology of the disease. INCB018424 is a potent and selective Janus kinase 1 (JAK1) and JAK2 inhibitor. We conducted a phase 1−2 trial of INCB018424 in patients with JAK2 V617F−positive or JAK2 V617F−negative primary myelofibrosis, post–essential thrombocythemia myelofibrosis, or post–polycythemia vera myelofibrosis. A total of 153 patients received INCB018424 for a median duration of more than 14.7 months. The initial dose-escalation phase established 25 mg twice daily or 100 mg once daily as maximum tolerated doses, on the basis of reversible thrombocytopenia. A dose-dependent suppression of phosphorylated signal transducer and activator of transcription 3 (STAT3), a marker of JAK signaling, was demonstrated in patients with wild-type JAK2 and in patients with the JAK2 V617F mutation. We studied additional doses and established that a 15-mg twice-daily starting dose, followed by individualized dose titration, was the most effective and safest dosing regimen. At this dose, 17 of 33 patients (52%) had a rapid objective response (≥50% reduction of splenomegaly) lasting for 12 months or more, and this therapy was associated with grade 3 or grade 4 adverse events (mainly myelosuppression) in less than 10% of patients. Patients with debilitating symptoms, including weight loss, fatigue, night sweats, and pruritus, had rapid improvement. Clinical benefits were associated with a marked diminution of levels of circulating inflammatory cytokines that are commonly elevated in myelofibrosis. INCB018424 was associated with marked and durable clinical benefits in patients with myelofibrosis for whom no approved therapies existed. (Funded by Incyte; ClinicalTrials.gov number, NCT00509899.)