Clinical Relevance Of JAK2 And CALR Mutation In Patients With BCR-ABL Negative Myeloproliferative Neoplasms

Background:In the revised 2008 WHO (World Health Organization) classification system, the term’chronic myeloproliferative diseases (CMPDs)’is replaced by’myeloproliferative neoplasms (MPNs).And BCR-ABL-negative polycythemia vera(PV)、essential thrombocythemia (ET)、primary myelofibrosis (PMF) were classified in classic myeloproliferative neoplasms (Classic MPNs) category.The myeloproliferative neoplasms are clonal disorders of hematopoietic stem cells characterized by increased proliferation of myeloid or peripheral blood cells and an increased risk of developing bone marrow fibrosis (MF) and/or acute myeloid leukemia (AML). In 2005, several groups identified JAK2 V617F (V617F) as a highly recurrent somatic mutation in patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).Aggregate studies indicate a V617F frequency of 95% to 98% in PV and 50% to 60% in ET and PMF.The discovery of the JAK2 mutation in Philadelphia-chromosome negative myeloproliferative neoplasm (MPNs) has heralded an era of rapid genetic discovery in the MPNs. This has lead to substantive gains in the understanding of the pathobiology of these diseases. Importantly, this has also lead to the first targeted therapy for these diseases in the form of JAK2 inhibitors.Individualized treatment for MPN patients was highly emphasized according to its different clinical phenotypes and risk stratification. Accordingly, the goal of therapy in PV and ET is primarily to reduce excessive blood cells and prevent thrombo-hemorrhagic complications. For PMF patients, it is based on the degree of bone marrow fibrosis and suppression. In the traditional treatment options, low-dose aspirin has been used for low-risk PV and ET in order to control the disease, and will combie with phlebotomy if necessary. For advanced PV and ET, radiation and alkylating agents have been used in order to reduce excessive blood cells. According to PMF patient’s condition, immunomodulator hematopoietic stimulate factors, component blood transfusion, alkylating agent, and other bone marrow depressants were used for therapy. Traditional therapy, to some extent can help reducing the symptoms for the comfort of the patient, but the effect was short-lived and sometimes can’t stop disease progression and contribute to leukemic transformation. At present, the new targeted drug JAK2 inhibitors is mainly used in PMF and a small number of patients with high-risk/refractory/drug-resistant ET and PV. Improvements in spleen size, symptom burden represent clinically meaningful advances with these agents. Given that current JAK inhibitors are challenged by their inability to achieve hematologic remissions and that reductions of BM fibrosis or V617F allele burden may occur in only a subset of patients and are generally modest in nature.The side effects and safety of these agents needs further evaluation and there clinical use are restricted. On the other hand, the clinical effectiveness of interferon (IFN)had been demonstrated nearly a quarter of a century ago in PV and ET patients.At present, it is still not determined whether IFN gains the therapy superiority for the PV and ET with or without positive JAK2V617F mutation. So, in the first part of our study,110 patients with an initial diagnosis of advanced MPN and received interferon or hydroxylurea treatment from December 2007 to June 2014 in our hospital were analysed. Our purpose was to evaluate the therapeutic effect and adverse reactions of IFN-a in MPN patients with JAK2V617F mutations. Those dates could provide evidence of choices of treatment for MPN, from which patients would benefit by the most optimal way to improve quality of life and clinical cure rate.However, the conundrum of how a single mutation in JAK2, which appears to be sufficient for MPN pathogenesis, could result in different phenotypically variable diseases is still unknown. Furthermore, there were a signficant proportion of patients (5%PV、50%ET,50%PMF) have no identifiable JAK2 mutations. In recent years, studies committed to the MPN pathogenesis suggest that potential somatic genetic alterations,germline variants, and epigenetic events might contribute to MPN development, influence the MPN disease phenotype,progression, and/or promote transformation.The most relevant mutations identified so far can be broadly classified into two main groups:1-Mutations associated with dysregulated JAK-STAT signal transduction pathway, including mutations in JAK2, MPL, c-CBL, SH2B3, SOCS; 2-Mutations in several epigenetic modifiers, including mutations in TET2, ASXL1, IDH1/2, DNMT3A, EZH2 and so on. Almost simultaneously at the end of 2013, two research groups discoveried the insertion and/or deletion mutations within the CALR gene in a signficant proportion of JAK2-and MPL-unmutated essential thrombocythaemia (ET) and primary myelfibrosis (PMF) patients. It was a sign that the development of MPN have a dramatic breakthroughs. The relatively high rate of CALR mutation in JAK2-and MPL-unmutated ET and PMF patients makes it as another specific molecular markers in these patients. There is still lack of related research in our country. So, in the second parts of our study, Polymerase Chain Reaction was performed on genomic DNA samples from 205 MPN patients to detect the mutation of exon 9 of CALR gene, and then we will discuss the relationship and clinical significance between CALR mutation and MPN patients. We hope it will help to the MPN stratified diagnosis, prognostic and individualized treatment.Objective:1. Whether interferon alpha (IFN-a) has special therapeutic effect for myeloproliferative neoplasm (MPN) patients with JAK2V617F mutations was not widely confirmed. Our purpose was to evaluate the therapeutic effect of IFN-a in MPN patients with JAK2V617F mutations.2. To evaluate the frequency of CALR gene mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPN), and analyze its correlation with clinical fea tures of essential thrombocytosis (ET)Methods:1. 110 patients (age:19-82 years old, the median age 54) with an initial diagnosis of advanced MPN from December 2007 to June 2014 in our hospital were analysed retrospectively.ET patients received interferon or hydroxylurea treatment and were divide into groups according to JAK2 mutation status.For PV, The curative effect of interferons alpha and hydroxyurea was only compared in JAK2V617F mutation positive patients for their mutation rate of more than 90%.PMF patients were not included in this study due to their Clinical features, risk stratification and therapeutic schedule. The blood routine、liver and kidney function of the patients enrolled in this study were regularly tested in order to assess the progress of the disease、The median follow-up of 32.0 (6.0 to 60.0) months.2. Polymerase Chain Reaction was performed on genomic DNA that was isolated from bone marrow and/or granulocyte-enriched peripheral blood s amples from 205 MPN patients (male 107 cases, female 98 cases; age 19-82 years old, the median age of 55. PV 59 cases, ET 129 cases, PMF 17 cases. JAK2V617F+ PV 55 cases, JAK2V617F+ET 69 cases, JAK2V617F+PMF 9 cases), to amplify the high mutation domain of exon 9 of CALR gene. Then, the amplified product was purified and sequenced in both direction. The homologous analysis was performed in combination of clinical data. Patient’s white blood cells, platelet count, hemoglobin level in peripheral blood and the size of spleen, thromboembolism and bleeding events were recorded when first diagnosed, and will combined with laboratory data for statistical analysis.Results:1. The overall response rate between IFN-a and Hydroxyurea therapy groups of essential thrombocytosis (ET) patients with JAK2V617F mutations had no significant difference((88.2% vs 85.0%,P>0.05),but the 5-year progression-free survival rate of IFN-a and Hydroxyurea therapy groups showed significant difference(88.2% vs 55.0%, P<0.05). The overall response rate (78.6% vs 82.4%) and 5-year progression-free survival rate (57.1% vs 58.8%) between IFN-a and Hydroxyurea therapy groups of ET patients without JAK2V617F mutations had no significant difference(P>0.05). The overall response rate between IFN-a and Hydroxyurea therapy groups of polycythemia vera (PV)patients with JAK2V617F mutations had no significant difference(80.0% vs 75%, P>0.05),but the 5-year progression-free survival rate of IFN-a and Hydroxyurea therapy groups showed significant difference(86.7% vs 50.0%, P<0.05). Six months after treatment, the ratio of Interferon-treated patients need to continue phlebotomy was significantly lower than hydroxyurea therapy group (8.3% vs 58.3%, P<0.05). The thromboembolic events, splenomegaly, bone marrow fibrosis of interferon treatment group were lower than hydroxyurea treatment group showed significant difference(P<0.05).The adverse reactions of IFN-a was moderate, most of the patients in this study could tolerate the therapy. The major side effect of hydroxyurea was hematologic adverse reactions (Grade 1-2) with mainly reduce of white blood cells and thrombocytopenia, which showed difference between IFN-α and hydroxyurea (P<0.05).2. Of the 205 patients, the total mutations was 178 (86.8%). JAK2 mutations were detected in 133 (64.9%), CALR in 45 (22.0%), both negative (JAK2, CALR) in 27 (13.2%).CALR mutational frequency was 30.2% among ET patients (n=39), and 65% among those not mutated for JAK2 (39/60). For PMF patients, the frequency of CALR mutation was (29.4%) (n=5), and 62.5% among those not mutated for JAK2 (5/8).Whereas one PV patient and one ET patint displayed both CALR and JAK2 mutations. There were 66.7%(30/45) type 1 (52 bp deletion) and 24.4%(11/45) type 2 (5 bp insert) mutations that contain 91.1% (41/45) of all the kind of types. Among the 127 study ET patients, the median ages (P=0.037) and risk stratification (P=0.04) of patients with JAK2 mutation were higher than those of patients with CALR mutations; the ET patients with JAK2 mutations had an even higher white blood cell count and hemoglobin than patients with CALR mutations (P=0.006, P=0.038) or both negative (JAK2, CALR) (P=0.000, P=0.015), and lower platelet count than patients with CALR mutations or both negative (JAK2, CALR) (P=0.013,P=0.024), with statistical significance. Furthermore, the number of patients with asymptomatic with CALR mutation were more than patients with JAK2 mutation (P=0.001); there were 25 (19.7%) patients were admitted to hospital because of the thrombotic events, and among these the number of patients with JAK2 mutation were more than patients whit CALR mutation (P=0.029);30 (23.6%) patients were discovered with splenomegaly at diagnosis, and the incidence of splenomegaly in patients with JAK2 mutation was higher than patients with CALR mutation (P=0.018), shows significant differencesConclusion:1. ET and PV patients with JAK2V617F mutations who were treated with IFN-α can get a better progression-free survival. And for PV patients with JAK2V617F mutations can get rid of phlebotomy more ideal.2. The mutation of exon 9 of CALR gene is a major molecular genetic marker for Chinese patients with BCR-ABL negative myeloproliferative neoplasms, and for ET patients, it may has a certain influence on the phenotype and clinical features.